The present invention relates to a nucleic acid molecule encoding a fusion protein, wherein the nucleic acid molecule comprises: (a) a first nucleic acid sequence encoding a transmembrane domain linked to a first biosensor, wherein said first biosensor is a first molecule capable of interacting with a second molecule to form part of a first inducible interaction module, and wherein said first biosensor is linked to the transmembrane domain such that the first biosensor is located intracellular^ upon expression of the fusion protein in a cell; (b) a second nucleic acid sequence encoding an effector-activating module, wherein the effector-activating module comprises: (i) a nucleic acid sequence encoding a first part of a protease, wherein said first part of the protease is capable of interacting with a second part of said protease to form an active form of said protease; or (ii) a nucleic acid sequence encoding a second biosensor, wherein said second biosensor is a first molecule capable of interacting with a second molecule to form part of a second inducible interaction module; (c) a third nucleic acid sequence encoding a third biosensor comprising a protease cleavage site, wherein the protease cleavage site is sterically occluded in the absence of a stimulus for said third biosensor and wherein the protease cleavage site becomes accessible in the presence of said stimulus; and (d) a fourth nucleic acid sequence encoding an effector molecule. The present invention further relates to a vector comprising the nucleic acid molecule of the invention, to sets of nucleic acid molecules, to the sets of nucleic acid molecules of the invention comprised in one or more vectors, to a cell expressing a set of nucleic acid molecules according to the invention as well as to a cell comprising the one or more vectors of the invention. Furthermore, the present invention relates to a method for inducing intracellular signaling, as well as to a method for monitoring intracellular signaling.

In this specification, a number of documents including patent applications and manufacturer's manuals is cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. One of the ongoing challenges in scientific research is the analysis of complex networks underlying cellular functions. In the last decade, a wide variety of methods to study protein- protein interactions ranging from biochemical to genetic or cell-based approaches, such as affinity purification, co-immunoprecipitation as well as yeast two-hybrid systems, have been developed. In addition, fluorescence-based methods for in-cell visualization of protein-protein- interactions have been introduced, including e.g. fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), both of which are based on the expression of fluorescently labeled proteins or fragments thereof. However, the analysis of live events in cells still remains a challenge.

In particular the visualization of brain function has been a long-lasting challenge. Imaging neuromodulation is as important as mapping neuronal activity in a brain. Visualizing neuromodulatory states in specific neural circuits is critical to understanding the diversity of animal behavior, sensation, and cognitive functions. In recent decades, a large array of genetic or optical techniques has been developed to label neuronal structure and activities

(Alvarez, V. A. & Sabatini, B. L. Anatomical and physiological plasticity of dendritic spines.

Annual review of neuroscience 30, 79-97 (2007); Bhatt, D. H., Zhang, S. & Gan, W. B.

Dendritic spine dynamics. Annual review of physiology 71 , 261-282, (2009); Chen, T. W. et al.

Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300, (2013)). However, no useful genetically-encoded neuromodulation mapping techniques have been developed so far and none of the previous efforts have led to useful methods for visualizing modulatory actions in the brain.

Also the identification and manipulation of active neural circuits that are indispensible for specific actions or perception has been a long-lasting challenge in modern neuroscience. The possible approach should label a subset of neurons selectively in the pool of thousands of neurons that have similar structural and functional characteristics. The recently developed genetically encoded calcium indicators (GECIs) enable the monitoring of active populations of neurons while animals are performing a particular task (Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300, (2013)). The degree of sensitivity of this approach is sufficient to detect a Ca ²⁺ rise elicited by a single action potential as well as at individual synapses (Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300, (2013)). However, all GECI signals decay back to the basal level within short time window of a few seconds and, therefore, only limited brain areas are available for visualization at a time. To supplement this limitation, a designed calcium integrator named CAMPARI has recently been developed (Fosque, B. F. et al. Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators. Science 347, 755-760, (2015)). This technique uses irreversible photo- conversion of the EOS protein upon high Ca ²⁺ and violet light, such that a group of active neurons can be captured at a specific time point with high temporal resolution. However, fluorescence can only be visualized under low calcium concentrations, so the sample needs to be incubated in the presence of a calcium buffer such as ethylene glycol tetraacetic acid (EGTA). This fact limits the applicability of this system to post-hoc analysis of sampled tissues or neuronal cultures. Furthermore, photo-conversion does not modify or amend downstream protein signaling or gene expression, and thus does not enable for the testing of a causal relationship between neuronal function and behavior.

As alternative approaches, a couple of activity-dependent gene expression systems have been developed to label functional neuronal ensembles. One method that is widely used is the visualization of immediate-early gene (IEG) expression such as c-fos or Arc (Barth, A. L, Gerkin, R. C. & Dean, K. L. Alteration of neuronal firing properties after in vivo experience in a FosGFP transgenic mouse. The Journal of neuroscience: the official journal of the Society for Neuroscience 24, 6466-6475, (2004); Inoue, M. et al. Synaptic activity-responsive element (SARE): A unique genomic structure with an unusual sensitivity to neuronal activity. Communicative & integrative biology 3, 443-446, (2010); Okuno, H. ef al. Inverse synaptic tagging of inactive synapses via dynamic interaction of Arc/Arg3.1 with CaMKIIbeta. Cell 149, 886-898, (2012); Smeyne, R. J. et al. fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system. Neuron 8, 13-23 (1992).).

IEG expression is induced by transcription factors converged from various calcium-dependent signaling cascades (Bito, H., Deisseroth, K. & Tsien, R. W. Ca2+-dependent regulation in neuronal gene expression. Current opinion in neurobiology ?, 419-429 (1997); Flavell, S. W. & Greenberg, M. E. Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annual review of neuroscience 31 , 563-590, (2008)). Taking advantage of the gene expression, this technique was further engineered to express various reporter genes under IEG promoters. Using these approaches, recent studies successfully triggered artificial memory by manipulating a labeled neuronal ensemble, thus demonstrating evidence of sufficiency for specific behavior (Garner, A. R. et al. Generation of a synthetic memory trace. Science 335, 1513-1516, (2012); Liu, X. ef al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381-385, (2012)). Furthermore, the magnitude of gene expression was highly enhanced by putting several activity-dependent transcription factors together (Kawashima, T. ef al. Functional labeling of neurons and their projections using the synthetic activity-dependent promoter E-SARE. Nature methods 10, 889-895, (2013)). Although I EG system and its derivatives have the capability of gene manipulation, all these activity-dependent gene markers are nonetheless not able to faithfully reflect individual neuronal activity as precisely as GECIs, and the quantitative correlation with neuronal activity is weak.

Furthermore, there exists a plethora of general systems based on reporter activation for investigating protein-protein interactions and compounds affecting same. For example, US Patent No. 8,574,865 describes general methods and assays for identifying a compound that modulates a protein-protein interaction. These methods and assays make use of an inactive reporter activating protein, which becomes activated as the consequence of protease- mediated cleavage of a protease cleavage site interposed between two portions of the inactive reporter activating protein. If protein-protein interaction is induced by a test compound, this interaction will lead to activation of the reporter activating protein and the respective signal produced by the reporter can be detected.

Light-inducible gene expression is another approach that is currently employed in the art. For example, W092/19724 describes light-regulated DNA sequences that are capable of promoting the expression of heterologous genes in transgenic plants. The method makes use of light-inducible promoters, in particular the chalcone synthase promoter,

Kennedy et a/. 2010 (Kennedy, M. J. er a/. Rapid blue-light-mediated induction of protein interactions in living cells. Nature methods 7, 973-975, (2010)) describe the use of genetically encoded light-inducible protein interaction modules based on Arabidopsis thaliana cryptochrome 2 (CRY2) and CIB1 , in combination with split effector molecules, such as the Gal4 transcription factor or Cre recombinase. Upon light induction, the photosensor CRY2 binds to the HLH protein CIB1 without the need for an exogenous chromophore, thereby reconstituting the split parts of the respective effector molecule. otta-Mena er a/. 2014 describe an inducible promoter system based on the EL222 bacterial transcription factor. This transcription factor contains two minimal elements: a photosensory LOV domain and a helix-turn-helix DNA-binding domain. In the dark, both domains are bound to each other, which results in the covering of the HTH-DNA-binding domain, thereby preventing dimerisation and DNA-binding. Blue light illumination leads to the formation of protein/flavin adduct within the LOV domain, which disrupts the inhibitory LOV/HTH interaction and allows EL222 to dimerise and bind DNA. Guntas et al. 2015 (Guntas, G. et a/. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proceedings of the National Academy of Sciences of the United States of America 112, 112-117, (2015)) describe the engineering of the AsLOV2 domain to make it more light-sensitive, i.e. reducing the amount of activity in the dark (leakiness) and, thus, increasing the effect seen upon light-activation. The AsLOV2 domain is used as a fusion construct with the SsrA peptide. In the dark, binding to the binding partner SspB is prevented by steric occlusion, but upon light activation, SsrA becomes sterically available due to the conformational change in AsLOV2 and binds to SspB, making this system a light-inducible heterodimer pair.

WO 2015/120548 describes proteins derived from fluorescent proteins that are photocleavable, i.e. they dissociated into two distinct fragments or release one end of an internal loop upon illumination. As a consequent, the proteins change their fluorescence or become non-fluorescent.

The currently best gene-based method among the known methods suitable for neuromodulation mapping is the so-called "Tango" system, a tool for visualizing brain states by labeling neuromodulator-sensitive neuronal populations (Barnea, G. et al. The genetic design of signaling cascades to record receptor activation. Proceedings of the National Academy of Sciences of the United States of America 105, 64-69, (2008); Inagaki, H. K. er al. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583-595, (2012); Jagadish, S., Barnea, G., Clandinin, T. R. & Axel, R. Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace. Neuron 83, 630-644, (2014)). This genetic-based labeling technique was originally designed to monitor metabotropic G-coupled receptor activation (Barnea, G. er al. The genetic design of signaling cascades to record receptor activation. Proceedings of the National Academy of Sciences of the United States of America 105, 64-69, (2008)), but has also been used to map neuromodulation in Drosophila (Inagaki, H. K. er al. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583-595, (2012); Jagadish, S., Barnea, G., Clandinin, T. R. & Axel, R. Identifying functional connections of the inner photoreceptors in Drosophila using Tango- Trace. Neuron 83, 630-644, (2014)), examine lipid metabolism (Kono, M. et al. Sphingosine- 1 -phosphate receptor 1 reporter mice reveal receptor activation sites in vivo. The Journal of clinical investigation 124, 2076-2086, (2014)), and perform drug screens of human GPCRs (Kroeze, W. K. ef al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nature structural & molecular biology 22, 362-369, (2015)). However, the Tango system has never been used in a mammalian brain, mainly because of high level of ligand-independent background signals and poor signal-to-noise ratio. These technical limitations as well as the inability of specific antagonists to block ligand-induced gene expression (Inagaki, H. K. et al. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583-595, (2012)) have limited the application of the Tango system to studies of phasic neuromodulatory states.

Thus, despite the fact that a lot of effort is currently being invested into the development of methods for monitoring and modulating cell signaling, in particular for neuromodulation mapping, these methods typically suffer from poor signal-to-noise ratios, usually because of ligand-independent background signals. Accordingly, there is still a need to provide alternative methods for monitoring and/or modulating cell signaling, for tagging neuromodulatory action, and in particular for monitoring and/or modulating behaviorally-related neuromodulatory action. Such methods would represent valuable research tools and would offer tremendous value to the field.

This need is addressed by the provision of the embodiments characterized in the claims.

Accordingly, the present invention relates to a nucleic acid molecule encoding a fusion protein, wherein the nucleic acid molecule comprises: (a) a first nucleic acid sequence encoding a transmembrane domain linked to a first biosensor, wherein said first biosensor is a first molecule capable of interacting with a second molecule to form part of a first inducible interaction module, and wherein said first biosensor is linked to the transmembrane domain such that the first biosensor is located intracellular^ upon expression of the fusion protein in a cell; (b) a second nucleic acid sequence encoding an effector-activating module, wherein the effector-activating module comprises: (i) a nucleic acid sequence encoding a first part of a protease, wherein said first part of the protease is capable of interacting with a second part of said protease to form an active form of said protease; or (ii) a nucleic acid sequence encoding a second biosensor, wherein said second biosensor is a first molecule capable of interacting with a second molecule to form part of a second inducible interaction module; (c) a third nucleic acid sequence encoding a third biosensor comprising a protease cleavage site, wherein the protease cleavage site is sterically occluded in the absence of a stimulus for said third biosensor and wherein the protease cleavage site becomes accessible in the presence of said stimulus; and (d) a fourth nucleic acid sequence encoding an effector molecule.

In accordance with the present invention, the term "nucleic acid molecule", also referred to as nucleic acid sequence or polynucleotide herein, includes DNA, such as cDNA or genomic DNA, and RNA. It is understood that the term "RNA" as used herein comprises all forms of RNA including mRNA. Both, single-strand as well as double-strand nucleic acid molecules are encompassed by this term.

The nucleic acid molecules of the invention can e.g. be synthesized by standard chemical synthesis methods, produced semi-synthetically, i.e. by combining parts synthesized by chemical synthesis with parts that are isolated from natural sources, or produced recombinantly, i.e. by combining parts that are isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods, such as restriction digests, ligations and molecular cloning.

In accordance with the present invention, the nucleic acid molecule encodes a fusion protein. The term "fusion protein", as used herein, relates to a construct in which (poly)peptides are fused together that do not naturally occur in such a combination. For example, the (poly)peptides may naturally occur as separate molecules. Such a fusion is achieved by the joining of two or more nucleic acid sequences that originally coded for separate molecules, i.e. the fusion protein of the invention is produced by recombinant DNA technology, i.e. genetic engineering. Translation of these fused nucleic acid sequences results in a fusion protein, with functional properties derived from each of the original molecules. Suitable methods for creating such fused nucleic acid sequences by recombinant DNA technology as well as suitable vectors for expression of the fusion proteins are well established in the art, e.g. in Molecular Cloning: A laboratory Manual (Fourth Edition) by Michael R. Green and Joseph Sambrook. In accordance with the present invention, the term "fusion protein" does not encompass conjugate proteins obtained by chemically linking two (or more) separate (poly)peptides, i.e. by expressing the separate (poly)peptides and, after their expression, chemically linking them to form a conjugate. The term "comprising", as used herein, denotes that further components and/or steps can be included in addition to the specifically recited components and/or steps. However, this term also encompasses that the claimed subject-matter consists of exactly the recited components and/or steps.

In those embodiments where the nucleic acid molecule encoding the fusion protein includes more than the recited sequences, additional sequences may include for example sequences introduced for purification, to ensure correct intracellular translocation or to enable appropriate processing of the transcript as well as peptide linker sequences. Purification sequences typically encoding peptides that confer on the resulting fusion protein an affinity to certain chromatography column materials. Typical examples for such peptides include, without being limiting, the 17 tag, the Xpress tag, myc tags, oligohistidine-tags, Strep-iags, FLAG-tags, glutathione S-transferase, maltose-binding protein or the albumin-binding domain of protein G. Particularly preferred sequences are the 17 tag (having the sequence MASMTGGQQMG (SEQ ID NO:26)), the Xpress tag (having the sequence DLYDDDDK (SEQ ID NO:27)) or the myc tag (having the sequence EQKLISEEDL (SEQ ID NO:28)). Non-limiting examples of sequences that ensure correct intracellular translocation include nuclear export sequences, such as e.g. the sequence LQLPPLERLTLE (SEQ ID NO: 29) or the IgK leader sequence (e.g. METDTLLLWVLLLWVPGSTGD; SEQ ID NO:30) employed in the appended examples. Non-limiting examples of sequences that enable appropriate processing of the transcript e.g. in bicistronic systems include a P2A, such as e.g. the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 31 ) employed in the appended examples. Linker sequences are sequences that connect the individual amino acid sequences encoded by the nucleic acid molecule with each other. A peptide linker as envisaged by the present invention is a linker of at least 1 amino acid in length. Preferably, the linker is 1 to 100 amino acids in length. More preferably, the linker is 2 to 50 amino acids in length and even more preferably, the linker is 5 to 20 amino acids in length. Preferably, the linker is a flexible linker comprising or consisting of the amino acids glycine, serine, arginine, leucine and/or glutamic acid. Preferably the linker sequences comprise or consist of a sequence selected from the group consisting of GGGGSGGGGSGGGGRSGGS (SEQ ID NO:32), GGSGGLEG (SEQ ID NO:33), GGGGRSGGGGS (SEQ ID NO:34), GGGGSGGGGSGGGG (SEQ ID NO:35) and GSGSG (SEQ ID N0.36). More preferably, the linker comprises or consists of the sequence GSGSG or GGGGSGGGGSGGGG. The length and sequence of a suitable linker depends on the composition of the respective fusion protein. Methods to test the suitability of different linkers are well known in the art and include e.g. the comparison of the binding affinity or the protein stability or the production yield of the fusion protein of the invention to the same fusion proteins that comprise different linkers.

