Field of Invention

The present invention relates to nucleic acid constructs for use in transcription assay and to transcription assays employing such constructs, which are designed to be used to facilitate the identification of compounds which modulate, such as inhibit, DNA transcription, by targeting a specific DNA sequence.

Background to the Invention

Transcription is the critical first step in the flow of genetic information. Central to this process is an RNA polymerase (RNAP) which catalyses the synthesis of an RNA molecule with a nucleotide sequence complementary to its corresponding DNA template. The initiation and elongation phases of transcription are highly regulated and involve an orchestrated series of protein-protein and protein-nucleic acid interactions in order to guide the RNAP to its promoter, to initiate the synthesis of a target RNA molecule. The large number of proteins involved in transcription initiation, promoter release and active elongation provide multiple targets for inhibitory small molecules in anti-cancer and anti-microbial therapies (1-3).

Although transcription is fundamental to all living organisms, there is a dearth of methods available to assay direct transcriptional inhibition in real-time (2). Whilst gel electrophoresis-based footprinting, chromatin immunoprecipitation (DNA ChIP) and deep-sequencing provide valuable information concerning the potency and selectivity of transcriptional inhibition, they are typically low-throughput and labour-intensive. A more direct method to detect small molecule transcriptional inhibitors would be one which correlates the synthesis of a specific RNA molecule with a fluorescent read-out.

A recent application of a direct fluorescence-based reporter of transcription is the Spinach TART assay (4). In this assay, a fluorogenic Spinach aptamer sequence installed downstream to an RNA sequence of interest is used to directly correlate target RNA synthesis with an increase in fluorescence emission of the small molecule DFHBI when bound to the Spinach aptamer. In addition, the Spinach-TART assay was used to evaluate the dose-dependence of T7 RNAP inhibition using heparin, wherein a loss of DFHBI fluorescence is directly proportional to the heparin concentration. Summary of the Invention

The present teaching is based on the development of a variant of the Spinach TART assay in order to identify compounds which modulate transcription through sequence selective DNA binding.

In a first aspect there is provided an assay construct for identifying a compound, which inhibits transcription through DNA binding, the construct comprising in sequence (5’-3’): an RNA promoter sequence, a compound DNA target sequence and a transcription reporter sequence.

A feature of the present invention is the ability to identify compounds which are capable of modulating, for example an inhibitory or activating effect, on DNA transcription by virtue of a compound binding to a specific DNA sequence, rather than effecting transcription by other means, such as binding to an RNA polymerase or non- specifically binding to DNA. Non-specific binding to DNA may include DNA inter chelating compounds.

In order to distinguish compounds which bind to DNA in a targeted or sequence selective manner, from compounds which may affect transcription by other means, it may additionally be necessary or appropriate to test any compounds using a different assay construct which is designed to help distinguish a compound’s mode of action. For example, in order to ascertain if a compound modulates transcription, such as by directly inhibiting RNA polymerase or non-specifically binding DNA, a similar construct, to that defined above may be provided, in which the compound DNA target sequence is replaced with a non-specific or random DNA sequence. In this manner upon conducting assays using both types of constructs, the user will be able to ascertain whether or not a particular compound is modulating transcription by specifically binding to the DNA target sequence, or if transcription is being modulated by another mode of action, such as by way of acting directly upon the RNA polymerase, or by non-specific DNA binding.

The target DNA sequence may be a wild-type or mutant sequence and this may allow a user to identify compounds which differentially or preferentially bind to a wild-type or mutant, for example a single nucleotide polymorphism (SNP), sequence. By employing constructs with varying DNA sequence selective binding sequences, it may be possible to identify compounds, which, for example, are capable of inhibiting the transcription of a mutant, but not a wild-type sequence. Such compounds may find utility in treating diseases where transcription, typically at a low level, of mutant sequences is known or expected to be undesirable. The present inventors have shown that minor alterations in the target DNA sequence, such by only one or two nucleotide alterations, can lead to detectable and optionally quantifiable changes in the degree/level of transcription. Thus, based on the findings by the inventors, it is appreciated that the constructs and assays of the present invention can be used in order to observe the effects minor DNA sequence changes can have in terms of a compound’s ability to modulate transcription. Thus, it is possible to observe an effect a compound has in terms of modulating transcription of wild-type and mutant target DNA sequences. This may be attractive, for example, in terms of identifying compounds, which are capable of activating or inhibiting transcription of wild-type or mutant sequences, or to identify compounds which are capable of selectively modulating transcription of only a wild-type or mutant sequence.

The evidence also supports the possibility that minor changes to a compound structure may lead to differences in a compounds ability to modulate transcription of a wild-type or mutant sequence. Thus, for example, if a compound is known to modulate transcriptional activity of a wild-type sequence, rational design and alteration of the compound structure can be carried out in order to identify compounds which are capable of modulating transcription of a mutant sequence in a similar or alternative manner. Thus, it is relatively easy for the skilled addressee to test structurally similar compounds and observe their effect on modulating transcription of a wild-type or mutant sequence.