In accordance with the present invention, the nucleic acid molecule encoding the fusion protein comprises at least the specifically recited elements (a) to (d), preferably in the recited order, i.e. (a), (b), (c) and (d). This order can be either starting from the N-terminal residue and ending with the most C-terminal residue (N->C) or vice versa, i.e. starting from the C- terminal residue and ending with the most N-terminal residue (C- N). Preferably, the order is from N- to C-terminal, i.e. N->C. More preferably, the nucleic acid molecule of the invention comprises additional linker sequences between each of (a), (b), (c) and (d).

In accordance with the present invention, the nucleic acid molecule encoding the fusion protein comprises a first nucleic acid sequence encoding a transmembrane domain linked to a first biosensor, wherein said first biosensor is linked to the transmembrane domain such that the first biosensor is located intracellularly upon expression of the fusion protein in a cell.

The "transmembrane domain", in accordance with the present invention, relates to an amino acid sequence, typically a hydrophobic amino acid sequence, that forms a polypeptide that spans once or several times through the phospholipid membrane of cell. In those cases where the transmembrane domain spans only once through the membrane, one part of the transmembrane domain is located on the outside of the cell and another part of the transmembrane domain is located at the inside of the cell, i.e. intracellularly. There is no particular restriction as to which part of the transmembrane domain is intracellular and which part is extracellular. However, typically, the N-terminal part is located extracellularly and the C-terminal part is located intracellularly. Preferably, the intracellular part is the C-terminus. In those cases where the transmembrane domain spans several times through the membrane, situations can arise wherein both ends of the transmembrane domain are located on the same side of the cell, i.e. either intra- or extracellularly. In that case it is necessary that a transmembrane domain is chosen for which, upon expression in a cell, both the N- and the C- terminus are located intracellularly.

The transmembrane domain can be a functional, naturally occurring molecule, such as e.g. a receptor or channel. Alternatively, the transmembrane domain can be an amino acid sequence that does not naturally occur as such in a cell but that forms a polypeptide that spans through the membrane, thereby forming an anchor at the internal surface of the cell.

Non limiting examples of transmembrane domains include G-protein coupled receptors (GPCR), such as e.g. DRD2 or part of the PDGF receptor as shown in the appended examples, as well as e.g. the CD28 transmembrane domain (represented in SEQ ID NO:37 and 38) or the CD8a transmembrane domain (represented in SEQ ID N0.39 and 40). A preferred transmembrane domain including parts of the PDGF receptor is shown in SEQ ID NOs:41 and 42, including an IgK leader sequence, a myc tag, parts of the PDGF receptor and a C-terminal linker sequence. The main function of the transmembrane domain is to keep the fusion protein encoded by the nucleic acid molecule of the invention at a particular location within the cell by anchoring same in the membrane. In this way, the effector molecule is also kept at this location until its release, as described further below.

Linked to the part of the transmembrane domain that is located intracellularly upon expression in a cell is a first biosensor. The biosensor can be linked to any portion of the transmembrane domain that, after expression in a cell, is located intracellulary, preferably to the intracellular terminus of the amino acid sequence representing the transmembrane domain. If both termini are present inside the cell upon expression, the biosensor is preferably linked to the C- terminal end of the transmembrane domain.

The term "linked", as used herein, refers to the covalent connection of two sequences, either directly via a peptide bond between one amino acid of the transmembrane domain and one amino acid of the first biosensor, or indirectly via a peptide linker, as described above. Preferably, a linking sequence is encompassed between the transmembrane domain and the first biosensor. Non-limiting examples of linking amino acid sequences have been provided above. Additional examples include the sequence

ACGGGGSGGGGSGGGGRSGGSMLQLPPLERLTLE (SEQ ID NO:43) as employed in the construct shown in SEQ ID NO:1 , the sequence LQLPPLERLTLGGSGGLE (SEQ ID NO:44) as employed in the construct shown in SEQ ID NO:3, the sequence LQLPPLERLTLGGSGGLEG (SEQ ID NO:45) as employed in the construct shown in SEQ ID NO:5 or the sequence ACGGGGSGGGGSGGGGR (SEQ ID NO:46) as employed in the construct shown in SEQ ID NO:8.

The term "biosensor", as used herein, relates to a molecule capable of sensing, i.e. detecting, a biological stimulus. Non-limiting examples of such stimuli are light, pharmacological drugs, chemicals, the binding of an interaction partner such as e.g. a ligand, as well as the presence of molecules such as e.g. ions, in particular calcium.

In the presence of the respective stimulus, said first biosensor interacts with a second molecule to form part of a first inducible interaction module. Such interaction modules are referred to herein as "inducible" interaction modules, because the interaction between the respective individual molecules only occurs in the presence of the respective stimulus, i.e. it is induced in the presence of the stimulus. It will be appreciated that this interaction is reversible, i.e. in the absence of the stimulus the interaction partners can separate again and, thus, can subsequently again be induced to interact should the stimulus be present again. Examples of such inducible interaction modules are well known in the art and include, without being limiting, light inducible interaction modules, such as e.g. CRY2PHR and CIBN, PIF and PhyB or LOV and LOV; ligand-inducible interaction modules, such as e.g. G protein-coupled receptors or parts thereof, such as e.g. V2tail, and -arrestin2; calcium-inducible modules such as e.g. calmodulin with M13 or M13 variants with different calcium affinities; as well as drug inducible systems, such as e.g. rapamycin or kinase/phosphatase-inducible systems, e.g. MAPK or receptor tyrosine kinases. These systems have been described in the art, e.g. in Chen, T. W. ef a/. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300, doi:10.1038/nature12354 (2013); Zhang, K. & Cui, B. Optogenetic control of intracellular signaling pathways. Trends in biotechnology 33, 92-100, doi: 10.1016/j.tibtech.2014.11.007 (2015); Barnea, G. et al. The genetic design of signaling cascades to record receptor activation. Proceedings of the National Academy of Sciences of the United States of America 105, 64-69, doi:10.1073/pnas.0710487105 (2008). In accordance with the present invention, the nucleic acid molecule encoding the fusion protein further comprises a second nucleic acid sequence encoding an effector-activating module.

The term "effector-activating module" relates to a molecule that is capable of activating the effector molecule of (d) under specific, pre-determined circumstances and when the protease cleavage site of (c) is accessible. Depending on the nature of the effector-activating module, these conditions leading to effector activation differ, as detailed in the following.

In the first option (i), the effector-activating module comprises a first part of a protease, wherein said first part of the protease is capable of interacting with a second part of said protease to form an active form of said protease.

The choice of protease is not particularly limited, as long as the protease can be split into two molecules that are capable of exerting the enzymatic activity of the protease upon interaction of the two molecules. Preferably, the protease is the Tobacco Etch Virus nuclear-inclusion-a endopeptidase (TEV).

Also with regard to the length and composition of the first part of the protease (and consequently of the resulting second part of the protease), there is not particular limitation. It will be appreciated that the two parts of the protease should be stable and should be able to interact with the respective other part. With regard to TEV, preferred N-terminal and a C- terminal TEV molecules as employed in the appended examples are shown in SEQ ID NO:24 and SEQ ID NO:25, respectively. There is further no particular limitation as to which part of the protease is included in the effector-activating module. For example, for TEV protease, either the N-terminal part or the C-terminal part can be included in the effector-activating module. Preferably, the N-terminal part of TEV is included in the effector-activating module, as shown in the appended examples.

In accordance with this first option (i), the conditions that enable effector activation when the protease cleavage site of (c) is accessible are conditions that enable the interaction of the two parts of the protease. For example, where the first inducible interaction module is e.g. a light- inducible interaction module, the presence of a corresponding light stimulus leads to the interaction of the first biosensor with its respective second molecule. By providing within the cell a further fusion protein comprising said second molecule fused to the second part of the protease, the interaction of the first biosensor with this second molecule results in that the two parts of the protease can interact and, thus, will be able to enzymatically cleave the fusion protein at the protease cleavage site of (c). This approach is described herein below in more detail with regard to the BLITz system, /Tango2 system and Cal-Light system of the invention.

In the second option (ii), the effector-activating module comprises a second biosensor, wherein said second biosensor is a first molecule capable of interacting with a second molecule to form part of a second inducible interaction module.

The definitions and preferred embodiments provided herein above with regard to the first biosensor apply mutatis mutandis. It will be appreciate that the second biosensor has to be different from the first biosensor. Preferably, the first biosensor is part of a ligand- or calcium- inducible interaction module (such as e.g. a GPCR or part thereof, such as V2tail, or a calcium-sensor, such as e.g. CaM) and the second biosensor is part if a light-inducible interaction module, such as e.g. CIBN.

In accordance with this second option (ii), the conditions that enable effector activation when the protease cleavage site of (c) is accessible are also conditions that enable the interaction of the two parts of the protease. However, as opposed to option (i) above, the binding of two interacting molecules is required, each carrying one part of the protease. Here, the first inducible interaction module brings one part of the protease into the vicinity of the protease cleavage site of (c) and the second inducible interaction module brings the second part of the protease into the vicinity of the protease cleavage site of (c). Once both modules are induced, the two parts of the protease interact and are able to enzymatically cleave the fusion protein at the protease cleavage site of (c). This approach is described herein below in more detail with regard to the /Tango system.

In accordance with the present invention, the nucleic acid molecule encoding the fusion protein further comprises a third nucleic acid sequence encoding a third biosensor.

Again, the definitions and preferred embodiments provided herein above with regard to the first biosensor apply mutatis mutandis, unless otherwise defined. In accordance with the invention, the third biosensor is capable of undergoing a conformational change in its 3D structure in response to a stimulus. Thus, the protease cleavage site is sterically occluded in the absence of a stimulus for said third biosensor, i.e. it is protected from cleavage, but becomes accessible in the presence of said stimulus, i.e. the cleavage site can now serve as a target for the protease. It will be appreciated that the protease cleavage site is a cleavage site for the protease to be used in the intended system, i.e. the protease formed by the interaction of the individual modules described herein. Thus, where the protease employed e.g. in option (b)(i) is TEV, the cleavage site is a TEV cleavage site. Moreover, where the first and second inducible interaction module bring a first and a second part of TEV in the vicinity of the cleavage site, as described above for option (b)(ii), the cleavage site is a cleavage site for TEV protease. Suitable cleavage sites for a variety of proteases of choice are well known in the art and can be selected by the skilled person without further ado. Preferably, the third biosensor is a biosensor derived from Avena sativa phototropinl light-oxygen-voltage 2 (AsLOV2).

The stimulus for the third biosensor can be the same or different than the stimuli for the first and/or second biosensor. Preferably, the stimulus for the third biosensor is chosen such as to enable the use of two different stimuli when employing the nucleic acid molecule of the invention. Accordingly, where the effector-activating module is as defined in (b)(i), it is preferred that the stimulus for the third biosensor differs from the stimulus for the first biosensor. Most preferably in this regard, the stimulus for the first biosensor is either ligand binding or calcium binding and the stimulus for the third biosensor is light. Where the effector- activating module is as defined in (b)(ii), biosensors for two different inducible interaction modules requiring two different stimuli are already present, as discussed above. In that case, it is preferred that the stimulus for the third biosensor is identical to one of the stimuli that induce either the first or the second biosensor. Most preferably in this regard, the stimulus for the first biosensor is either ligand binding or calcium binding and the stimulus for the second and for the third biosensor is light. Further in accordance with the present invention, the nucleic acid molecule encoding the fusion protein additionally comprises a fourth nucleic acid sequence encoding an effector molecule.

The term "effector molecule", as used herein, relates to a molecule capable of eliciting a desired and detectable effect within a cell. Non-limiting examples include the activation of gene transcription, e.g. of a reporter gene, the inactivation of expression of a constitutively active gene; or the modulation of the genome within the cell, e.g. by inducing strand breaks in the DNA and subsequent homologous or non-homologous recombination; or protease induced protein inactivation, e.g. by employing a dormant N-degron as described in more detail herein below. The effector molecule can also be e.g. a reporter molecule or an enzyme.

Due to the incorporation of the effector molecule into the fusion protein encoded by the nucleic acid molecule of the invention, the effector molecule is kept at a location within the cell where it cannot exert its action. For example, by keeping a transcription or genome modulator anchored at the cell membrane, it cannot exert its effect on the genome, which is located in the cell nucleus. Upon cleavage of the protease cleavage site of (c), the effector molecule is released and can translocate to the relevant subcellular compartment. It will be appreciated that additional sequences, such as e.g. a nucleus localizing signal (NLS) can be fused to the effector molecule to ensure its translocation to the correct compartment. Preferably, an effector molecule is chosen that translocates on its own, i.e. that does not requires such additional sequences. In accordance with the present invention, a novel approach to converting signaling events into a detectable output, such as e.g. gene expression, is provided. This approach offers a high spatiotemporal resolution while at the same time having a reduced signal-to-noise ratio. Previous approaches, such as e.g. the Tango system, suffered from the drawback of high levels of ligand-independent background signals and poor signal-to-noise ratios. These technical limitations have severely limited the application of these systems in the past, in particular when the aim was the study of phasic neuromodulatory states.

These fundamental problems have been solved herein by developing a dual control system, named Blue-Light Inducible TEV protease (BLITz), which makes use of the nucleic acid molecule of the invention. In short, based on TEV as an exemplary protease, the TEV protease recognition sequence has been made photocleavable and the reconstitution of split TEV protease has been rendered either light-inducible or inducible by other stimuli, including ligands or calcium. The original split TEV system was first published in Nature Methods (Wehr, M. C. et al. Monitoring regulated protein-protein interactions using split TEV. Nature methods 3, 985-993, (2006)) and was widely used in various fields including neuroscience and cell biology. However, so far no light-inducible split TEV system has been developed. By providing a light-inducible split TEV system for the first time, a noninvasive, fast reactive, spatially precise and also reversible system is provided that does not require any pharmacological drugs for its regulation. By implementing this BLITz system into the original Tango system, an induced Tango system (referred to as "/Tango") is provided herein with significantly improved spatiotemporal resolution. In /Tango2-transfected neurons, background signals were nearly undetectable, but light- and ligand-inducibility was very robust, with a SNR corresponding to roughly 900 % fold change. The same experiments using the conventional Tango system yielded only a 50 % fold change (Djannatian, M. S., Galinski, S., Fischer, T. M. & Rossner, M. J. Studying G protein-coupled receptor activation using split-tobacco etch virus assays. Analytical biochemistry 412, 141 -152, (2011 ))

Due to the improvement of spatiotemporal resolution and signal-to-noise ratio the new techniques can now be applied in a timely precise way to mammalian brains (e.g. in transgenic animals), which are typically difficult to investigate because subtle neuromodulatory signals are constantly flowing in and out. Thus, the present invention allows to investigate the neuromodulation code in a complex neuronal network, which has not been attainable before. Furthermore, the present invention also provides a good template for a light-inducible G-protein coupled signaling monitoring platform. Simply exchanging GPCR parts from the system shown in the appended example allows to build whole library of GPCRs and to image individual's action as shown in a recent study (Kroeze, W. K. et al. PRESTO- Tango as an open-source resource for interrogation of the druggable human GPCRome. Nature structural & molecular biology 22, 362-369, (2015)). So far, no technique linking GPCR activation to gene expression in a light-dependent way has been described. Moreover, the nucleic acid molecule of the present invention further enables a novel activity- dependent labeling system that reliably and quantitatively relays neuronal activity to gene expression. The basic principle of this technique is to render gene expression dependent on calcium and light. For example, whenever neurons fire, calcium enters neurons, but gene expression will not begin until a light stimulus is provided. When light is illuminated, gene expression will be initiated only in active neurons because calcium influx will occur in active neurons. Because this labeling system is designed to turn on gene transcription initiation when both light and calcium present, this technique was named Calcium and Light-Induced Gene Handling Tookit, "Cal-Light". This Cal-Light system reliably translates functions to gene expression, so its application will be diverse in circuit level studies. As is shown in the appended examples (see example 4), the Cal-Light system expresses reporter genes in a calcium- and light- dependent manner in cultured neurons. When Cal-Light was introduced in vivo, a functional neuronal group in the motor cortex could be selectively labeled. Successful visualization of long-range axonal trajectory in the basal ganglia area can also be achieved. It will be further possible to control the behavior of an animal by activating labeled neurons selectively by the Cal-Light constructs.