Based on the above, it may be possible to identify compounds which may find application in treating diseases which are associated with mutant sequences and which lead to lower or elevated levels of transcription as compared to a wild-type sequence.

In certain embodiments, the compound may be obtained from a wide variety of sources including iibraries of synthetic or naturai compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of naturai compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. In addition, known pharmacological agents may be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs.

Suitable compounds may encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds. Small organic compounds generally have a molecular weight of more than 50 yet less than about 3,500, typically less than about 2000. Candidate agents typically comprise functional chemical groups necessary for structural interactions with DNA, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two or more of said functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the aforementioned functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof, and the like. One class of molecules which may be suitable for use in accordance with the present invention, are pyrrole-imidazole polyamides (i5).

Compounds shown to modulate transcription through sequence selective binding may provide valuable reagents or precursors to the pharmaceutical and agricultural industries for cellular, plant, field crop, animal and/or human applications. For animal and/or human applications, any suitable compounds which are identified to modulate transcription through sequence selective binding, may find use in treating disease.

The RNA promoter sequence may be a eukaryotic or prokaryotic promoter sequence.

The terms "eukaryotic promoter" or "prokaryotic promoter" as used herein refer to promoters recognized by the transcription machinery of a eukaryotic or a prokaryotic cell, respectively. Suitable prokaryotic promoter sequences may be recognizable by T7, T3, SPC or E.coli RIMA polymerases, for example. For eukaryotic RNA promoters, the sequence mat be recognizable by any of RNA polymerase I - V. However, in certain embodiments, the RNA promoter sequence is recognizable by R A polymerase li(6). in one embodiment an assay may initially be conducted using a construct employing a prokaryotic promoter in order to identify a limited number of compounds and such limited number of compounds thereafter provided to a construct employing a eukaryotic promoter, such as an RNAP P promoter, as it may be more complex and/or expensive to conduct all tests using a eukaryotic system.

The transcription reporter sequence typically encodes a nucleic add aptamer that can bind selectively to conditionally fluorescent molecules (“fluorophores”) to enhance the fluorescence signal of the fluorophore upon exposure to radiation of suitable wavelength. Molecular complexes formed between the aptamers and fluorophores and their signal generation is, also discussed herein.

Aptamers are nucleic add molecules characterized by a single-strand and having a secondary structure that may possess one or more stems (i.e., base-paired regions) as well as one or more non base-paired regions along the length of the stem. These non- base-paired regions can be in the form of a bulge or loop (e.g., internal loop) along the length of the siem(s) and/or a loop at the end of the one or more stem(s) (e.g , hairpin loop). These nucleic add aptamers possess specificity in binding to a particular target molecule, and they non-covaiently bind their target molecule through an interaction such as an ion-ion force, dipole-dipole force, hydrogen bond, van der Waals force, electrostatic interaction, stacking interaction or any combination of these interactions.

WO/2010/096584 to Jaffrey and Paige describes a number of RNA aptamers that bind to conditionally fluorescent molecules derived from the chromophore of green fluorescent protein. Other suitable sequences include Genetically encoded "Spinach" RNA Is an aptamer capable of binding to, and turning on, a cell-permeable, non-toxic ligand, to emit GFP-iike fluorescence (7). More recently aptamers such as "Broccoli" RNA (8) and "Mango" RNA (Dolgosheina E.V. et al., ACS Chem. Biol., 2014, Vol. 9(10), pages 2412- 2420) were successfully used in live-cell imaging of small molecules and metabolites and may also be used. In one embodiment iSpinach RNA aptamer (Autour et al) may be used.

The fluorophores recognized by the nucleic acid aptamers of the present invention include those that possess a methyne (also known as methine) bridge between a substituted aromatic ring system and a substituted imidazol(thi)one, oxazol(thi)one. pyrrolin(thi)one, or furan(thi)one ring. Importantly, the methyne bridge contains a single carbon that is double-bonded to a ring carbon of the substituted imidazol(thi)one, oxazoi{thl)one, pyrrolin(thi)one, or furan(thi)one ring. Thus, these conditionally fluorescent compounds are unlike cyanine dyes characterized by a poiymethyne bridge.

Exemplary fiuorophores identified in the above-referenced PCT Application Pubi. No. WO/2Q 10/096584 to Jaffrey and Paige include, without limitation, 4-(3,4.5~ trimethoxybenzylidene)-1 ,2-dimethy!-imidazol-5-one (“T BG); 4-{4-hydroxy-3,5~ dimethoxybenzyjidene)-1 ,2-dimethyj-imidazoj-5-one (“DMHBi”). In some embodiments, the signal-emitting ligand is hydroxy benzyiideneimidazolinone (HBI), a derivative of HBI, thiazole orange (TO), or a derivative of TO. In some embodiments, the signal- emitting ligand is 3,5-difluoro-4-bydroxy benzyiideneimidazolinone (DFHBi), DFHBI-1T, DFHBI-2T or TOI-Blotin.

The fiuorophores used in the present invention, are characterized by a low quantum yield at a desired wavelength in the absence of aptamer binding in certain embodiments, the quantum yield of the fluorophore, in the absence of specific aptamer binding, is iess than about 0.01 , more preferably less than about 0.001 , most preferably less than about 0.0001.