In a preferred embodiment of the nucleic acid molecule of the invention, the first inducible interaction module and the second inducible interaction module are independently selected from a light-inducibie interaction module, a ligand-inducible interaction module and a calcium- inducible module.

The term "are independently selected", as used herein, relates to the fact that the first inducible interaction module and the second inducible interaction module have to be different. Nonetheless, both may be selected from the same class, as long as they respond to a different stimulus. For example, the first inducible interaction module may respond to blue light, while the second inducible interaction module may be responsive to red light. Also the use of different ligands is explicitly envisaged herein. Most preferably, however, the first inducible interaction module and the second inducible interaction module are selected from different groups, e.g. one is a light-inducible interaction module and the other is either a ligand-inducible interaction module or a calcium-inducible module; or one is a ligand-inducible interaction module and the other is either a light-inducible interaction module or a calcium- inducible module; or one is a calcium-inducible module and the other is either a ligand- inducible interaction module or a light-inducible interaction module.

The module is inducible by the respective stimulus, i.e. light, ligand or calcium, if the presence or absence of the stimulus is capable of initiating a modification of the module. Preferably, the individual parts of the module interact in the presence of the stimulus and do not interact in the absence of the stimulus, or vice versa, or the molecules of the module undergo a conformational change in the presence of the stimulus and return to the original confirmation in the absence of the stimulus.

It will be appreciated that any kind of light capable of inducing such a modification might be used in the light-inducible interaction module. Preferably, the light is selected from blue light, UV light or red/far red light. Most preferably, the light is blue light. It is further preferred that light-inducible interaction module is CIBN/CRY2PHR.

It will further be appreciated that any kind of ligand capable of inducing such a modification might be used in the ligand-inducible interaction module. Preferably, the ligand is a ligand for a GPCR. It is further preferred that the ligand-inducible interaction module is GPCR/β- arrestin2.

It will also be appreciated that in those cases where the module is a calcium-inducible module, such that the modification is induced by calcium, any method of increasing intracellular calcium might be used. For example, as is well known, upon stimulation of neurons, they become depolarized. This depolarization in turn leads to the opening of voltage- sensitive calcium channels, thereby resulting in a calcium influx into neurons. Accordingly, one approach is to stimulate neurons, for example by electrical stimulation, optogenetic activation of neurons, blocking inhibition by GABA receptor antagonists, KCI application, or any pharmacological application that causes neuronal depolarization. In in vivo conditions, neurons fire spontaneously or are activated by synaptic inputs, which does not need extrinsic stimulation. Behaviorally relevant neurons will fire and calcium will enter into neurons while animals are behaving. It is particularly preferred that the calcium-inducible module is CaM/ 13.

In a further preferred embodiment of the nucleic acid molecule of the invention, the effector molecule is a transcriptional modulator, a genome modulator, a reporter molecule, an enzyme, or degron. The term "transcriptional modulator", as used herein, relates to one or more molecules capable of activating or inactivating the transcription of a particular gene, or capable of altering the amount of transcription of a particular gene. Transcriptional modulators are well known in the art and have been described e.g. in Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences of the United States of America 89, 5547-5551 (1992). Non- limiting examples of transcriptional modulators in accordance with the present invention include TetR-VP16(tTA), as well as TetR-VP15, TetR-VP64, GAL4-VP16 or GAL4-VP64. Preferably, the transcriptional modulator is the TetR-VP16(tTA) molecule employed in the appended examples (see e.g. SEQ ID NO:10 and 11 ).

The term "genome modulator", as used herein, relates to one or more molecules capable of altering the genomic constitution within a target cell, for example by inducing double-strand breaks and, subsequently, inducing non-homo!ogous or homologous recombination. Genome modulators and gene editing mediated by such modulators are well known in the art and have been described e.g. in Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262-1278, (2014); Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99-109 (2000). Non- limiting examples of genome modulators in accordance with the present invention include the Crispr-Cas9 system as well as the Cre recombinase. A schematic overview of these system incorporated into the nucleic acid molecule of the present invention is presented in Figure 21 below.

The effector molecule can also be a reporter molecule, such as e.g. a fluorescent or bioluminescent molecule fused to a nuclear localization sequence. Upon release of the reporter molecule from the fusion protein, i.e. after cleavage of the protease cleavage site, the reporter molecule can translocate to a different site within the cell. This translocation can be observed and used as a read-out. Suitable fluorescent proteins include, without being limiting, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP) or infrared fluorescent protein (IFP) as well as fluorescent dyes such as e.g. Fluorescein, Alexa Fluor or Cy dyes. Suitable bioluminescent proteins include, amongst others, luciferase, in particular bacterial luciferase (luxAB), Photinus luciferase and Renilla luciferase.

Furthermore, the effector molecule can also be an enzyme. For example, the enzyme can be an enzyme capable of catalyzing chromogenic, chemiluminescent or fluorescent reactions, such as e.g. horseradish peroxidase (HRP), luciferase, β-galactosidase and alkaline phosphatase (AP). Also envisaged herein are genetically engineered enzymes, such as for example, a procaspase that has been modified to contain a TEV cleavage site (TEVseq), such that the procaspase is inactive. . Upon cleavage of the TEVseq the procaspases switches to an active caspase, causing apoptosis (Gray, D. C, Mahrus, S. & Wells, J. A. Activation of specific apoptotic caspases with an engineered small-molecule-activated protease. Cell 142, 637-646, (2010)).

A further type of molecule suitable for use as an effector molecule in accordance with the present invention is degron, in particular N-degron. N-degrons are natural or artificial tags that can be added to the N-terminal end of a protein of interest. Because N-degrons are proteolytically processed, amino acids other than methionine form the N-terminus of the protein, which serve as recognition signal for poly-ubiquitinylation and the subsequent proteasomal degradation through the N-end rule pathway in eukaryotes. By attaching a further protein to the N-terminal end of the N-degron, the N-degron can be rendered dormant, i.e. it is protected from proteolytic processing. In accordance with the nucleic acid molecule of the present invention, said further protein is the nucleic acid sequence of (a) to (c). Upon cleavage at the protease cleavage site of (c), the N-terminal end of N-degron becomes exposed, thus initiating the degradation of the protein of interest. This method is well known in the art and has been described, e.g. in Taxis, C, Stier, G., Spadaccini, R. & Knop, M. Efficient protein depletion by genetically controlled deprotection of a dormant N-degron. Molecular systems biology 5, 267, (2009). Because this method regulates protein level by accelerating protein degradation, the use of degron can be beneficial in defining cellular functions that require sustained protein activity.

Yet another effector molecule suitable for use as an effector molecule in accordance with the present invention is Tetanus Toxin Light Chain (TeTxLC). TeTxLC cleaves synaptobrevin 2, an essential protein mediating vesicle release. Therefore, when TeTxLC is expressed in a cell, all vesicular fusion events in that cell are prevented, such that neurotransmitters such as glutamate and GABA are not released. TeTxLC is a non-toxic protein that retains the enzymatic activity encoded by the holotoxin and that is - as a protein - unable to gain access to intracellular targets without microinjection. Moreover, there is no chance of contamination by the heavy chain since only the light chain is encoded and expressed.

By employing TeTxLC as an effector molecule in accordance with the invention, it becomes possible to investigate functions associated with vesicular activity within a selective group of cells without killing or damaging these cells.

Furthermore, it is envisaged that the effector molecule is composed of a split system, preferably making use of the SpyTag/SpyCatcher system. This system is well known in the art and has been described e.g. in Bedbrook, C. N. et al. Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo. Chemistry & biology 22, 1108-1121 , (2015). By combining the one part of an effector molecule, such as of e.g. Cre recombinase or Cas9, with a SpyTag, the activity of the effector molecule can be further controlled. This is because the effector molecule will only be active once the SpyTag interacts with the SpyCatcher, to which the second part of the effector molecule will be attached. Thus, particularly envisaged is the use of the fusion protein SpyTag-Cre-N (i.e. the N-terminal part of Cre recombinase) as part (d) of the nucleic acid molecule of the invention, wherein the fusion protein SpyCatcher-Cre-C (i.e. the C-terminal part of Cre recombinase) is to provided to the cell as the interaction partner. Alternatively envisaged is the use of the fusion protein SpyTag-Cas9-N (i.e. the N-terminal part of Cas9) as part (d) of the nucleic acid molecule of the invention, wherein the fusion protein SpyCatcher-Cas9-C (i.e. the C-terminal part Cas9) is to provided to the cell as the interaction partner. A schematic overview of such an approach is provided in Figure 22 below.

Incorporation of the SpyTag/SpyCatcher system is particularly advantageous for use in combination with Cre recombinase or the C ISPR Cas system, because in theory, a single protein of Cre or Cas9 can initiate gene editing. Thus, even the smallest amount of light-independent Cas9 or Cre protein release can cause non-specific gene editing. To prevent this possibility, split Cas9 and Cre proteins can be employed as described, such that these proteins are not functional on their own. In the presence of the respective stimulus, sufficiently large amounts of the split proteins will be released and will relocate to the nucleus, where they interact with the second half of the protein to form a functional molecule. By employing the SpyTag/SpyCatcher system, the binding of the two halves of the split protein to each other is facilitated.

In a further preferred embodiment of the nucleic acid molecule of the invention, the third nucleic acid sequence comprises (i) a nucleic acid sequence encoding the N-terminal amino acids 1 to 138 or 1 to 139 of Avena sativa phototropinl light-oxygen-voltage 2 (AsLOV2), linked at its C-terminus to (ii) a protease cleavage site.

The N-terminal amino acids 1 to 138 of AsLOV2 are represented in the sequence listing as SEQ ID NO:49 and the corresponding nucleic acid sequence is represented in SEQ ID NO:50. Further, the N-terminal amino acids 1 to 139 of AsLOV2 are represented in the sequence listing as SEQ ID NO:51 and the corresponding nucleic acid sequence is represented in SEQ ID NO:52. These sequences are fused at their C-terminal end to a protease cleavage site, preferably a TEV cleavage site as described below. AsLOV2 acts as a biosensor for light which changes its conformation upon a light stimulation, thereby making the protease cleavage site accessible. The two preferred AsLOV2 constructs according to this preferred embodiment have been identified in the appended examples as particularly useful light-sensors with a low back-ground activation in the absence of a light stimulus and a high activation in the presence of the stimulus, i.e. a particularly good signal- to-noise-ratio. In a particularly preferred embodiment of the nucleic acid molecule of the invention, the protease cleavage site is a TEV protease cleavage site.

Any site that can be cleaved by TEV protease can be employed in accordance with this embodiment. Thus, the TEV protease cleavage can be a naturally existing cleavage site for TEV protease, or a modified cleavage site, as long as the TEV protease can still cleave said site. Preferably, the cleavage site is specific for TEV protease, i.e. it is only cleaved by TEV protease, but not by any other protease. Preferably, the TEV cleavage site has the amino acid sequence ENLYFQG (SEQ ID NO:53) or EALYFQG (SEQ ID NO:54). Furthermore, the most C-terminal G in these sequences can be replaced by any one of the other 19 proteinogenic amino acids, i.e. by S, A, M, C, N, H, Y, K, D, Q, F, T, W, R, L, E, I, V, or P (Kapust, R. B., Tozser, J., Copeland, T. D. & Waugh, D. S. The ΡΓ specificity of tobacco etch virus protease. Biochemical and biophysical research communications 294, 949-955, (2002). The nucleic acid sequences encoding the preferred two TEV protease cleavage sites ENLYFQG and EALYFQG, are represented by SEQ ID NOs: 55 and 56, respectively.

In another preferred embodiment of the nucleic acid molecule of the invention, the nucleic acid molecule comprises (i) a nucleic acid sequence encoding a transmembrane domain linked to a nucleic acid sequence selected from: SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:8 such that the nucleic acid sequence selected from: SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:8 is located intracellularly upon expression of the protein in a cell; and (ii) a nucleic acid sequence encoding an effector molecule, wherein the effector molecule is selected from TetR-VP16(tTA), Cas9 or Cre recombinase. In accordance with this embodiment, a nucleic acid sequence encoding a transmembrane domain of interest is linked with one of the nucleic acid sequences cited, wherein SEQ ID NO:1 represents a nucleic acid sequence comprising, in the recited order, CIBN - TEV-N - AsLOV2 - TEVseq; SEQ ID NO:3 represents a nucleic acid sequence comprising, in the recited order, V2tail - TEV-N - AsLOV2 - TEVseq; SEQ ID NO:5 represents a nucleic acid sequence comprising, in the recited order, V2tail - CIBN - AsLOV2 - TEVseq; and SEQ ID NO:8 represents a nucleic acid sequence comprising, in the recited order, CaM - TEV-N - AsLOV2- TEVseq. Accordingly, SEQ ID NO:1 represents a sequence suitable for use in the BLITz system, SEQ ID NO:3 represents a sequence suitable for use in the /Tango system, SEQ ID NO:5 represents a sequence suitable for use in the /Tango2 system and SEQ ID NO:8 represents a sequence suitable for use in the Cal-Light system. The amino acid sequences encoded by these nucleic acid sequences are represented in SEQ ID NOs:2, 4, 6 and 9.

The effector, in accordance with this embodiment, is selected from TetR-VP16(tTA), Cas9 or Cre recombinase. Suitable nucleic acid sequence encoding such effectors, as well as the respective amino acid sequences, are represented in SEQ ID NO: 10 and 11 for TetR- VP16(tTA), SEQ ID NO:12 and 13 for Cas9 and SEQ ID NO:14 and 15 for Cre recombinase.

In a more preferred embodiment of the nucleic acid molecule of the invention, the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane domain linked to a nucleic acid sequence selected from SEQ ID NO: 16 (CIBN - TEV - N-AsLOV2 -TEVseq - TetR-VP16(tTA)), SEQ ID NO: 18 (V2tail - TEV-N - AsLOV2 -TEVseq - TetR-VP16(tTA)), SEQ ID NO:20 (V2tail - CIBN - AsLOV2 -- TEVseq - TetR-VP 6(tTA)) and SEQ ID NO:22 (CaM - TEV-N - AsLOV2 -TEVseq - TetR-VP16(tTA)), such that the nucleic acid sequence selected from: SEQ ID ΝΟ. 6, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22 is located intracellularly upon expression of the protein in a cell. The amino acid sequences corresponding to these nucleic acid sequences are represented in SEQ ID NOs: 17, 19, 21 and 23, respectively.

The present invention further relates to a vector comprising the nucleic acid molecule of the invention.

Usually, the vector is a plasmid, cosmid, virus, bacteriophage or another vector used conventionally e.g. in genetic engineering. Preferably, the vector is a plasmid, more preferably a plasmid based on the multipurpose expression vector pCS2+ (Addgene), which is suitable for expressing proteins in Xenopus embryos, zebrafish embryos as well as a wide variety of mammalian and avian cells. Another suitable mammalian expression vector is the pCS4+ vector derived from pCS2+ as described in Yeo C and Whitman M, 2001 (Nodal signals to Smads through Cripto-dependent and Cripto-independent mechanisms. Mol Cell 7(5):949- 957), which has been employed in the appended examples.

Alternative vectors include, without being limiting, plasmid vectors, such as pQE- 2, the pUC- series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11 , pJOE, the pBBR1-MCS series, pJB861 , pBSMuL, pBC2, pUCPKS, pTACTI and vectors compatible with expression in mammalian cells like E- 027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), p C neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV- , pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, plZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI , pcDNA3 (Invitrogen), pcDNA3.1 , pSPORTI (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Ciontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Non-limiting examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pA0815, pPIC9K and pPIC3.5K (all Invitrogen).

Generally, vectors can contain one or more origins of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes. In addition, the coding sequences comprised in the vector can be ligated to transcriptional regulatory elements and/or to other amino acid encoding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, without being limiting, regulatory sequences ensuring the initiation of transcription, internal ribosomal entry sites (IRES) (Owens, G.C. et al. [2001] Proc. Natl. Acad. Sci. U.S.A. 98:1471-1476) and optionally regulatory elements ensuring termination of transcription and stabilization of the transcript. Non-limiting examples for such regulatory elements ensuring the initiation of transcription comprise promoters, a translation initiation codon, enhancers, insulators and/or regulatory elements ensuring transcription termination, which are to be included downstream of the nucleic acid molecules of the invention. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals or, depending on the expression system used, signal sequences capable of directing the expressed protein to a cellular compartment or to the culture medium. The vectors may also contain an additional expressible polynucleotide coding for one or more chaperones to facilitate correct protein folding. Suitable bacterial expression hosts comprise e. g. strains derived from JM83, W31 10, KS272, TG1 , BL21 (such as BL21 (DE3), BL21 (DE3)PlysS, BL21 (DE3)RIL, BL21 (DE3)PRARE) or Rosettaa. For vector modification, PCR amplification and ligation techniques, see Sambrook & Russel [2001] (Cold Spring Harbor Laboratory, NY). Examples of suitable origins of replication include, for example, the full length ColE1 , truncated ColE1 , the SV40 viral and the M13 origins of replication, while examples of suitable promoters include, without being limiting, the cytomegalovirus (CMV) promoter, in particular the CMV IE94 promoter employed in the appended examples, SV40-promoter, the tetracycline promoter/operator (tef'°), which is chemically inducible with anhydrotetracycline, RSV-promoter (Rous sarcome virus), the lacZ promoter, chicken β-actin promoter, CAG- promoter (a combination of chicken β-actin promoter and cytomegalovirus immediate-early enhancer), the gai10 promoter, human elongation factor 1a-promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, the lac, trp or tac promoter, the T7 or T5 promoter, the lacUV5 promoter, the Autographs californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or a globin intron in mammalian and other animal cells. One example of an enhancer is e.g. the SV40-enhancer. Non-limiting examples for regulatory elements ensuring transcription termination include the SV40-poly-A site, the tk-poly-A site, the rho- independent Ipp terminator or the AcMNPV polyhedral polyadenylation signals. Non-limiting examples of selectable markers include the ampicillin-resistance gene (β-lactamase), dhfr, gpt, neomycin, hygromycin, blasticidin or geneticin.

Preferably, the vector of the present invention is an expression vector. An expression vector according to this invention is capable of directing the replication and the expression of the nucleic acid molecule of the invention and, accordingly, of the fusion protein of the present invention encoded thereby.

The nucleic acid molecules and/or vectors of the invention as described herein above may be designed for introduction into cells by e.g. chemical based methods (calcium phosphate, liposomes, DEAE-dextrane, polyethylenimine, nucleofection), non chemical methods (electroporation, sonoporation, optical transfection, gene electrotransfer, hydrodynamic delivery or naturally occurring transformation upon contacting cells with the nucleic acid molecule of the invention), particle-based methods (gene gun, magnetofection, impalefection) phage vector-based methods and viral methods. For example, expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, Semliki Forest Virus or bovine papilloma virus, may be used for delivery of the nucleic acid molecules into targeted cell population. Additionally, baculoviral systems can also be used as vector in eukaryotic expression system for the nucleic acid molecules of the invention. Preferably, the nucleic acid molecules and/or vectors of the invention are designed for viral infection methods. The present invention further relates to a set of nucleic acid molecules comprising: (a) the nucleic acid molecule of the invention, wherein the effector-activating module comprises a first part of a protease, wherein said first part of the protease is capable of interacting with a second part of said protease to form an active form of said protease; and (b) a second nucleic acid molecule encoding a second fusion protein, the second nucleic acid molecule comprising (i) a first nucleic acid sequence encoding a molecule that represents the corresponding second molecule of the first inducible interaction module according to the nucleic acid molecule of the invention; and (ii) a second nucleic acid sequence encoding the second part of a protease, capable of interacting with the first part of said protease to form an active form of said protease. This set of nucleic acid molecules is also referred to herein as the "first set of nucleic acid molecules of the invention".

The term "set", as used herein, relates to a combination of at least the recited nucleic acid molecules. In other words, the set of the present invention requires that more than one molecular species of nucleic acid molecules is present. However, the term "set" does not require the presence of any other compounds, vials, containers, manuals and the like. The term "comprising" in the context of the set(s) of the invention denotes that further components can be present in the set. The various components of the set may be present in isolation or combination. For example, the recited nucleic acid molecules of the set may be packaged in one or more containers such as one or more vials.

The set in accordance with this embodiment comprises at least the nucleic acid molecule of the invention, i.e. a nucleic acid molecule encoding a fusion protein as described herein above. The effector- activating module is as defined in the first alternative, i.e. it is a first part of a protease that is capable of interacting with a second part of said protease to form an active form of said protease. As described herein above, a particularly preferred protease is TEV protease and said first part of the protease is preferably the N-terminal part of TEV (TEV-N) and the second part is preferably the C-terminal part of TEV (TEV-C). The set in accordance with this embodiment further comprises a second nucleic acid molecule encoding a second fusion protein. The nucleic acid molecule encoding said second fusion protein comprises two nucleic acid sequences: a first sequence that encodes the counterpart of the first inducible interaction module; and a second sequence that encodes the second part of the protease, such as e.g. TEV-C. The order of the first and second sequence within the second nucleic acid molecule from 5' to 3' is not particularly limited and may be first (i) and then (ii) or, alternatively, first (ii) and then (i).

As described herein above, the first inducible interaction module may be chosen from a plethora of suitable inducible systems. Depending on the choice made in constructing the nucleic acid molecule of the invention, the respective corresponding interaction partner has to be included in this nucleic acid molecule encoding the second fusion protein of the invention. For example, if CIBN is present in the nucleic acid molecule of the invention as the first biosensor, then CRY2PHR has to be present in the nucleic acid molecule encoding the second fusion protein in accordance with the present embodiment. Alternatively, if either PIF or PhyB is present in the first fusion protein, the respective partner (i.e. either PhyB or PIF) has to be present in the second fusion protein. Further combinations have been recited above, e.g. LOV and LOV, GPCR and -arrestin2, CaM and M13 and so forth.

Exemplary nucleic acid sequences encoding for such a second fusion protein are represented by SEQ ID NOs:57, 59 and 61 , wherein SEQ ID NO:57 represents a fusion protein of CRY2PHR with TEV-C (for use when the first fusion protein is e.g. the protein encoded by the nucleic acid molecule of SEQ ID NO:1); SEQ ID NO:59 represents a fusion protein of β- arrestin2 with TEV-C (for use when the first fusion protein is e.g. the protein encoded by the nucleic acid molecule of SEQ ID NO:3) and, additionally, TdTomato (the same sequence without TdTomato is represented in SEQ ID NO:63 and 64); and SEQ ID NO:61 represents a fusion protein of TEV-C with M13 (for use when the first fusion protein is e.g. the protein encoded by the nucleic acid molecule of SEQ ID NO:8). The corresponding amino acid sequences are represented by SEQ ID NOs: 58, 60 and 62, respectively.

Also in accordance with the second nucleic acid molecule of this embodiment, the term "comprising" means that additional nucleic acid sequences may be encompassed in said nucleic acid molecule. Such additional sequences include for example transfections control sequences, such as the TdTomato sequence employed in the appended examples and as shown in combination with p-arrestin2 and TEV-C in SEQ ID NO:59. The set of nucleic acid molecules according to this embodiment provides suitable nucleic acid molecules for carrying out methods of monitoring or inducing intracellular signaling, as described in more detail herein below. Thus, the methods described herein as the BLITz system, the /Tango2 system and the Cal-Light system can all be carried out by employing the set of nucleic acid molecules in accordance with this embodiment. Exemplary schematic representations of first and second nucleic acid molecules in accordance with this embodiment are shown e.g. in Figure 1(e) for the BLITz system, in Figure 3(b) for the /Tango2 system and in Figure 19 for the Cal-Light system.

The present invention further relates to a set of nucleic acid molecules comprising: (a) the nucleic acid molecule according to the invention, wherein the effector-activating module is as defined in option (b)(ii); (b) a second nucleic acid molecule encoding a second fusion protein, the second nucleic acid molecule comprising (i) a first nucleic acid sequence encoding a molecule that represents the corresponding second molecule of the first inducible interaction module according to option (a); and (ii) a second nucleic acid sequence encoding a first part of a protease, wherein said first part of the protease is capable of interacting with a second part of said protease to form an active form of said protease; and (c) a third nucleic acid molecule encoding a third fusion protein, the third nucleic acid molecule comprising (i) a first nucleic acid sequence encoding a molecule that represents the corresponding second molecule of the second inducible interaction module according to option (b)(ii); and (ii) a second nucleic acid sequence encoding the second part of a protease, capable of interacting with the first part of the protease in accordance with (b)(ii) to form an active form of said protease.

This set of nucleic acid molecules is also referred to herein as the "second set of nucleic acid molecules of the invention".

The definitions and preferred embodiments provided herein above for the term "set" and the meaning of "comprising" apply mutatis mutandis also to this alternative set of nucleic acid molecules. This set of nucleic acid molecules comprises, in accordance with the present invention, at least the nucleic acid molecule of the invention, i.e. a nucleic acid molecule encoding a fusion protein as described herein above. The effector- activating module is as defined in the second alternative, i.e. it is a second biosensor, wherein said second biosensor is a first molecule capable of interacting with a second molecule to form part of a second inducible interaction module. Preferred molecules for this second biosensor and this second inducible interaction module have been described herein above and include, without being limiting, CIBN and CRY2PHR, PIF and PhyB or LOV and LOV. It will be appreciated that the second inducible interaction module is composed of different molecules than the first inducible interaction module.

The set in accordance with this embodiment further comprises a second nucleic acid molecule encoding a second fusion protein. The nucleic acid molecule encoding said second fusion protein comprises two nucleic acid sequences: a first sequence that encodes for the counterpart of the first inducible interaction module; and a second sequence that encodes a first part of a protease that is capable of interacting with a second part of said protease to form an active form of said protease. As described herein above, a particularly preferred protease is TEV protease and said first part of the protease is preferably the N-terminal part of TEV (TEV-N) and the second part is preferably the C-terminal part of TEV (TEV-C).

The set in accordance with this embodiment further comprises a third nucleic acid molecule encoding a third fusion protein. The nucleic acid molecule encoding said third fusion protein also comprises two nucleic acid sequences: a first sequence that encodes the counterpart of the second inducible interaction module; and a second sequence that encodes the second part of the protease, such as e.g. TEV-C. The order of the first and second nucleic acid sequence within the nucleic acid molecule encoding the second fusion protein and within the nucleic acid molecule encoding the third fusion protein from 5' to 3' is not particularly limited and may be first (i) and then (ii) or, alternatively, first (ii) and then (i). As described herein above, the first as well as the second inducible interaction module may be chosen from a plethora of suitable inducible systems, with the proviso that two different modules are chosen. Depending on the choice made in constructing the nucleic acid molecule of the invention, the respective corresponding interaction partners have to be included in the second and third fusion proteins in accordance with the invention. For example, if GPCR (in particular V2tail as shown in the appended examples) is present in the nucleic acid molecule of the invention as the first biosensor, then -arrestin2 has to be present in the second fusion protein. To continue with this example, -arrestin2 would be present in said second fusion protein with a first part of a protease, preferably TEV-N. The third fusion protein would then comprise the second part of said protease, e.g. TEV-C, together with the second part of the first inducible interaction system. If the first part of said first inducible interaction system present in the nucleic acid molecule of the invention is e.g. CIBN, then CRY2PHR will have to be present in the nucleic acid molecule encoding the third fusion protein.

An exemplary nucleic acid sequence encoding for such a second fusion protein is represented by SEQ ID NO:65, which represents a fusion protein of P-arrestin2 with TEV-N and, additionally, TdTomato (the same sequence without TdTomato is represented in SEQ ID NO:67 and 68). An exemplary nucleic acid sequence encoding for such a third fusion protein is represented by SEQ ID NO:69, which represents a fusion protein of CRY2PHR with TEV-C. These two exemplary sequences may e.g. be employed together with the nucleic acid molecule of the invention represented in SEQ ID NO:5. The amino acid sequence encoded by these nucleic acid sequences are represented in SEQ ID NOs: 66 and 70, respectively. This set of nucleic acid molecules according to the invention also provides suitable nucleic acid molecules for carrying out methods of monitoring or inducing intracellular signaling, as described in more detail herein below. More specifically, the method described herein as the /Tango system can be carried out by employing this set of nucleic acid molecules. An exemplary schematic representation of first, second and third nucleic acid molecules in accordance with this embodiment are shown e.g. in Figure 2(c) for the /Tango2 system.

In a preferred embodiment of the set of nucleic acid molecule, the set of nucleic acid molecules is comprised in one or more vectors.

All definitions and preferred embodiments provided herein above regarding the term "vectors" apply mutatis mutandis also with regard to this embodiment. The term "one or more", as used herein, refers to exactly one but also to more than one, such as e.g. two, three, four, five, six, seven and so on. Moreover, the term "one or more" does not define the actual number of one type of molecule present, but refers to the number of distinct molecules of the recited class. For example, the term "one or more vectors" refers to exactly one vector, i.e. one vector carrying for example the first nucleic acid molecule encoding the first fusion protein, the second nucleic acid molecule encoding the second nucleic acid molecule as well as, where applicable, the third nucleic acid molecule encoding the third fusion protein. The term, however, also refers to more than one vector, such as e.g. two or three different vectors, carrying the different nucleic acid molecules. The present invention further relates to a host cell or host expressing the set of nucleic acid molecules of the invention. The present invention further relates to a host cell or host comprising (and expressing) the one or more vectors of the invention. Preferably, the host is a non-human host. The "host cell", in accordance with the present invention, can be any cell in which signal transduction events are to be investigated. In a preferred embodiment, the host cell is/are (an) isolated cell(s) which may be part of a cell culture.

For example, suitable mammalian host cells include, without being limiting, HEK293, Hela, H9, Per.C6 and Jurkat cells, mouse NIH3T3, NS0 and C127 cells, COS 1 , COS 7 and CV1 , quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells. Also within the scope of the present invention are primary mammalian cells or cell lines. Primary cells are cells which are directly obtained from an organism. Suitable primary cells are, for example, human dermal and pulmonary fibroblasts, human epithelial cells (nasal, tracheal, renal, placental, intestinal, bronchial epithelial cells), human secretory cells (from salivary, sebaceous and sweat glands), human endocrine cells (thyroid cells), human adipose cells, human smooth muscle cells, human skeletal muscle cells, human leucocytes such as B-cells, T-cells, NK-cells or dendritic cells and stable, immortalized cell lines derived thereof (for example hTERT or oncogene immortalized cells), mouse neuronal cells, mouse embryonic fibroblasts (MEF), mouse primary hepatocytes, cardiomyocytes as well as mouse muscle stem cells (satellite cells).

Other suitable eukaryotic host cells are e.g. chicken cells, such as e.g. DT40 cells, or yeasts such as Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe and Kluyveromyces lactis. Insect cells suitable for expression are e.g. Drosophila S2, Drosophila Kc, Spodoptera Sf9 and Sf21 or Trichoplusia Hi5 cells. Suitable zebrafish cell lines include, without being limiting, ZFL, SJD or ZF4.

Particularly preferred host cells in accordance with the present invention are HEK293T cells and neuronal cells, such as e.g. the primary hippocampal cells employed in the appended examples.

Appropriate culture media and conditions for the above described host cells are known in the art. For example, for HEK293T cells, an exemplary suitable medium is high glucose Dulbecco's Modified Eagle Medium (DMEM) comprising 10% fetal bovine serum and 1% penicillin-streptomycin. Suitable culture conditions include e.g. incubation at 37°C under 10% C0 ₂ conditions. For neuronal cells, in particular hippocampal cells, an exemplary suitable medium is neurobasal medium (Invitrogen) comprising: 1 % (v/v) FBS, 1 % (v/v) Glutamax supplement, 2 % (v/v) B27 supplement, and 1 % (v/v) penicillin-streptomycin. Exemplary culture conditions for primary hippocampal neuron culture are 37°C at 10 % C0 ₂ conditions with a media-change for fresh FBS-free medium every four days.