The fiuorophores are substantially unable to exhibit increases in quantum yield upon binding or interaction with molecules other than the aptamer(s) that bind specifically to them. This includes other molecules in a cell or sample besides those aptamer molecules having a polynucleotide sequence that was selected for binding to the fluorophore.

The fiuorophores are preferably water soluble, non-toxic, and ceil permeable. Preferably, the fluorophore is soluble in an aqueous solution at a concentration of 0.1 pM, 1 mM, more preferably 10 pM, and most preferably 50 m or higher. Preferably, incubating a cell with these concentrations of the fluorophore does not affect the viability of the cell. The fiuorophores are preferably capable of migrating through a cell membrane or cell wall into the cytoplasm or periplasm of a cell by either active or passive diffusion. Preferably, the fluorophore is able to migrate through both the outer and inner membranes of gram-negative bacteria, the cell wail and membrane of gram- positive bacteria, both the ceil wall and plasma membrane of plant cells, cell wall and membrane of fungi and molds (e.g yeast), the capsid of viruses, and/or the plasma membrane of an animal cell. As used herein, the terms“enhance the fluorescence signal" or“enhanced signal" (i.e., upon specific aptamer binding) refer to an increase in the quantum yield of the fluorophore when exposed to radiation of appropriate excitation wavelength, a shift in the emission maxima of the fluorescent signal (relative to the fluorophore emissions in ethanol glass or aqueous solution), an increase in the excitation coefficient, or two or more of these changes. The increase in quantum yield is preferably at least about 1.5- foid, more preferably at least about 5 to 10-fold, at least about 20 to 50-foid, more preferably at least about 100 to about 200-fold. Fold increases in quantum yield exceeding 500-fold and even 1000-fold have been achieved with the present invention.

The radiation used to excite the fluorophore may be derived from any suitable source, preferably any source that emits radiation within the visible spectrum or Infrared spectrum. The radiation may be directly from a source of radiation (e.g., a light source) or indirectly from another fluorophore (e.g., a FRET donor fluorophore). The use of FRET pairs is discussed more fully hereinafter.

Directly attaching the DNA target sequence to the sequence encoding the aptamer sequence may be less than desirable. Thus, in some embodiments a sequence encoding a ribozyme sequence, such as a hammerhead ribozyme sequence, is positioned between the DNA target sequence and the sequence encoding the aptamer sequence in this manner after transcription occurs, ribozyme cleavage of the DNA sequence selective binding sequence occurs, leaving the ribozyme sequence attached to the aptamer sequence. Suitable ribozyme sequences are disclosed in (11) and (12), to which the skilled reader is directed.

In a further aspect there is provided a method for identifying a compound, which inhibits transcription through targeted DNA binding, the method comprising: providing a compound to a transcription assay mixture comprising an assay construct in accordance with the present invention and described herein and detecting any signal generated by a reporter molecule binding to a transcribed reporter sequence.

The reporter molecule and reporter sequence may typically be the aptamer and fluorophore molecules as described above.

In addition to the assay constructs, reporter molecule and test compound as defined herein, the transcription assay generally includes one or more additional reagents, such as salts, buffers, etc. to facilitate optimal protein-nucleic add binding. A variety of other reagents may also he included in the mixture. These include reagents like detergents which may be used to reduce non-specific or background protein~substrate _: nucleic acid-substrate, protein-protein and protein-DNA interactions, etc. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The assay method may be performed at any temperature which facilitates optimal binding, typically between 4° and 40° C., more commonly between 15° and 4G ^C C. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening. Typically, the reagents are coincubated between 0.1 and 10 hours, preferably less than 5 hours, more preferably less than 2 hours each; of course, the incubations may and preferably do run simultaneously.

The assay methods of the present invention may be suited to automated high throughput compound screening. In a preferred embodiment, the individual sample incubation volumes are less than about 500 ui, preferably less than about 250 ul, more preferably less than about 100 ul. Such small sample volumes minimize the use of often scarce candidate compounds, and expensive transcription complex components.. Furthermore, the methods provide for automation, especially computerized automation. Accordingly, the method steps are preferably performed by a computer-controlled electromechanical robot. The computer is loaded with software which provides the instructions which direct robotic operations and provides input (e.g. keyboard and/or mouse) and display (e.g. monitor) means for operator interfacing.

The assays are conducted in viino, and may be celi based or ceil free assays if ceil based, the assay is carried out in a suitable ceil and the relevant RNA polymerase may be provided by the cell. In cell free systems, it will be necessary to provide a source of the relevant RNA polymerase.

Throughout ibis specification reference is made to a number of citations and/or patent applications, the entire contents of which are hereby incorporated by way of reference.

Detailed Description

The present invention will now be further described by way of example and with reference to the figures, which show: Figure 1 shows Representative plasmid maps of constructs used for STI assay a. DNA4. b. DNA5. Plasmids purchased from IDT Inc. and linearised via endonuclease cleavage prior to use in assay.