It will be appreciated that the "host cell comprising the one or more vectors of the invention", in accordance with the present invention, expresses the fusion proteins encoded by the nucleic acid molecules comprised in said one or more vectors. Transgenic (non-human) animals as hosts transfected with and/or expressing the set of nucleic acid molecules of the invention or the one or more vector(s) of the present invention also lie within the scope of the invention. In a preferred embodiment, the transgenic animal is a mammal or a fish, more preferably the transgenic animal is selected from the group consisting of a mouse, rat, hamster, cow, cat, pig, dog, horse, rabbit, monkey or fish. Methods for the production of a transgenic animal include for example methods for the production of transgenic mice or other mammals, which usually comprise introduction of the nucleic acid molecule or targeting vector(s) of the present invention into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. The DNA of the embryonic membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe. A general method for making transgenic animals is described in the art; see for example WO 94/24274. For making transgenic organisms (which include homologously targeted non-human animals), embryonal stem cells (ES cells) are preferred. Murine ES cells, such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62:1073-1085 (1990)) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112) may be used for homologous gene targeting. Other suitable ES lines include, but are not limited to, the E14 line (Hooper et al., Nature 326:292-295 (1987)), the D3 line (Doetschman et al., J. Embryol. Exp. Morph. 87:27-45 (1985)), the CCE line (Robertson et al., Nature 323:445-448 (1986)), the AK-7 line (Zhuang et al., Cell 77:875-884 (1994)). The success of generating a mouse line from ES cells bearing a specific modification depends on the pluripotency of the ES cells (i, e., their ability, once injected into a host developing embryo, such as a blastocyst or morula, to participate in embryogenesis and contribute to the germ cells of the resulting animal). The blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant non- human females and are born, e.g. as chimeric mice. The resultant chimeric transgenic mice are backcrossed and screened for the presence of the correctly targeted transgene(s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify heterozygous transgenic mice.

The host cells or hosts in accordance with these embodiments may e.g. be employed in the methods of monitoring or inducing intracellular signaling described herein below. The present invention further relates to a method for inducing intracellular signaling, the method comprising: (a-i) providing a cell expressing the first set of nucleic acid molecules according to the invention; (a-ii) applying a first stimulus to the cell of (a-i), wherein the first stimulus is capable of inducing the first inducible interaction module; and (a-iii) applying a second stimulus to the cell of (a-i), wherein the second stimulus is capable of inducing the third biosensor such that the protease cleavage site becomes accessible; or (b-i) providing a cell expressing the second set of nucleic acid molecules according to the invention; (b-ii) applying a first stimulus to the cell of (b-i), wherein the first stimulus is capable of inducing the first inducible interaction module; (b-iii) applying a second stimulus to the cell of (b-i), wherein the second stimulus is capable of inducing the second inducible interaction module; and (b-iv) applying a third stimulus to the cell of (b-i), wherein the third stimulus is capable of inducing the third biosensor in accordance with claim 1(c) such that the protease cleavage site becomes accessible; thereby effecting a biological response due to the activation of the effector molecule.

The term "intracellular signaling", as used herein, is not particularly limited and encompasses various kinds of signal transduction events that occur within a particular cell of interest. Non- limiting examples of such signal transduction events are receptor activation-dependent activation of downstream signaling pathways, protein translocation, ion-dependent activation of downstream signaling. By employing the method of the present invention, these signals can be activated in a targeted manner due to high temporal blue light sensitivity and will result in gene expression only in the group of cells targeted, such as cells in an experiment in vitro setting as well as cells involved in vivo in an animal's perception, action, or emotion. Once cells express reporter genes, their expression can be monitored. Moreover, their expression can be used to create e.g. artificial behavior, sensation, emotion, or memory within a live animal.

In accordance with this method of the invention, a cell is provided that expresses either the first set of nucleic acid molecules of the invention (option (a)) or the second set of nucleic acid molecules of the intention (option (b)). The term "a cell", as used herein, refers to one type of cells, such as e.g. HEK293 cells or hippocampal neurons, but is not limited to one cell, but also encompasses a plurality of cells of this one cell type.

In those cases where the cell expresses the first set of nucleic acid molecules of the invention, a first and a second stimulus are then applied to the cell. The first stimulus is chosen such that it is capable of inducing the first inducible interaction module. In other words, where the first inducible interaction module is e.g. a light-inducible interaction module, the first stimulus to be applied is light; where the first inducible interaction module is e.g. a ligand-inducible interaction module, the first stimulus to be applied is a ligand and where the first inducible interaction module is e.g. a calcium-inducible interaction module, the first stimulus to be applied is calcium, e.g. via providing a stimulus that leads to an increase in intracellular calcium. Preferably, the first stimulus is a ligand or calcium.

In addition, either simultaneously or prior or after the first stimulus, a second stimulus is applied to the cell, wherein this second stimulus is chosen such that it is capable of inducing the third biosensor in the nucleic acid molecule of the invention (i.e. the third biosensor encoded by the nucleic acid molecule of (c)), such that the protease cleavage site becomes accessible. Preferably, the second stimulus is light.

In those cases where the cell expresses the second set of nucleic acid molecules of the invention, three stimuli are applied to the cell. The first stimulus is chosen such that it is capable of inducing the first inducible interaction module, as described above for the first alternative of this method. Preferably, the first stimulus is a ligand or calcium.

In addition, either simultaneously or prior or after the first stimulus, a second stimulus is applied to the cell, wherein this second stimulus is chosen such that it is capable of inducing the second inducible interaction module of the nucleic acid molecule of the invention (i.e. according to option (b)(ii) in the nucleic acid molecule of the invention). If the second inducible interaction module is inducible by a different stimulus than the first inducible interaction module, which is preferred as detailed above, then it will be appreciated that the second stimulus is different from the first stimulus. Preferably, the second stimulus is light, most preferably blue light.

Furthermore, simultaneously or prior or after the first stimulus and/or the second stimulus, a third stimulus is applied to the cell. This third stimulus is chosen such that it capable of inducing the third biosensor in the nucleic acid molecule of the invention (i.e. the third biosensor encoded by the nucleic acid molecule of (c)), such that the protease cleavage site becomes accessible.

Preferably, the third stimulus is identical to either the first or the second stimulus. More preferably, the third stimulus is identical to the second stimulus, and most preferably the third stimulus is light. It will be appreciated that where the third stimulus is identical to the first or second stimulus, said stimulus does not have to be provided separately.

Particularly preferred is that the first stimulus is a ligand or calcium and the second and third stimulus are a light stimulus, preferably blue light. Once a cell expressing either the first or the second set of nucleic acid molecules of the invention has been exposed to the recited stimuli, all interaction partners will interact with each other, thereby bringing the individual parts of the protease together, such that the protease can exert its enzymatic action on the protease cleavage site that has become accessible due to the respective stimulus. As a consequence, the effector molecule is released from the fusion protein and can now exert its action.

All other definitions and preferred embodiments provided herein above with regard to the nucleic acid molecule and the sets of nucleic acid molecules of the invention, in particular with regard to the individual components of the fusion protein and their combinations, apply mutatis mutandis to this aspect of the invention.

The present invention further relates to a method for monitoring intracellular signaling, the method comprising: (a-i) providing a cell expressing the first set of nucleic acid molecules according to the invention; (a-ii) applying a first stimulus to the cell of (a-i), wherein the first stimulus is capable of inducing the first inducible interaction module; and (a-iii) applying a second stimulus to the cell of (a-i), wherein the second stimulus is capable of inducing the third biosensor such that the protease cleavage site becomes accessible; or (b-i) providing a cell expressing the second set of nucleic acid molecules according to the invention; (b-ii) applying a first stimulus to the cell of (b-i), wherein the first stimulus is capable of inducing the first inducible interaction module; (b-iii) applying a second stimulus to the cell of (b-i), wherein the second stimulus is capable of inducing the second inducible interaction module; and (b-iv) applying a third stimulus to the cell of (b-i), wherein the third stimulus is capable of inducing the third biosensor such that the protease cleavage site becomes accessible; and (c) detecting the biological response effected by the effector molecule.

Signaling events that can be monitored have been described herein above.

The term "monitoring" refers to tracking the events within the cell upon a stimulus based on the activity of the effector molecule. Depending on the choice of effector molecule, said monitoring can for example be carried out by observing the cells by microscopy, e.g. two- photon or confocal microscopy imaging by use of fluorescent labeling; by detecting gene expression levels in e.g. western blots, chemiluminescent assays, or immunohistochemical staining; as well as by measuring receptor-mediated currents in e.g. electrophysiological recordings. The steps (a) and (b) in accordance with this method of the invention of monitoring intracellular signaling are identical to the steps (a) and (b) in accordance with the method of the invention of inducing intracellular signaling. Thus, the definitions and preferred embodiments provided above with regard to the method of the invention of inducing intracellular signaling apply mutatis mutandis to this method of the invention of monitoring intracellular signaling.

In addition, in a further step (c), the biological response effected by the effector molecule employed is detected.

Detection of the biological response can be carried out by any of a plethora of methods well known in the art. For example, if the biological response is an increase (e.g. in case of using a transcriptional activator) or decrease (e.g. in case of employing degron) in transcription or a change in transcription due to genome modulation (e.g. in case of employing Cre recombinase or the CRISPR/Cas system), detection can be carried out on the nucleic acid level or on the amino acid level. Methods for detecting transcription on the nucleic acid level include, but are not limited to, northern blotting, PCR, RT-PCR or real time RT-PCR. These methods are well known in the art. Methods for the detecting transcription on the amino acid level include but are not limited to western blotting or polyacrylamide gel electrophoresis in conjunction with protein staining techniques such as Coomassie Brilliant blue or silver- staining. Also of use in protein quantification is the Agilent Bioanalyzer technique. Where the biological response is an increase in the transcription of a fluorescently or bioluminescently labeled reporter molecule, or a change in the location of such a molecule within the cell, the detection can, in addition to the above techniques, also be carried out by microscopy or electrophysiological approaches such as whole-cell current or voltage clamp recording in combination with two-photon photolysis, or electrical or optogenetic neuronal stimulation. Electrophysiological recording will in particular be used when monitoring ionotrophic or metabotrophic receptor level changes. Furthermore, immunohistochemistry offers a further suitable approach for detecting the biological response effected by the effector molecule employed. Further aspects that can be monitored, in particular when the method is carried out in vivo in a host animal, include, without being limiting, sensory perception, motor planning/execution, emotional expression, psychiatric disorder symptoms such as schizophrenia or depression, as well as higher level cognitive functions such as motivation or learning.

Preferably, the method is carried out in vitro, most preferably in (an) isolated cell(s). As is shown in the appended examples, the provision of the nucleic acid molecule of the invention now enables the performance of various improved methods for inducing and/or monitoring intracellular signaling events, in particular the iTango or /Tango2 system or the Cal-Light system. Due to the two-step verification system employed in these methods, they represent ideal templates for multi-protein interaction induction and/or monitoring platforms. The methods of the present invention have an improved spatiotemporal resolution and signal- to-noise ratio compared to established techniques in the art and, thus, they can be applied even in environments such as the brain, that are difficult to investigate because of the subtle neuromodulatory signals that are constantly flowing in and out.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the patent specification, including definitions, will prevail.

All the sequences accessible through the Database Accession Numbers cited herein are within the scope of the present invention and also include potential future updates in the database, in order to account for future corrections and modifications in the entries of the respective databases, which might occur due to the continuing progress of science.

All amino acid sequences provided herein are presented starting with the most N-terminal residue and ending with the most C-terminal residue (N->C), as customarily done in the art, and the one-letter or three-letter code abbreviations as used to identify amino acids throughout the present invention correspond to those commonly used for amino acids.

Regarding the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise. Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The above considerations apply mutatis mutandis to all appended claims. To give a non- limiting example, the combination of claims 12, 9 and 4 is clearly and unambiguously envisaged in view of the claim structure. The same applies for example to the combination of claims 12, 9 and 7, etc..

The figures show: Figure 1 : Development of BLITz system, (a) Schematic drawing of BLITz proteins and simple diagram of light-induced gene expression. The major protein body, TM— CIBN— TEV- N— AsLOV2— TEVseq— TetR-VP16 (tTA), is targeted to a membrane with a transmembrane domain (TM). CRY2PHR— TEV-C is localized in the cytosol. Blue light causes interaction of CRY2PHR with CIBN, and TEV-C and TEV-N subsequently interact with each other. At the same time, TEVseq is unmasked due to conformational changes of AsLOV2 Joe-helix. When these two light-dependent processes are satisfied, TEVseq cleaves TetR-VP16, which translocates to the nucleus and causes targeted gene expression, (b) Design of TEVseq insertion into Joe-helix. The C-terminal end of Ja-helix was serially deleted and replaced by the TEV cleavage sequence (TEVseq; sequence ENLYFQG (SEQ ID NO:24)). The TEV cleavage site (after "Q" and before "G") is labeled by arrowheads. Note that numbering of different BLITz constructs is independent of deletion length, (c) Average of SEAP activity assay when transfected with different types of BLITz constructs. ^** indicates p < 0.01. (d) Fold induction changes when blue light was illuminated. * indicates p < 0.05 (e) Schematic of DNA plasmids transfected into cells (top). EGFP and TdTomato expression when different BLITz constructs were transfected. When TEVseq was not hidden, a significant amount of EGFP expression is visible even in dark condition. When BLITz-1 and -6 were transfected, EGFP signals were detected only when blue light was illuminated. The control without tTA (No tTA) did not trigger any gene expression even in the light condition. TdTomato is a transfection marker, (f) Light exposure time-dependent gene expression fold changes, (g) Representative images of region-specific gene expression controlled by light. Scale bars, 200 μηι (e) and 1 mm (g).

Figure 2: Development of the /Tango platform, (a) Graphical illustration of the /Tango system. DRD2-V2tail (Vasopressin 2 tail)— CIBN— AsLOV2— tTA functions as the main platform. Two more modules co-operate together to cleave TEVseq by reacting to either light or ligand. DRD2 activation by an agonist leads to -3Π·β8ίίη-2— TEV-N fusion protein translocation and binding to V2tail. The other part of TEV, TEV-C, is recruited by blue light via CRY2PHR— CIBN binding. When all /Tango modules combine, released tTA translocates into the nucleus to cause gene expression, (b) The level of gene expression quantified by SEAP assay. Concentration of quinpirole (1 nM ~ 10 μΜ) and period of blue light was varied. SEAP activity was proportionally increased by the concentration of quinpirole and the duration of light exposure, (c) The DRD2-/Tango system monitored by EGFP expression. EGFP expression was prominent only when both light and ligand are present. No EGFP was expressed even by high concentration of quinpirole if blue light was off. Blue light only was not sufficient to induce EGFP expression. TdTomato is used as a transfection marker. Scale bar, 200 μτη.

Figure 3: Light-and ligand-inducibility of gene expression in neurons using /Tango2. (a)

Schematic design of /Tango2 system. Dopamine binding to DRD2 causes -arrestin-2 recruitment to V2tail. Light exposes TEVseq, leading to the release of tTA and subsequent gene expression, (b) Light- and ligand-dependent gene expression pattern tested in hippocampal culture neurons. /Tango2 constructs and EGFP reporter genes were transfected and gene expression levels were compared between the dark and blue light conditions. Two representative neurons from each condition are magnified for clear visualization of expression, (c) Summary graph showing that EGFP expression is increased depending on the blue light exposure time and the presence of the DRD2 agonist, quinpirole. Gene expression level was measured by calculating the ratio of green to red fluorescence intensities, (d) Cumulative plot of EGFP expressing neurons. Blue light shifted the curve to the right direction significantly, indicating that more neurons display high level of green fluorescence. Note that blue light alone could not elicit any EGFP expression. Quinpirole without blue light also failed to cause gene expression at all, while a short period of blue light robustly enhanced EGFP expression. Scale bar, 100 μητι (b).

Figure 4: A general strategy for a ligand-mediated gene expression system gated by light. Schematic illustration of a ligand-mediated gene expression system gated by blue light. The strategy is similar to "AND logic gate". Ligand binding event or blue light provide independent signaling inputs to the system and the final output (target gene expression) is only turned on when both signals are concomitantly triggered. Gene expression level becomes proportional to the strength and the duration of ligand-mediated signaling in the presence of blue light.

Figure 5: Optimization of BLITz in various condition of transfection ratio, (a) Schematic experimental procedures in HEK293T cells. Right before blue light activation, media was replaced by fresh DME containing 0% FBS. (b) Summary graph of SEAP assay at different ratios of BLITz constructs. The best fold change was observed for the 8:8:2 (TM-CIBN-TEV- N-AsLOV2-tTA: CRY2-TEV-C: TetO-SEAP) ratio. Error bar represents ± S.D. of three independent experiments.