Figure 2 shows - Overview of the Spinach Transcription Inhibition (STI) assay. PT7 = T7 RNAP promoter; SOI = sequence of interest; HHR = hammerhead ribozyme sequence; Reporter = fluorogenic iSpinach aptamer (10);

Figure 3 shows Optimisation of STI assay a. Linearized DNA constructs b. Comparative fluorescence emission for three Spinach variants. Normalised to emission of DNA1 (negative control). Influence of 100 mM K+ on iSpinach fluorescence as a function of [Mg2+] d. Influence of 100 mM Na+ on iSpinach fluorescence as a function of [Mg2+] All results based on n=3 ± 1 s.d.;

Figure 4 shows Analysis of transcriptional inhibition using the STI assay a. Inhibitor structures b. Concentration-dependent decrease of iSpinach/DFHBMT fluorescence in presence of known transcriptional inhibitors. Data fit using a variable slope sigmoidal dose response curve (GraphPad Prism V.7) for n=3 ± 1 s.d. c. IC50 values for known inhibitors calculated from data in (b) for n=3 ± SEM. MOI: Mechanism of Inhibition;

Figure 5 shows Probing sequence-selective transcriptional inhibition of PIPs using the STI assay a. Structures of PA1 and PA2. b. SOI sequences used to explore PIP sequence selectivity c. Concentration-dependent decrease of iSpinach/DFHBMT fluorescence emission in the presence of PA1. Data fit using a variable slope sigmoidal dose response curve (GraphPad Prism V.7) for n=3 ± 1 s.d. IC50 values calculated from data for n=3 ± SEM. d. Effect of SOI position on transcription inhibition. % transcriptional activity normalised against basal inhibitory activity of 175 nM PA1 against DNA4. Results based on n=3 ± SEM. e. Concentration-dependent decrease of iSpinach/DFHBMT fluorescence emission in the presence of PA2. Data fit using a variable slope sigmoidal dose response curve (GraphPad Prism V.7) for n=3 ± 1 s.d. IC50 values calculated from data for n=3 ± SEM.

Procedures for Transcription Assays General

Reagents and solvents were purchased from commercial sources and were used without further purification. Chlorambucil was purchased from Fluorochem. DFHBI-1T was purchased from Lucerna, Inc. T7 RNA polymerase (50,000 units/mL), ribonucleotide solution mix (25 mM), and EcoRV-HF ^® (100,000 units/mL) were purchased from New England BioLabs. Ndel (10,000 units/mL) was purchased from Thermo-Fisher. All other reagents were purchased from Sigma-Aldrich. Incubation and shaking was performed on a Heidolph Instruments Titramax 1000 fitted with a Heidolph Incubator 1000. Fluorescence measurements were obtained in black 96 well ½-area microplates (Greiner Bio-One) using a Perkin Elmer Wallac Victor Multilabel plate reader fitted with 485/535 nm filters.

DNA Construct Preparation

Plasmids containing designed DNA sequences were purchased from Integrated DNA Technologies, Inc. (Fig. 1). Plasmids were isolated on a large scale from 250 ml_ XL1- Blue cultures via alkaline lysis using laboratory-prepared buffers. Ethanol-precipitated plasmid DNA was linearised with either Ndel (DNA1 -4) or EcoRV-HF ^® (DNA5-11). Linearised DNA was then purified by phenol:chloroform:isoamyl alcohol (25:24:1) extraction and ethanol precipitation and quantified by UV absorption at 260 nm.

DNA sequences of fluorescent aptamers explored for STI assay.

Optimised Assay

For a given experiment (n=1):

Transcription assay buffer was prepared in RNAse-free water (6.7 mM 1 ,4- Dithiothreitol, 53 mM HEPES, 133 mM KCI, 13.3 mM MgCI2). The solution was heated in a water bath to 40 °C and buffered to pH 7.5 using 1 M NaOH.

The final volume for an individual well in a black 96 well ½-area microplate was 40 mI_. Final concentrations of components/well are given in the table below.

A 0.67 mM NTP-supplemented buffer solution was prepared by dissolving 25 mM NTPs (ATP, CTP, GTP, UTP) in assay buffer. A 40 nM plasmid solution was prepared by dissolving the plasmid in NTP-supplemented buffer. 10 pL of the 40 nM plasmid solution was transferred to each well. The dilution series of the inhibitor was prepared in RNAse-free water and 10 pL of these dilutions were transferred to the appropriate wells in the 96-well plate. Finally, a solution of T7 RNAP (3.35 units/pL) and 2 mM DFHBI-1T in NTP-supplemented buffer was prepared and 20 L was transferred to each well. The mixtures were gently pipetted and the plate was covered with a plastic cover, wrapped in plastic wrap, and placed in a sealed bag covered with aluminium. The plate was then incubated at 40 °C with shaking at 350 rpm for 45 minutes. Upon completion, the plate was transferred to the plate reader without any covering and fluorescence was measured (Ex/Em: 485 nm/535 nm). Optimisation Aptamer Optimisation

Experiments were performed using 50 nM DNA1-4. 200 nM solutions of DNA1-4 were prepared in RNAse-free water and 10 pL were added to a 96-well plate in triplicate. NTP-supplemented buffer was prepared as described and 10 pL was added to the wells. The T7 RNAP/DFHBI-1T solution was prepared as described and 20 pL were added to the wells. The plate was then incubated and measured as described. This experiment was repeated three times.