Figure 6: Optimization of DRD2-iTango1 at various transfection ratios, (a) Schematics of DNA constructs of DRD2-iTango1. (b) SEAP assay shows different gene expression level of DRD2-iTango1 at various transfection ratios. To test DRD2-iTango1 , 10 μΜ Quinpirole were treated and a 10-second pulsed blue light was given for 12 hrs. The summary graph is represented by means + S.D. of three independent measurements.

Figure 7: Blue light- and ligand-dependent gene expression of DRD2-/Tango2 in HEK293T cells, (a) Simplified illustration of DNA constructs containing individual protein modules, (b) Summary graph of dose-dependent gene expression pattern of DRD2-/Tango2 in HEK293T cells. Various concentration of quinpirole was applied in the presence of blue light (5 sec on/55 sec off) for 20 min.

Figure 8: Schematic figure representing a workflow to clone CMV::TM-CIBN-NES-TEV-N- BLITz-1-tTA and a primer list (SEQ ID NOs: 75 to 88) used in the cloning process.

Figure 9: Schematic figure represents a workflow to clone CMV::NES-CRY2PHR-TEV-C and a primer list (SEQ ID NOs: 89 and 90) used in the cloning process. Figure 10: Schematic figure represents a workflow to clone CMV::HA-DRD2-V2tail-CIBN- BLITz-6-tTA and a primer list (SEQ ID NOs: 91 to 94) used in the cloning process. Figure 11 : Schematic figure represents a workflow to clone C V::P-Arr2-TEV-N P2A TdTomato and a primer list (SEQ ID NOs: 95 to 100) used in the cloning process.

Figure 12: Schematic figure represents a workflow to clone CMV::HA-DRD2-V2tail-TEV-N- BLITz-1-tTA and a primer list (SEQ ID NOs: 101 and 102) used in the cloning process.

Figure 13: Schematic figure represents a workflow to clone CMV:: -Arr2-TEV-C P2A TdTomato and a primer list (SEQ ID NOs: 103 and 104) used in the cloning process.

Figure 14: Schematic figure represents a workflow to clone both TetO-SEAP and TetO-EGFP and a primer list (SEQ ID NOs: 105 and 106) used in the cloning process.

Figure 15: Synthetic nucleotide backbone to develop the BLITz and /Tango systems. Backbone "A" (SEQ ID NO:7) (IgK leader sequence: bold, Myc sequence: solid line, PDGFR transmembrane domain: dashed line, TEV-N sequence: bold italic) is used to clone CMV::TM- CIBN-NES-TEV-N-BLITz-1-tTA as described in Fig. 8; Backbone "B" (SEQ ID NO:47) (NES sequence: solid line, TEV-C: bold) is used to clone CMV::NES-CRY2PHR-TEV-C as described in Fig. 9; Backbone "C" (SEQ ID NO:48) (HA + FLAG sequences: solid line, V2tail: dashed dotted line, NES sequence: dashed line) is used to clone CMV::HA-DRD2-V2tail- CIBN-BLITz-6-tTA as described in Fig. 10.

Figure 16: In vivo labeling of DA-sensitive neuronal population, (a) Schematic figure of virus injection. A mixture of DRD2-/Tango2 viruses including EGFP reporter were injected into left and right NAc areas in DAT-Cre mouse. At the same mouse, AAV-dFlox-ChR2(H134R)- mCherry was injected to the right side of VTA area to control DA release selectively. Coronal section view of virus injection and fiber optic implantation. Both NAs were injected by /Tango2 viruses and an optic fiber was implanted in both hemispheres, (b) Representative confocal images of DRD2-/Tango2 expression by blue light (10 s ON and 50 s OFF, one hour). Red, green, and merged channel images were presented at upper, middle, and bottom row, respectively. Note that TdTomato expression is robust in both NAs but EGFP signals are very high only in the right NAc. High magnification images of left and right NAc were presented for further clarification, (c) Summary graph of G/R ratio in NAc (Dark: 0.045 ± 0.008, 5 mice, Light only: 0.069 ± 0.017, 6 mice; Dopamine and Light: 0.341 ± 0.061 , 6 mice; p<0.01 ). (d) The percentage of red, green, and yellow positive neurons, (e) Coronal section view of (a) from same mouse. AAV-dFlox-ChR2(H134R)-mCherry was injected in the right hemisphere of VTA. mCherry expression distinctively visualizes DA releasing neurons in VTA region of DAT- Cre mice (left). High magnification image of VTA area (right), (f) Schematic drawing of a ball maze. Head fixed mouse navigates the ball maze to find hidden spot for water rewards. The ball maze consists of 4 sections and each quadrant surface has different textures, plain, grooved, grid, and striped. Reward spot is hidden at the center of grid surface section. Mouse movement is monitored by bluetooth motion sensor imbedded in the center of ball, (g) Average graph for the number of water reward successes. Animal training was performed for consecutive 7 days (Day 7: Water Restricted: 53.3 ± 8.09, 7 mice, Satiated: 20.3 ± 3.78, 7 mice, p<0.01 ). (h) Blue light was illuminated starting at training day 2 for 6 days. Blue light was shined for 3 seconds each time mice get water rewards, (i) Representative TdTomato and EGFP signals from M2 region. Yellow square boxes on the top images are magnified and presented in the bottom, (j) Summary graph of G/R from individual neurons in water restricted with and without rewards, satiated, and 6-OHDA injected mouse group (Water Restricted + Reward: 0.297 ± 0.027, 1252 neurons, 7 mice; Water Restricted + No Reward: 0.200 ± 0.009, 703 neurons, 7 mice; Satiated: 0.124 ± 0.007, 892 neurons, 6 mice; Water Restricted + 6- OHDA: 0.080 ± 0.006, 452 neurons, 5 mice), (k) Representative M2 area images from 6- OHDA injected mice. (I) TH staining of ipsilateral and contralateral side of M2 after 6-OHDA injection. Red signals indicate TH staining and blue signals indicate DAPI staining. Scale bars, 100 prn (b), (e), (i, bottom), (k), and (I), and 1 mm (i, top). * and ** indicates p < 0.05 and p < 0.01 , respectively. Error bar represents s.e.m. Figure 17: In vivo manipulation of locomotion- and reward-related DA-sensitive neuronal populations in the central striatum, (a) Schematics of virus injection. DRD2- /Tango2 viruses and TRE-ChR2-EYFP were injected into central striatum bilaterally, (b) Graphical illustration of experiments. Blue light (473 nm) was delivered for 3 seconds when mice begin locomotion (speed greater than 0.5 ms ^"1 for at least 2 s) or rest with a reward, for 50 trials per day over 3 days to induce the expression of ChR2-EYFP reporter in locomotion- or reward-related DA-sensitive neuronal populations, respectively. Two days after induction, a probe test was conducted by delivering blue light when mice were rest to activate ChR2- expressing populations, (c) Coronal sections of the central striatum from mice not exposed to blue light (dark control, top), mice received blue light during rest with a reward (reward-DA, middle), and mice received blue light during locomotion (locomotion-DA, bottom), (d) Locomotion speed aligned on onset of laser stimulation (light blue) during a representative probe session from reward-DA (top) or locomotion-DA (bottom) groups. Each row represents a single trial from rest (left). Individual (thin gray line) and average (thick black and red) traces of locomotion speed over time are plotted (right), (e) Superimposed average speed of locomotion aligned on onset of laser stimulation (light blue box) from mice of reward-DA and locomotion-DA groups. (Locomotion-DA, 6 mice; Reward-DA, 6 mice; Repeated measures ANOVA, F _{group x time} = 5.58, ***p < 0.001 ).

Figure 18: In vivo manipulation of DA-sensitive neuronal populations in the NAc. (a) Schematics of virus injection, (b) Coronal sections of the NAc from Cre-negative (left), D1-Cre (middle) or D2-Cre (right) mice, exposed to blue (473 nm) light for 60 minutes after a cocaine injection (bottom) or dark controls (top), (c) Quantification of infected neurons expressing EYFP alone, TdTomato alone or co-expressing both fluorescent proteins, (d) Ex vivo validation that amber (561 nm) light inhibits spike activity of halorhodopsin-expressing MSNs (F _current = 1325.42, p < 0.0001 , F, _aser = 90.73, p < 0.001 F _{current x} i _aser = 12.815, p = 0.03). (e) Schematic of experiment; blue light exposure with injection of cocaine (Day 1 ) to induce the expression of halorhodopsin, which was subsequently activated with amber light during the cocaine challenge, (f) Locomotor activity of mice after initial and challenge cocaine injections. Cre-negative mice showed robust sensitization to the challenge injection of cocaine (n = 13, p < 0.0001 ); this sensitized response was absent in D1-Cre mice (n = 8, p = 0.565), while D2- Cre mice (n = 8, p = 0.006) also showed robust sensitization (F _ge neotype = 39.86, p < 0.0001 , Fgeneotype x time = 7.96, p = 0.002). (g-j) Dark/light controls, (g) Schematics of experimental design. Dark controls received an injection of cocaine, but were not exposed to light, while light controls were exposed for 60 minutes to blue light prior to the cocaine injection, (h) Coronal NAc sections from Cre-negative (left) and D1-Cre (right). Dark controls (top) or mice exposed to blue light for 60 minutes prior to cocaine injection (bottom), (i) Quantification of infected neurons expressing EYFP alone, TdTomato alone or co-expressing both fluorescent proteins, (j) Both dark controls (Cre-negative: t ₅ = 4.79, p = 0.005, D1-Cre: t ₅ = 6.15, p = 0.002) and light controls (Cre-negative: t ₅ = 4.31 , p = 0.008, D1-Cre: t ₅ = 2.66, p = 0.045) exhibited a sensitized response to the second cocaine injection (n = 6 / group). *p < 0.05, **p < 0.01 , ***p < 0.001.

Figure 19: Principles of Cal-Light. Schematic overview showing the principle of calcium-and light-induced signaling in the Cal-Light system. Figure 20: Cal-Light test in dissociated culture neurons. Cal-Light constructs and EGFP reporter plasmid were transfected to hippocampal culture neurons. After 5 days of expression, a short pulse of blue light (5 sec on/55 sec off) was illuminated for 30 min in the presence or absence of tetrodotoxin (TTX). Two days later, slices were fixed and images were taken by confocal microscopy. EGFP expression was robustly increased only in a condition when blue light was illuminated and neuronal activity was not inhibited by TTX.

Figure 21 : Cal-Light test in cortical slice culture. AAV expressing Cal-Light constructs and EGFP reporter were infected to cortical slice culture at DIV3 (Days in vitro). After 12 days of expression, a short pulse of blue light (8.5 sec on/51.5 sec off) was illuminated for 30 min while a short burst of electrical stimulation was given. Examples of action potentials were plotted at the top left panel. Electrical stimulation reliably triggered action potentials measured in layer 2/3 pyramidal neurons. Two days later, slices were fixed and images were taken by confocal microscopy. EGFP expression was robustly increased in a condition when blue light and high frequency stimulation were given. Blue light or high frequency stimuli alone was not able to increase the level of EGFP expression.

Figure 22: In vivo labeling of an active neuronal population in awake behaving mice with Cal-Light. 2 weeks expression of AAV Cal-Light (400nl) with GFP reporter (100 nl). (a) AAV injection and cranial window site. AAV-GCaMP6s was injected into mouse motor cortex to monitor neuronal activity by Ca2+ imaging., (b) Task schematics. A mouse is head-fixed sitting on the air-floating styrofoam ball. In this setup, the mouse can freely run on the ball. Blue light (473 nm) is delivered for 5 sec whenever the mouse start to run. (c) Calcium imaging (plotted as Delta F/F) from individual layer 2/3 neurons in motor cortex while a mouse runs on the ball. Whenever mice move, many neurons fire simultaneously as detected by calcium imaging. Because we illuminated blue light only while mice move, only active neuronal population related movements is supposed to be labeled by Cal-Light constructs, (d) In vivo imaging of Cal-light positive neurons. R0: reference plane (to identify exactly same brain area before and after blue light from the same mice, we imaged the landmark on the brain surface). Fluorescent intensity of cells in two regions (R1 and R2) is compared before and after (48 hours) blue light exposure, (e) Fluorescent intensity of cells before and after blue light exposure.

Figure 23: Labeling active population of neurons during complex behavior, (a) Bilateral injection of Cal-Light viruses in M1 area. To label learning-related neuronal population, we trained water restricted mice to learn repetitive lever pressing behavior to get water rewards. (b) Schematic mouse training schedule and Cal-Light labeling with blue light, (c) Fiber optics were implanted both hemispheres of M1 area and blue laser was programmed to be switched on for 5 sec whenever mice press the lever. Once light was on for 5 sec, the next blue light was prohibited for the following 25 sec even though mice press the lever, (d) Number of rewards were gradually increased during learning. Lever press learning was same regardless of blue light (8 mice trained without light, 11 mice trained with light). Shaded blue box indicates labeling sessions by blue light, (e) The total number of lever press per minute was also significantly increased while mice were under training (8 mice trained without light, 1 mice trained with light), (f) Summary graph of lever press per minute at different conditions. Note that the number of lever press was significantly reduced by muscimol injection at M1 region. Lever press learning was not impaired after one month or together with fiber optic implantation surgery (CRF: 2.5 ± 0.6, 11 mice; FR-12: 13.7 ± 1.3, 11 mice, p < 0.005; FR-5: 6.6 ± 0.7, 11 mice, p < 0.01 ; Saline: 6.4 ± 1.3, 4 mice; Muscimol: 0.12 ± 0.09, 3 mice, p < 0.05; Light-: 7.1 ± 1.2, 8 mice; Remote: 6.4 ± 2.1 , 5 mice; Remote + Surgery: 5.1 ± 1.3, 4 mice), (g) Representative images from mouse brains trained with or without blue light. Scale bars, 100 pm. (h) Cumulative distribution of G/R at each condition (Light only: n = 448 cells / 8 mice; Activity only: n = 585 cells / 9 mice; Light + Activity: n = 504 cells / 11 mice), (i) Summary box plot chart of G/R (Light only: 0.32 ± 0.09, n = 448; Activity only: 0.36 ± 0.11 , n = 585, p = 0.34; Light + Activity: 1.1 ± 0.97, n = 504, p < 0.005). Error bars represent s.d. ^* , ^** and *** indicate p < 0.05, p < 0.01 and p < 0.005, respectively. Error bars represent s.e.m. except for (i).

Figure 24: In vivo manipulation of learning related neuronal population, (a) Cal-Light viruses were injected into M1 bilaterally. eNpHR reporter was injected in order to inhibit activity from selective neuronal population labeled during lever pressing behavior, (b) 589 nm light efficiently inhibited the neuronal activity. APs were measured in a current clamp mode before and after 589 nm light from eNpHR expressing neurons. Average number of APs and representative traces were displayed (Light OFF at 350 pA, 15.4 ± 2.1 , n = 9; Light ON at 350 pA, 8.8 ± 1.8, n = 9, p < 0.005). (c) In a condition of either light or activity alone did not cause high eNpHR reporter gene expression. G/R from individual neurons and an average mean were plotted in a box chart (Blue light only: 0.24 ± 0.12, n = 228; Activity only: 0.23 ± 0.11 , n = 115, p = 0.56; Light + Activity: 0.79 ± 0.43, n = 165, p < 0.005). Error bars represent s.d. (d) Illustration of experimental procedures. Neuronal population related to successive lever pressing learning was labeled by blue light and the learned behavior was selectively inhibited by yellow light, (e) Summary learning curve plotted by the number of lever press per minute. Each group of mice were symbolized by different colors or shapes. "589 only" indicates a mouse group who received the same number and duration of yellow light, but not labeled by blue light, (f) Average numbers of lever press per minute at different experimental conditions were plotted (Blue label + 589 OFF: 15.9 ± 1.1 , n = 7; Blue label + 589 ON: 5.1 ± 0.6, n = 5, p < 0.005; Blue label + 589 OFF (1d after): 14.4 ± 2.1 , n = 7, p < 0.01 compared to Blue label + 589 ON; No Blue label + 589 ON (589 only): 16.3 ± 0.9, n = 6; Blue label + 589 OFF: 15.1 ± 2.6, n = 5; Blue label + Random 589 ON: 14.7 ± 1.7, n = 3, p = 0.884 compared to Blue label + 589 OFF; Random Blue label + 589 ON: 14.4 ± 1.9, n = 6, p = 0.5 compare to Random Blue label + 589 OFF [15.9 ± 1.1 , n = 6, not shown in a graph]), (g) The frequency of lever pressing behavior over time, (h) Cumulative lever press number over time (Blue label + 589 OFF: 13.3 ± 1.1 minutes to reach 240 lever press, n = 7; Blue label + 589 ON: 39.2 ± 2.9, n = 5; Blue label + 589 OFF (1d after): 16.9 ± 3.1 , n = 6, p < 0.005 compared to Light OFF). ^* * and ^*** indicate p < 0.01 and p < 0.005, respectively. Error bars represent s.e.m. except for (c).