Buffer Optimisation

Experiments were performed using 25 nM DNA4 and final salt concentrations of 100 mM KCI/NaCI and 10, 17.5, 25 mM MgCh. Transcription assay buffers were prepared as described above with either 133 mM KCI or NaCI and varying [MgCh] (13.3, 23.3, 33.3 mM). A 100 nM DNA4 solution was prepared in RNAse-free water and 10 pL were added to a 96-well plate in triplicate. NTP-supplemented buffers were prepared as described and 10 pL was added to the wells. The T7 RNAP/DFHBI-1T solution was prepared as described and 20 pL were added to the wells. The solutions were mixed by pipetting and fluorescence was measured initiating t=0 min. The plate was then covered as described and incubated at 40 °C with shaking. Measurements were then taken at 15, 30, 45, 60, 90, 120 minutes. Following each measurement, the plate was re-covered and returned to the incubator. This experiment was repeated three times.

Assay Conditions

Experiments were performed using final DNA4 concentrations of 10, 20, 30 40 and 50 nM. A dilution series of DNA4 was prepared in RNAse-free water to ensure that the final experimental concentrations were as described. 10 pL of each dilution was added to a 96-well plate in triplicate. NTP-supplemented buffer was prepared as described and 10 pL was added to the wells. The T7 RNAP/DFHBI-1T solution was prepared as described and 20 pL were added to the wells. The solutions were mixed by pipetting and fluorescence was measured initiating t=0 min. The plate was then covered as described and incubated at 40 °C with shaking. Measurements were then taken at 15, 30, 45, 60, 90, 120 minutes. Following each measurement, the plate was re-covered and returned to the incubator. This experiment was repeated three times.

General Inhibitors

a-Amanitin

A 1.1 mM stock solution of a-amanitin was prepared by dissolving a-amanitin in RNAse-free water. A dilution series of a-amanitin was then prepared in RNAse-free water to provide an experimental concentration range of 0.1-100 mM. 10 pl_ of each dilution was added to a 96-well plate in triplicate. A solution of T7 RNAP (6.7 units/pL) was prepared by dissolving T7 RNAP in assay buffer and 10 pL were added to the wells. The solutions were mixed by pipetting and the plate was covered and incubated at room temperature for 20 minutes. A solution of 40 nM DNA4 and 2 mM DFHBI-1T was prepared in 1 mM NTP-supplemented buffer and 20 mI_ were added to the wells. The plate was then incubated and measured as described. This experiment was repeated three times.

Actinomycin D

A 10 mM stock solution of actinomycin D was prepared by dissolving actinomycin D in DMSO. A dilution series of actinomycin D was then prepared in RNAse-free water to provide an experimental concentration range of 0.03-100 mM. 10 mI_ of each dilution was added to a 96-well plate in triplicate. A 40 nM DNA4 solution was prepared in NTP-supplemented buffer as described and 10 pL were added to each well. The T7 RNAP/DFHBI-1T solution was prepared as described and 20 mI_ were added to the wells. The plate was then incubated and measured as described. This experiment was repeated three times.

Chlorambucil

A 100 mM stock solution of chlorambucil was prepared by dissolving chlorambucil in DMSO. A dilution series of chlorambucil was then prepared in RNAse-free water to provide an experimental concentration range of 1-1000 mM. A 40 nM DNA4 preincubation mixture was prepared by dissolving stock DNA4 and 10 mI_ of the chlorambucil dilutions in RNAse-free water to give a final volume of 50 pL/dilution. The preincubation mixtures were then incubated at 37 °C with shaking at 350 rpm for 2.5 hours. Upon completion of preincubation, 10 pL of each dilution was added to a 96-well plate in triplicate. 10 pL NTP-supplemented buffer was added to the wells. The T7 RNAP/DFHBI-1T solution was then prepared as described and 20 mI_ was added to the wells. The plate was then incubated and measured as described. This experiment was repeated three times.

Heparin

A 4 mM stock solution of heparin was prepared by dissolving heparin sodium salt in RNAse-free water and filtering through a 0.2 pm filter. A dilution series of heparin was then prepared in RNAse-free water to provide an experimental concentration range of 0.001-100 pM. 10 pL of each dilution was added to a 96-well plate in triplicate. A 40 nM DNA4 solution was prepared in NTP-supplemented buffer as described and 10 pL were added to each well. The T7 RNAP/DFHBI-1T solution was prepared as described and 20 pL were added to the wells. The plate was then incubated and measured as described. This experiment was repeated four times.