Figure 25: Schematic representation of incorporation of alternative effector molecules.

A) Schematic representation of a combination with the CRISPR-Cas system. B) Schematic representation of a combination with the Cre recombinase system. Figure 26: Schematic representation of light-inducible, Spy-tagged CRISPR-Cas9 and CRE recombinase system. Both CRISPR-Cas9 and Cre systems are gene editing system. Cas9 or Cre proteins are split, so that N- or C-terminal fragment are obtained that are not functional on their own. In the scheme, Cre-N or Cas9-N have been designed such that they are expressed in the cytosol, while Cre-C or Cas9-C are expressed in the nucleus, in order to avoid stimulus-independent activation of the CRISPR-Cas9 or CRE system. When the Cre-N or Cas9-N is released by TEV cleavage, they can bind to the C-terminal part of the respective protein, because each N- and C-terminal fragment are linked to SpyTag and SpyCatcher, respectively. SpyTag and SpyCatcher can interact by themselves without any additional triggers.

Figure 27: Schematic overview of the various methods. The following examples illustrate the invention:

Example 1 : General methods Design and construction of plasm id vectors

All plasmid vectors were constructed using a mammalian expression pCS4+ vector containing CMV IE94 promoter and ampicillin resistant sequence. Construction strategies and primers are fully described in Figures 8 to 15. Sequences encoding TEV-N, TEV-C, and V2tail were chemically synthesized by Eurofin Genomics (Huntsville, AL, USA) and their full sequences are provided in Figure 15.

To generate CMV::TM-CIBN-NES-TEV-N-BLITz-1-tTA (Fig. 8), CIBN, AsLOV2, and tTA sequences were amplified from pCIBN (ANLS)-pmGFP (Addgene #26867), pLL7.0: Venus- ILID-CAAX (Addgene #60411 ), and pSAM200 (provided by Dr. Wilfried Weber, University of Freiburg, Germany; tTA can be obtained from the pUHD15-1 vector described in Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences of the United States of America 89, 5547-5551 (1992), which is commercially available from Clontech, Cat No 631017), respectively. Amplified PCR products were digested by suitable combination of restriction enzymes and each PCR product was sub-cloned into synthesized TEV-N backbone as described in Fig. 15.

CMV::NES-CRY2PHR-TEV-C (Fig. 9) was generated by ligating the synthesized TEV-C backbone and the amplified CRY2PHR from Pcry2PHR-mCherryN1 (Addgene #26866).

CMV::HA-DRD2-V2tail-CIBN-BLITz-6-tTA (Fig. 10) was produced by a series of ligation of V2tail, AsLOV2-tTA, and DRD2. The sequence encoding HA signal and V2tail backbone originated from Presto-Tango sequences except for modifications of several restriction enzyme sites (Kroeze, W. K. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nature structural & molecular biology 22, 362-369, doi:10.1038/nsmb.3014 (2015)). DRD2 was amplified from pcDNA3.1-D2-YFP (Addgene #44194) with BamHI and EcoRI sites.

CMV:-.p-Arrestin2-TEV-N-P2A-TdTomato (Fig. 11) was produced with a simple subcloning method. p-Arrestin2 sequence was amplified from β-Arrestin GFP WT (Addgene #35411). All cloning enzymes and reagents were purchased from New England Biolabs (MA, USA). All plasmid vectors generated in this study were confirmed once again by DNA sequencing (Eurofin Genomics). Site-directed mutagenesis for BLITz constructs

To generate BLITz variants, small nucleotides were inserted or deleted from the original AsLOV2 sequence through site-directed mutagenesis. Whole amplification of vector was performed by KOD polymerase (Cat #71086-3, EMD Millipore, Billerica, MA, USA) which allows high speed and accurate PGR amplification. To remove parental templates, Dpnl (Cat #R0176, New England Biolabs) restriction enzyme was used at 37°C for 1 hr. This mixture was directly added to competent cells (E. coli DH5a strain, Zymo Research, CA, USA). Primers used for mutagenesis are described in Fig 8 and shown in SEQ ID NOs: 75 to 88. HEK293T cell culture and DNA transfections

HEK293T cells were grown in high glucose Dulbecco's Modified Eagle Medium (DMEM) (Gibco, CA, USA) containing 10% fetal bovine serum (Cat# 10438-018, Gibco) and 1% penicillin-streptomycin (Invitrogen, NY, USA). Cells were incubated at 37°C and under 10% C0 ₂ conditions. For the experiment, all dishes and coverslip were pre-coated with a 1 mg/ml Poly-D-Lysine hydrobromide (Cat# P0899, Sigma-Aldrich, St. Louis, MO, USA) solution for 2 hrs. After 0.25% trypsin (Cat# 25200, Gibco) treatment for 2 min, detached cells were collected and the total cell numbers were counted. Dissociated cells were plated at 2x10 ⁵ cells per 12 mm coverslip. 24 hrs later, DNA plasmid vectors were transfected using the calcium phosphate transfection kit (Clontech, CA, USA). The mixture of the DNA solution was slowly added into 2x Hepes Buffered Saline. After 1 hr incubation, precipitated solutions were added into each well.

Preparation of dissociated hippocampal cultures and DNA transfections

Primary hippocampal neuron culture was performed as previously described (Lee, D. et al. Inositol 1 ,4,5-trisphosphate 3-kinase A is a novel microtubule-associated protein: PKA- dependent phosphoregulation of microtubule binding affinity. J Biol Chem 287, 15981-15995, doi:10.1074/jbc.M112.344101 (2012)). Briefly, rat hippocampus (embryonic 18 days) was rapidly dissected and digested with 0.25% trypsin-EDTA (Invitrogen) for 10 m at 37°C. After trypsin-EDTA was removed, trypsinized cells were carefully triturated with 1 ,000 μL-sized pipet tip for 10 times. Dissociated cells were counted and plated at 10 ⁵ cells onto 12 mm PDL- coated coverslips. Plating media consisted of neurobasal medium (Invitrogen) and the following reagents: 1 % (v/v) FBS, 1 % (v/v) Glutamax Supplement (Gibco), 2 % (v/v) B27 supplement (Gibco), and 1 % (v/v) penicillin-streptomycin. Primary hippocampal neuron culture was grown in 37°C temperature and 10 % C0 ₂ conditions. Every 4 days, one third of the volume of media was replaced with fresh maintaining media lacking FBS. DNA was transfected by using a neuronal calcium phosphate transfection method as previously described (Jiang, M. & Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat Protoc , 695-700, doi:10.1038/nprot.2006.86 (2006)). Three days later (DIV 10), a short period of blue light was illuminated for 2 hrs (5 s ON / 55 s OFF) and quinpirole and/or haloperidol were added into media when needed. After 2 hrs of incubation, the media were replaced to fresh ones. Neurons were fixed at DIV 12 for image acquisition.

Blue-light illumination

Blue light was illuminated by 465 nm wavelength blue LED array (LED wholesalers, Hayward, CA, USA) that was controlled by a high-accuracy digital electronic timer (Model 451, GraLab, Centerville, OH, USA). The LED array was installed inside the 37°C and 10% C0 ₂ incubator. One transparent blank plate with 2 cm height was inserted between the LED source and the sample to inhibit potential undesirable heating caused by the direct contact of the LED. In the experimental setup, the power of blue light at the specimen was 1.7 mW, measured by a power meter (PM100D, ThorLabs, Newton, NJ, USA). To make a dark condition, all lights were prevented by wrapping culture plates with an aluminum foil, and all experimental procedures were carried out under dim red light.

SEAP chemiluminescent assay

For the quantification of gene-expression levels, a secreted embryonic alkaline phosphatase (SEAP) chemiluminescent assay was used. All reagents for the SEAP assay were purchased from InvivoGen (San Diego, CA, USA). 40 μΙ of sample was collected from the medium of each well and transferred into 96-wells plate. Samples were pre-heated in a 60°C incubator for 10 m to inhibit the activity of endogenous alkaline phosphatase. All mixtures were added into a single master tube including SEAP substrates and L-homoarginine. The mixed solutions were carefully added into each sample onto 96-wells plate without bubbles. The chemiluminescence of each sample was measured by a micro-plate reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA, USA) at 37°C at 405 nm. Assays were made at every 30 s for 2 hrs. All data and the calculation of V _max were acquired by a SoftMax Pro 5.4.1 (Molecular Devices).

Preparation and acquisition of Images

Cells were fixed by pre-warmed 4% paraformaldehyde (Santa Cruz Biotechnology, Dallas, TX, USA) for 10-15 min. Fixed cells were rinsed with PBS three times. Coverslips were mounted using mounting solution (Electron Microscopy Science, PA, USA). Imaging was performed using an upright confocal laser-scanning microscope (LSM780, Zeiss, Oberkochen, Germany) with 20x/0.8 M27 objective lens. Pharmacological drugs and statistics

Quinpirole and haloperidol were purchased from Tocris Bioscience (Minneapolis, MN, USA). Statistical significance was calculated by one-way ANOVA with post hoc Games Howell test using SPSS 12.0 (IBM) software.

Example 2: Design of the Blue-Light Inducible TEV protease (BLITz) system.

Initial experiments showed that most photoactivatable proteins employed, such as CRY2 (cryptochrome 2) and CIB1 (cryptochrome-interacting basic-helix-loop-helix 1 ) (Zhang, K. & Cui, B. Optogenetic control of intracellular signaling pathways. Trends in biotechnology 33, 92-100, (2015)), provided a certain degree of light-independent background signals. Without wishing to be bound by theory, it is believed that this was due to the intrinsic promiscuity of protein-protein interactions. As this would prove problematic in an environment like the brain, where only subtle amounts of neuromodulators are flowing in and out, a novel two-step light switch control system was developed and named Blue-Light Inducible TEV protease (BLITz) (Fig. 1a). The BLITz system consists of two synthetic proteins and a single reporter vector with a tetracycline response element (TRE). The first synthetic protein is a membrane- tethered protein consisting of multiple light-sensitive modules: 1 ) CIBN (a truncated form of CIB1) (Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nature methods 7, 973-975, (2010)); 2) TEV-N (N-terminal region of TEV protease) (Wehr, M. C. et al. Monitoring regulated protein-protein interactions using split TEV. Nature methods 3, 985-993, (2006)); 3) TEV protease cleavage sequence (TEVseq) inserted in a truncated form of Avena sativa phototropinl light-oxygen-voltage 2 domains (AsLOV2) (Guntas, G. et al. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proceedings of the National Academy of Sciences of the United States of America 112, 112-117, (2015)); and 4) TetR-VP16 (tetracycline-controlled transcriptional activator) (Fig. 1a). The second synthetic protein is a fusion protein of TEV-C (C-terminal region of TEV protease) and CRY2PHR (cryptochrome 2 photolyase homology region) ((Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nature methods 7, 973-975, (2010); Wehr, M. C. ef al. Monitoring regulated protein-protein interactions using split TEV. Nature methods 3, 985-993, (2006)) (Fig. 1a). In this set-up, TEV-C and TEV-N are separated into two different proteins and cannot bind each other in the absence of light (dark state). In the presence blue light, CRY2PHR and CIBN interact causing TEV-C and TEV-N to interact, regain protease function, and cleave TEVseq. Although the split TEV system reduced light-independent noise signals, some background cleavage was nonetheless observed due to the diffusion-mediated interaction between TEV-C and TEV-N (Williams, D. J., Puhl, H. L, 3rd & Ikeda, S. R. Rapid modification of proteins using a rapamycin-inducible tobacco etch virus protease system. PloS one 4, e7474, (2009)). To abolish spontaneous TEVseq cleavage, the crystal structure of AsLOV2 protein was consulted, which shows that the Ja-helix is tightly associated with the Per-ARNT-Sim (PAS) core domain in the dark state, but becomes released upon blue light illumination (Harper, S. M., Neil, L. C. & Gardner, K. H. Structural basis of a phototropin light switch. Science 301 , 1541-1544, (2003)). Thus, to prevent access by TEV protease in the dark state, but to allow complete access upon blue light illumination, the C-terminal region of the Ja-helix on AsLOV2 with the TEVseq was modified (Fig. 1a, b). To screen for light-inducible molecules with optimal SNR, serial deletion mutations were generated in the C-terminal region of the Ja-helix, including a point mutation within the TEVseq (Fig. 1 b). It was observed that background gene expression levels were high when TEVseq was inserted close to the Ja-helix C-terminus (BLITz-3 and -4) (Fig. 1 c), indicating TEVseq was accessible to TEV protease. Removing two more amino acids from the Ja-helix (BLITz-1 and -6), completely abolished baseline gene expression, while maintaining high light-induced gene expression. Removing amino acids 137 and 138 (BLITz-2) restored the baseline gene expression, suggesting those amino acids are tied to the light-dependent conformational changes (Fig. c). When the intact TEVseq was tested without a fusion to the AsLOV2 protein, similar to the classical Tango system, basal gene expression was high, as expected, and subsequent fold change was minimal (~1.4 fold) (Fig. 1c, and 1f,). Upon removal of TetR-VP16 (No tTA), signals were nearly undetectable, suggesting background gene expression did not originate from the reporter itself (Fig. 1 c). Based on a secreted embryonic alkaline phosphatase (SEAP) assay, BLITz-1 and -6 were found to be the best light-induced constructs, both with over 20-fold gene expression by blue light (Fig. 1d). Since the BLITz system is operated by protein-protein interactions, fold changes were found to be variable by the ratio of individual modules (Fig. 5). The results of the SEAP assay were in agreement with EGFP expression reporter data (Fig. 1e). BLITz-6 was selected for use in the neuromodulation mapping system.

Gene expression was also dependent on duration of blue light exposure. When a short light pulse (10 sec ON/50 sec OFF) was repeated for 5 min, gene expression was significantly increased, and the fold change was dramatically increased with longer exposure times (Fig. 1f). Because the light cycle was 10 sec ON/50 sec OFF per minute, just 50 seconds of total light exposure was sufficient to induce high gene expression and 5 minutes light caused fold changes higher than 20 folds (Fig. 1f). Additionally, this light-dependency greatly improved spatial resolution, enabling the limitation of EGFP expression only to the cells exposed to blue light (Fig. 1g). Thus, a light-inducible gene-coupled reporter system representing transitory protein interaction with a high SNR and precise spatiotemporal resolution has been developed. Example 3: Design of the /Tango systems.

Unlike the BLITz system, the original Tango system lacks an external control switch, resulting in constitutive activation and poor temporal resolution. To overcome these drawbacks, the original Tango system was reengineered by combining it with the BLITz system, such that gene expression is initiated only when both ligand and light are present (Fig. 2a). In this inducible Tango system (referred to herein as /Tango), the binding of ligands to receptors causes -arrestin-2/TEV-N recruitment, but does not cause TEVseq cleavage. Blue light illumination then recruits CRY2/TEV-C to form a functional protease that cleaves TEVseq. The two-step verification system of /Tango makes it the ideal template for a multi-protein interaction monitoring platform. Furthermore, by simply exchanging the GPCR components of the /Tango a whole library of GPCRs can be build, as shown in a recent study (Kroeze, W. K. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nature structural & molecular biology 22, 362-369, (2015)).

To verify that the /Tango system reliably labels neuromodulatory actions, dopamine action was tested by using dopamine 2 receptor (DRD2). /Tango constructs were expressed in HEK293T cells, and a DRD2 agonist, quinpirole, was introduced into the culture media. In the dark state, there was no noticeable spontaneous gene expression, but upon blue light illumination, gene expression was observed in a dose-dependent manner. Conversely, a complete block was observed with the DRD2 antagonist haloperidol (Fig. 2b). Interestingly, after transfecting a high amount of p-arrestin-2-TEV-N plasmid, SNR was robustly increased, suggesting that a reserved pool of p-arrestin-2 protein in the cytosol is critical (Fig. 6). An EGFP expression reporter assay revealed the same quinpirole- and light-dependent pattern (Fig. 2c).

These results indicate that gene expression was very selective to DRD2 activation, and ligand-independent background signals are almost negligible as expected from the two-step activation design. The temporal resolution was increased by about ~100 fold (calculated by net light exposure time, 10 min) as compared to the classical Tango system, which requires 12~24 hours of ligand incubation (Inagaki, H. K. ei al. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583-595, (2012)). More importantly, the constitutive presence of quinpirole, even at high concentration, did not cause any background signals and only gated by light. Thus, the new /Tango system is a fast and reliable light-inducible technique with a high SNR for monitoring behaviorally-related phasic neuromodulatory action.

A further challenge is presented by neurons due to their morphology. For example, the probability of all three /Tango proteins coming together in a thin and long space (e.g., dendrites or axons) is much lower than that of a compact, round space (e.g., HEK293 cells). Additionally, even if tTA is successfully released after TEVseq cleavage, it must travel a long distance through the dendrite to the cell body. To overcome these limitations, a simplified version of /Tango was generated and called /Tango2. This system lacks the CRY2PHR/CIBN light switch, allowing easier formation of the light- and ligand-induced protein complex. Transfecting /Tango2 into HEK cells greatly increased the overall density of gene expression (Fig. 7). Background signals were slightly elevated due to the lack of one light-sensitive module, but both light- and ligand-inducible features were still preserved. In /Tango2- transfected neurons, background signals were nearly undetectable, but light- and ligand- inducibility was very robust, with a SNR corresponding to roughly 900 % fold change (Fig. 3b- d). The same experiments using the conventional Tango system yielded only a 50 % fold change (Djannatian, M. S., Galinski, S., Fischer, T. M. & Rossner, M. J. Studying G protein- coupled receptor activation using split-tobacco etch virus assays. Analytical biochemistry 412, 141-152, (2011)).

Thus, the /Tango systems enable visualization of neuromodulation codes in a precise time and space, which will allow to understand neural network topology of internal brain states underlying behavioral diversity. Example 4: In vivo labeling of neuronal population sensitive to DA

To test whether endogenous DA release is sufficient to trigger /Tango2-mediated gene expression in vivo, we injected AAV1-hSYN-DRD2-V2 tail-TEV-N-AsLOV2-tTA, AAV1 -hSYN- □-Arrestin2-TEV-C-P2A-TdTomato, and AAV1 -hSYN-TRE-EGFP (short, DRD2-/Tango2 viruses) bilaterally into the nucleus accumbens (NAc) and AAV-dFlox.hChR2(H134R)- mCherry to the right ventral tegmental area (VTA) of DAT-Cre mice (Fig. 16a). Under these conditions, coincident DRD2-/Tango2 activation and DA release would occur only in the right hemisphere, but DRD2-/Tango2 in the left hemisphere would remain as a "Light only" control, because DA neuron projections are mostly unilateral ²⁷ . As expected, blue light illumination (10 s ON and 50 s OFF, one hour) elicited robust EGFP expression exclusively in the right NAc (Fig. 16b-d). Minimal expression of EGFP in the left NAc confirmed DRD2-/Tango2 constructs do not cause background signals without selective neuromodulator release (Fig. 16c, d). ChR2 expression in DA neurons was also confirmed to a posterior coronal section from the same mouse (Fig. 16e). These results indicate that DRD2- Tango2 is sensitive enough to detect endogenous phasic DA release in vivo. To determine the time course of induction and degradation of reporter gene expression, we carried out time-lapse imaging of EGFP expression after induction.

Feasibility of /Tango2 in vivo motivated us to test whether DRD2-/Tango2 can be used for identifying behaviorally-relevant neuronal populations during specific animal behaviors. We first ensured that the target gene expression was mediated by concurrent presence of light and ligand. In cultured neurons, we sequentially shined blue light for 2 h, and then applied quinpirole for 2 h. In this case, we did not detect a significant increase in double positive (green and red) neurons. We next delivered light and reward concurrently or asynchronously in water-restricted mice with /Tango2 viruses injected in their NAc. When we repeated the light and ligand administration 20 times with 90-second intervals, we did not observe robust gene expression; in fact, the expression levels were significantly lower than when light and reward were delivered coincidently. Thus, our data show that /Tango2-induced labeling requires a concurrent presence of light and ligand and is positively correlated with the amount of ligand in vivo.

Next, we trained mice in a simple ball maze. This is similar to the classical Morris water maze, but instead of visual cues, we added sensory cues on the surface of a Styrofoam ball such that the mice would learn to associate one of the sensory cues with a reward. The ball was evenly divided into four sections and each quadrant surface had one of four textured surfaces: plain, grooved, grid, or striped (Fig. 16f). The reward spot was hidden at the center of the grid surface section, so whenever the mice reached the hidden spot, a water reward was provided and the behavior counted as a success. Water-restricted mice learned this task quickly over several days as indicated by increased successes per day (Fig. 16g). On the other hand, success rates did not increase when fully satiated mice were placed at the ball maze setup (Fig. 16g). These results suggest that water-restricted mice were motivated to explore the ball, and that the ball maze task is a good method for assessing reward-based learning.

To identify the neuronal population activated by DA during this task, we injected DRD2- /Tango2 into premotor cortex (M2) and shined blue light for 3 seconds whenever rewards were delivered (Fig. 16h). Satiated mice were also exposed to the same amount of blue light. Two days after the last training session (Day 7), we observed that the intensity of EGFP expression was significantly higher in water-restricted mice versus satiated mice or water restricted mice with no reward (Fig. 16i, j). The group treated with 6-hydroxydopamine (6- OHDA) did not express EGFP, indicating that EGPF expression was induced by DA released at the time of reward delivery (Fig. 16j, k). The efficacy of 6-OHDA was also verified by tyrosine hydroxylase (TH) antibody staining (Fig. 161). Thus, the /Tango system enabled the visualization of neuromodulation action with high spatiotemporal precision in awake behaving animals and can be used to identify novel DA-sensitive neuronal populations.

Example 5: Labeling and manipulation of behaviorally-relevant subpopulation of neurons in behaving animals

The /Tango system links gene expression to coincident stimulation by both a ligand and light, which could enable the manipulation of neuronal activity by optogenetic effectors. To determine whether /Tango could be used to establish causal effects of neuromodulators on neuronal circuits, we labeled two separate neuronal populations related to different behaviors. We injected DRD2-/Tango2 viruses and TRE-ChR2-EYFP into the central striatum and labeled either locomotion- or reward-related neuronal populations that were recently reported to be activated by the DA (Fig. 17a, b). Using the same behavioral paradigm as described above, we labeled neurons with a ChR2 reporter, subjected mice to a running task on a floating ball or gave them rewards (Fig. 17c), and then shined a blue light to selectively activate the locomotion-sensitive neuronal population (locomotion-DA). During the probe test, ChR2 activation was sufficient to cause locomotion (Fig. 17d, e). When we labeled the reward-related neuronal population (reward-DA), the same blue light did not cause locomotion (Fig. 17d, e). Thus, /Tango2 is a useful tool to distinguish behaviorally relevant subpopulations of neurons in behaving animals and to test the sufficiency of eliciting behaviors.

Example 6: Reversal of drug-induced locomotion sensitization using /Tango2

We also tested /Tango2 system to influence circuits in a model of cocaine-induced locomotor sensitization. We used the D2-coupled DA receptor sensing system to induce the expression of halorhodopsin (eNpHR): an amber-light activated proton pump that inhibits cell firing (Fig. 18a). First, we validated that there was no expression of the AAV1-hSYN-Flox-[]-Arrestin2- TEV-C-P2A-TdTomato construct in Cre-negative cells, indicated by an absence of signal in wild-type mice. We also confirmed that blue light was necessary to drive the expression of eNpHR-EYFP, since no EYFP signal was observed in the absence of blue light in all genotypes (Fig. 18b, c). Next, we took slices of the NAc of Cre-positive mice exposed to coincident blue light and cocaine, and used ex vivo slice electrophysiology to validate that 561 nm light inhibited firing of EYFP-positive cells (Fig. 18d). Finally, we performed a locomotor sensitization assay to demonstrate the functional relevance of this population of DA-sensitive NA neurons in vivo. Following three days of habituation to the test apparatus, mice received an intraperitoneal (i.p.) injection of cocaine (20 mg/kg) paired with blue light, which induced a robust locomotor response (Fig. 18e, f). After seven days of withdrawal, mice were returned to the test apparatus and a challenge dose of cocaine was given. During this challenge, 561 nm light was applied for the duration of the behavioral measurement. Adaptations in D1- medium spiny neurons (MSNs) in the NAc have been implicated in the expression of locomotor sensitization to cocaine. We selectively expressed the /Tango2 constructs in either wild type, D1- or D2-Cre mice, in order to drive the expression of eNpHR in subpopulations that are responsive to cocaine-evoked DA release.

Optogenetic inhibition of the D1-MSN subpopulation during the cocaine challenge significantly suppressed the sensitized locomotor response to cocaine, whereas no effect was observed by manipulating DA-responsive D2-MSNs (Fig. 18f). When cocaine was administrated without light (Dark control) or asynchronized with light (Light control), there was no effect of 561 nm light on locomotor sensitization (Fig. 18g-j). These results are consistent with the selective role of DI-MSNs in locomotor sensitization to cocaine, and provide a proof-of-concept that the /Tango2 system can be used to manipulate and thus assess the behavioral relevance of a temporally and genetically-identified population of neurons.

Example 7: The Cal-Light system

First, it was tested whether the Cal-Light system expresses reporter genes in a calcium- and light- dependent manner in cultured neurons. Cal-Light constructs (Figure 20) and Channelrhodopsin 2 (ChR2) were co-transfected into hippocampal culture neurons. After five days of expression, a short pulse of blue light (5 second on/55 second off) was repetitively illuminated for 30 minutes.

Two days later, neurons kept in a dark condition did not show high levels of EGFP expression although tdTomato expression (transfection marker) was confirmed to be very high (Figure 20). This result indicates that Cal-Light proteins did not cause a target reporter gene expression in the absence of light stimulation. When Blue light was shone onto the neurons, but neuronal activity was completely blocked by TTX, reporter EGFP gene expression level was kept low as similar as in the dark condition (Figure 20).

Robust EGFP expression was only observed in a condition when both blue light was given and when neuronal activity was not blocked (Figure 20). These data demonstrate that the Cal- Light system reliably converts neuronal activity to gene expression in a light-dependent manner. To control neuronal activity independent of blue light, action potentials were then triggered by electrical stimulation. Cortical slice culture were made at postnatal day 3 and AAV expressing Cal-Light constructs including an EGFP reporter were infected to slices at DIV 3 (Days in vitro). After 12 days of expression, a bipolar stimulation electrode was placed in layer 2/3. Three repeats of brief high-frequency electric pulses were delivered (5 pulses at 20 Hz) at 4 second intervals, while blue light was continuously shining for 8.5 seconds (Figure 21 ). This stimulation protocol was repeated 30 times, resulting in triggering about 300 action potentials in total. Two days later, slices were fixed and gene expression pattern was imaged by confocal microscopy. Similar to the ChR2 experiments above, it was found that either blue light or high-frequency stimulation alone did not lead to high levels of EGFP expression, but when both light and stimulation occurred together, EGFP expressing neurons were robustly increased and their expression level was also increased (Figure 18). Further increase of neuronal activity by bicuculine application also resulted in significant increase of EGFP expression (Figure 21 ).

Because the Cal-Light technique reliably label active neurons upon light exposure, it is now possible to identify a population of neurons that is active during a specific behavior period in vivo. Based on light- and activity-dependent gene expression in slice experiments, the Cal- Light technique can further label active population of neurons that are involved in specific animal behavior. To monitor the level of neuronal activity during behavior in awake behaving mouse, AAV expressing Cal-Lights and GCaMP6s were injected into motor cortex in mouse (Figure 22A). After 2 week of viral expression, a head fixed mouse was placed on a floating styroform ball, such that the mouse can freely run on the ball (Figure 22B). Whenever mice started running, many neurons in motor cortex were firing, which were monitored by calcium imaging (Figure 22C). Next, blue light was illuminated through a glass cranial window for 5 seconds whenever the mouse ran. In this experiment, active neurons during running should be labeled when blue light is applied. The same population of neurons was imaged before and after blue light exposure to directly compare how much the gene expression is increased in the same neurons (Figure 22D). A significant increase in EGFP expression was observed two days after blue light exposure (Figure 22E). These data indicate that Cal-Light is also functioning in vivo.

We also trained mice in patterned lever pressing. This successive lever-pressing behavior is ideal to test Cal-Light because current techniques, such as methods that detect lEGs, have not been able to label neural circuits responsible for temporally locked motor behaviors. We bilaterally injected Cal-Light viruses into layer 2/3 of the primary motor cortex (M1 ; Figure 23a). During the first few days of continuous reinforcement (CRF), water-restricted mice quickly learned that lever-pressing actions were associated with water rewards (Figure 23b). Once mice learned the link between lever pressing and water rewards, we used fixed-ratio (FR) training to progressively increase the required number of lever presses to 12 (Figure 23b). During these FR sessions, blue light was synchronously illuminated with the first lever- pressing action, thus labeling the neuronal population responsible for successive lever- pressing behaviors (Figure 23c). All mice reliably learned lever-pressing behaviors, as indicated by an increase in the number of rewards and lever presses per minute (Figure 23d,e). The learning curve was similar whether or not we used blue light, demonstrating that blue light itself did not cause any behavioral changes (Figure 23d,e). To determine whether M1 layer 2/3 neurons are critical for lever-pressing learning, we injected muscimol or saline into the M1 area of well-trained mice; only the muscimol-injected group showed complete loss of lever-pressing behaviors (Figure 23f). This lever-pressing learning was long lasting, with the learned behavior still maintained about one month later and even after fiber optic implantation surgery (Figure 23f).

Three days after the last blue light exposure, we examined the expression levels of tdTomato and EGFP in the M1 area. The mouse group trained with no light (activity only) showed robust tdTomato but minimal EGFP expression, confirming that Cal-Light is light dependent (Figure 23g-i). We anesthetized mice to reduce neuronal activity and delivered the same duration of light to compare light-induced EGFP expression. Similar to our in vitro data, EGFP expression was very low, indicating that light alone cannot induce gene expression in vivo (Figure 23g-i). Only the mouse group that received blue light during normal training neurons expressing robust EGFP (Figure 23g-i). We counted putative excitatory pyramidal neurons and interneurons by their morphology; the majority were pyramidal neurons, but the brightest neurons at each slice generally displayed interneuron morphology. Thus, these results suggest that a subpopulation of pyramidal neurons and interneurons were active while executing learned lever-pressing behaviors.

Example 8: Behavioral control by using Cal-Light system

The Cal-Light system links gene expression with activity and light, enabling the causal effects of labeled circuits to be testable by optogenetic effectors. To test whether a specific labeled neuronal population is necessary for lever-pressing learning, we injected Cal-Light viruses with a halorhodopsin (eNpHR) reporter with the goal of silencing neuronal activity (Figure 24a). We first verified that 589 nm yellow light diminished AP firing from eNpHR-expressing neurons (Figure 24b). eNpHR reporter expression also showed dependency on neuronal activity and blue light (Figure 24c). We shined yellow light to suppress the activity of labeled neurons (Figure 24d) and found that pulses of 589 nm yellow light (2 sec ON/1 sec OFF) during the 45 min probe test (see methods) strongly inhibited the learned lever-pressing behaviors (Figure 24e,f), indicating that labeled neurons were learning related. One day later, lever-pressing behaviors were restored in the same mice that showed severe inhibition (Figure 24e,f). Thus, behavioral inhibition was not because of phototoxicity-induced tissue damage nor changes in long-term circuit connectivity. Using the same yellow light protocol on a group of mice whose active neurons were not labeled with blue light did not show any behavioral impairment (Figure 24e,f). This result indicates that yellow light itself does not affect mouse behaviors. Randomized intermittent yellow light (5 sec ON/various intervals) desynchronized with lever-pressing behaviors did not significantly reduce the learned behavior, indicating that inhibiting neuronal activity during lever-pressing is critical (Figure 24e,f). In another control mouse group, we shined blue light when mice were walking around in a chamber to label a random population of neurons; pulses of 589 nm light (2 sec ON/1 sec OFF) during the 45 min probe test did not impair lever-pressing behaviors (Figure 24e,f). As indication of learned behavior, lever-pressing frequency and inter-reward interval were also analyzed (Figure 24g,h). Altogether, our results show that Cal-Light selectively labels learning-related behaviors with high temporal resolution.