Temozolomide

A 100 mM stock solution of temozolomide was prepared by dissolving temozolomide in DMSO. A dilution series of temozolomide was then prepared in RNAse-free water to provide an experimental concentration range of 2-40,000 pM. A 40 nM DNA4 preincubation mixture was prepared by dissolving stock DNA4 and 10 pL of the temozolomide dilutions in RNAse-free water to give a final volume of 50 pL/dilution. The preincubation mixtures were then incubated at 37 °C with shaking at 350 rpm for 2 hours. Upon completion of preincubation, 10 pL of each dilution was added to a 96-well plate in triplicate. 10 pL NTP-supplemented buffer was added to the wells. The T7 RNAP/DFHBI-1T solution was then prepared as described and 20 pL was added to the wells. The plate was then incubated and measured as described. This experiment was repeated three times.

Gel Electrophoresis

Dilution series of chlorambucil and temozolomide were prepared in RNAse-free water to provide experimental concentration ranges of 1-1000 pM (chlorambucil) and 2- 40,000 pM (temozolomide). A 40 nM DNA4 mixture was prepared by dissolving stock DNA4 and 10 pL of the inhibitor dilutions in RNAse-free water to give a final volume of 50 pL/dilution. The preincubation mixtures were then incubated at 37 °C with shaking at 350 rpm for 2.5 h (chlorambucil) and 2 h (temozolomide). Upon completion, 10 pL samples (530 ng DNA4) were run on 1.2% agarose gels in 1X TAE buffer at 110 V for 1 h. 1 kb and 100 bp ladders (Hyperladder™, Bioline) were used as references. Gels were stained with SYBR gold nucleic acid stain (Thermo-Fisher) for 30 minutes, destained 3x5 min in 1X TAE buffer and imaged on a FujiFilm FLA-5100 at 473 nm. Polyamides

PIP Stock Preparation and Storage

Polyamide stock solutions (1 mM) were prepared from ditrifluoroacetate salts in RNAse-free water. Solutions were kept for four days and discarded to prevent aggregate formations from affecting results. Stocks were stored at -20 °C when not in use over the experimental period. During the experiment, stocks were warmed to 40 °C and periodically sonicated to minimise aggregate concentrations.

Dilution Series

A dilution series of polyamide was prepared in RNAse-free water to provide experimental concentration ranges of 10-10,000 nM (PA1) and 10-1000 nM (PA2). 10 pl_ of each dilution was added to a 96-well plate. 40 nM solutions of DNA4-9 were prepared in NTP-supplemented buffer as described and 10 pL were added to each well. The T7 RNAP/DFHBI-1T solution was prepared as described and 20 pl_ were added to the wells. The plate was then incubated and measured as described. This experiment was repeated three times/polyamide/DNA construct.

Effect of SOI Position in DNA construct

40 nM dilutions of DNA4,5, 10-11 were prepared in NTP-supplemented buffer as described and 10 pL was added to a 96-well plate in triplicate. A 175 nM solution of PA1 was prepared in RNAse-free water and 10 pL was added to the wells. The T7 RNAP/DFHBI-1T solution was prepared as described and 20 mI_ were added to the wells. The plate was then incubated and measured as described. This experiment was repeated three times/DNA construct.

Data Analysis

Graphing and analysis was performed using Microsoft Excel 2016 and GraphPad Prism V.7 (La Jolla, CA).

For % inhibition, inhibition was calculated as % fluorescence compared to untreated sample. Inhibitor concentrations were converted to log[inhibitor] and data from 3-4 independent tests were fit using variable slope sigmoidal dose response curves. Standard deviations were calculated and plot within GraphPad. IC50 data for each independent curve (calculated in GraphPad) were averaged and used to calculate the reported SEM.

Pyrrole-lmidazole Polyamides

General

All reagents and solvents were obtained from commercial sources and used without further purification. NMR spectroscopy was carried out using a Bruker AV 500 MHz spectrometer. All chemical shifts (d) were referenced to the deuterium lock and are reported in parts per million (ppm) and coupling constants are quoted in hertz (Hz). Abbreviations for splitting patterns are s (singlet), d (doublet), t (triplet) and m (multiplet). NMR data was processed using MestReNova. HRMS spectra were measured on a Bruker microTOFq High Resolution Mass Spectrometer.

Polyamides were purified by semi-preparative HPLC on a 150x21.2 mm Kinetex 5 pm C18 column using a Dionex Ultimate 3000 series HPLC equipped with a VWD-3400 variable wavelength detector. Purifications were performed using aqueous 0.1 % trifluoroacetic acid as Solvent A and acetonitrile/0.1% trifluoroacetic acid as Solvent B and were run at a flow rate of 9 mL/min using the following method: absorbance detector set at 310 nm. 10% B for 5 minutes, 10-60% B over 20 minutes, 60-90% B over 3 minutes, 90% B for 2 minutes, 90-10% B over 1 minute, 10% B for 10 minutes. Polyamide purity was analyzed via HPLC on a 250x4.6 mm Aeris 3.6um Widepore XB- C18 column using a Shimadzu Prominence HPLC equipped with a SPD-M20A photodiode array detector. Analysis was performed using aqueous 0.1 % trifluoroacetic acid as Solvent A and acetonitrile/0.1 % trifluoroacetic acid as Solvent B and were run at a flow rate of 0.5 mL/min using the following method: absorbance detector set at 310 nm. 15% B for 5 minutes, 15-60% B over 20 minutes, 60% B for 5 minutes, 60-90% B over 1 minute, 90% B for 5 minutes, 90-15% B over 1 minute, 15% B for 5 minutes. Synthesis and Characterisation

Pyrrole-imidazole (Py-lm) polyamides were prepared by manual solid phase synthesis as previously described· see (5)

PA1

Yield: 26%

RESULTS

Design of the Spinach Transcriptional Inhibition (STI) Assay

The DNA construct used in our STI assay consists of four modules: (i) a T7 promoter, (ii) a DNA sequence of interest (SOI) to assess sequence-selectivity of DNA-binding, (Hi) a hammerhead ribozyme sequence (HHR) to cleave the fluorescence reporter and thus ensure that the fluorescence output can be benchmarked across constructs, and (iv) a Spinach reporter which is used to correlate RNA synthesis with a fluorescent output when bound to the small molecule fluorophore DFHBI-1T (Fig. 2).

Optimization of fluorescence emission properties of the Spinach reporter was determined by surveying the type of reporter construct and metal ion concentration. Transcription of 50 mM DNA1 -4 (Fig. 3a) at 40 °C revealed the iSpinach aptamer (DNA4) provided a 2.6-fold and 6.6-fold greater fluorescence emission than Broccoli (DNA3) and Spinach2 (DNA2), respectively (Fig. 3b). Exploration of the fluorescence emission properties of DNA4 as a function of K7Na ⁺ relative to [Mg ²⁺ ] (Fig. 3c-d) identified 10 mM MgCh and 100 mM KCI to be optimal in the presence of 10 nM [DNA4] and 1 pM [DFHBI-1T] at 45 minutes. A time point of 45 min was employed for this work as it is a point where fluorescence increase remains linear, producing DFH BI- IT fluorescence emission ( _em 535 nm) that is both robust and reproducible (Z' factor 0.91 ± 0.014). Application of STI Assay to explore small molecule transcriptional inhibitors

By way of background, the broader utility of our STI assay was then explored using RNAP inhibitors heparin and a-amanitin, the DNA intercalator actinomycin D, the DNA cross-linker chlorambucil, and the clinically-approved methylating agent temozolomide (TMZ). Whilst heparin and actinomycin D required no pre-incubation with their respective targets, a-amanitin was pre-incubated with T7 RNAP for 20 min and chlorambucil and TMZ were pre-incubated with DNA4 for 2.5 and 2 hours, respectively (Fig. 4b). A concentration curve for each inhibitor (n = 3) and an inhibition constant (IC5 ₀ ) value were determined as a function of inhibitor concentration inducing a 50% decrease in fluorescence emission at a 45 min time-point (Fig. 4d). The most potent inhibitor in this suite of compounds was the RNAP inhibitor heparin (IC5 ₀ 100 ± 5 nM), whereas a-amanitin did not induce any transcriptional inhibition, which is in agreement with its specificity for RNAPII over T7 RNAP.

Whilst the DNA intercalator actinomycin D inhibited transcription in the low micromolar range (IC5 ₀ 3.1 ± 0.1 mM) (47), the DNA cross-linker chlorambucil was an order of magnitude less potent (IC5 ₀ 40 ± 2 mM). Gel electrophoretic analysis of chlorambucil incubated with DNA4 revealed significant changes in the electrophoretic mobility of the construct at a chlorambucil concentration close to our calculated IC5 ₀ value, which suggests covalent modification of dsDNA is the likely mechanism of action for transcriptional inhibition. Finally, TMZ was the weakest transcriptional inhibitor out of the series (IC5 ₀ 820 ± 30 mM), consistent with its predominant mechanism of cytotoxicity being the inhibition of the DNA-damage repair machinery rather than transcription itself.

Transcriptional inhibition of polyamides correlates with their DNA binding profile

The potential to use our STI assay as a tool to assess the sequence-selectivity of inhibiting transcriptional elongation using PIPs, for example, was then explored (Fig. 5a). PIPs are programmable minor groove binders which bind to target DNA sequences 7-24 base-pairs in length with nanomolar binding affinity and have demonstrated efficacy as gene selective transcriptional modulators ((13-18)). Two PIPs (PA1-2) were investigated: PA1 binds to the core 5'-WWGWWCW (where W = A/T) sequence represented in the Androgen Response Element (5'-AGAACANNNTGTTCA where N = any nucleotide; Fig. 5b), which is a well-characterised PIP target sequence, whereas PA2 targets the more G _* C-rich target sequence 5'-WWGGCWW (Fig. 5a) (19- 23)). An IC ₅₀ value of 490 ± 20 nM was determined for PA1 with the control construct DNA4, which lacked an SOI module. This provided a basal level of transcriptional inhibition across the series. The potency of transcriptional inhibition was then determined using DNA5 which contained its matched binding sequence (5'-AAGAACA) compared to DNA constructs containing mismatched sequences (DNA6-9) incorporated within the SOI module (Fig. 5b). Consistent with the highest binding affinity of PA1 for its general target sequence, the most potent transcriptional inhibition (IC ₅₀ 189 ± 12 nM, Fig. 5c) was observed for DNA5 relative to the mismatched sequences DNA6-9.

In order to probe the potency of transcription as a function of the position of the SOI, DNA constructs (DNA10-11) were prepared and compared with DNA5 (Fig. 5d). In the presence of PA1 , strongest inhibition of fluorescence was observed when the SOI was upstream of the HHR and iSpinach modules (DNA5, Fig. 5d). Significantly lower levels of transcriptional inhibition were observed when the SOI is placed in downstream positions (DNA10-11 , Fig. 5d).

Finally, the potency and sequence-selectivity of PA2 was investigated using our STI assay (Fig. 5e). The DNA8 construct contains the target binding sequence (5'- AAGGCAA), with the remaining constructs (DNA5-7.9) containing sequence mismatches in their SOI module (Fig. 5b). Using DNA4, the baseline IC50 value for PA2 was 596 ± 25 nM (Fig. 5e). PA2 exhibited potent transcriptional inhibition (IC50 103 ± 4 nM) using DNA8, which contained its target sequence relative to mismatched constructs DNA5-7.9 (Fig. 5e). Taken collectively, our results show that the STI assay is a robust platform to correlate transcriptional inhibition and the sequence selectivity of DNA-binding PIPs.

DISCUSSION

Our STI assay was designed as a real-time tool to assess the potency of inhibitors of T7 RNAP-mediated transcription. We contextualize several key outcomes of the work below.

STI assay identifies different types of transcriptional inhibitory mechanisms

The STI assay identified the strongest transcriptional inhibitors as heparin (IC50 100 ± 5 nM) and actinomycin D (IC50 3.1 ± 0.1 mM). These inhibitors have distinct mechanisms of action. Heparin inhibits transcriptional initiation of T7 RNAP by binding to the palm and finger subdomains, whereas actinomycin D indirectly inhibits transcriptional elongation by DNA intercalation i.e., indirect T7 RNAP inhibition by disrupting formation of the ternary transcriptional complex. Our method is also able to respond to differences in mechanistic specificity: a-amanitin is known to inhibit RNAPII but not T7 RNAP, which is reflected by the absence of an effect in our assay. The DNA-damaging agents chlorambucil and TMZ exhibit far weaker levels of transcriptional inhibition. This observation is aligned with the primary mechanisms of cytotoxic action of both chlorambucil and TMZ being the inhibition of DNA replication and the DNA-damage repair pathway machinery respectively, which occur at much lower concentrations in cells than reported in our in vitro assay.

Sequence selective inhibition of transcriptional elongation by DNA binding polyamides generally correlates with the binding affinity for a target dsDNA sequence

The results of our STI assay show that the most potent transcriptional inhibition, which presumably is due to the inhibition of transcriptional elongation, observed in this series is when the SOI module contained the target binding sequence. This is aligned with the established PIP pairing rules as reasonable predictors of sequence selective dsDNA binding. Binding of PA1 with DNA4 suggests that PIPs can bind to DNA in a non- selective complex and have a functional impact on the DNA-processing machinery at approximately 0.5 mM in vitro. Previous studies have shown that this non-selective effect on transcription is not necessarily recapitulated in cells, nevertheless, it is worth noting the potential for off-target consequences highlighted by this technique. Insertion of a target PIP DNA sequence in the SOI results In an improved effect as shown by PA1 inhibiting transcription D A5 which contains its target ARE more effectively than DIMA4. Significantly, our assay was able to show discrimination in functional output when more subtle changes in sequence are introduced through single mismatches in D A7-9. Again, these results illustrate that sequence-selective dsDNA binding by PIPs produces refined differences in the magnitude of the functional effect, as shown by the concentration-response curves in Figure 5c.

A non-linear relationship between PIP binding to the target sequence and its effect on fluorescence emission is suggested by the results of PA1 with D A5 and DNA10-11. Here, the same target sequence is embedded in the DNA, but the SOI is in different positions in the plasmid. The most effective inhibition of transcription occurs when the SOI is between the T7 RNAP promotor sequence and the Spinach reporter, suggesting that change in the level of fluorescence emission is highly sensitive to a PA binding site juxtaposed to the T7 promoter sequence.

The complexities of this link between binding and mechanistic impact is further demonstrated by the unexpected observation concerning the more potent transcriptional inhibitory activity of PA2 when presented with its target sequence (DNAS, ICso 103 ± 4 nM) relative to PA1 with DNA5 (IC50 189 ± 12 nM) This was unexpected as the reported binding affinity (K _s ) of PA2 for its target 5 -AAGGCAT sequence (4 x 10 ⁸ M ¹ ) (59,60) is ~1Q0-foid less effective than PA1 for its target 5 - TAGTACT ( a 3 x 10 ¹⁰ M ¹ ) (24)). We initially hypothesised that PIP binding affinity for its target dsDNA sequence would be the predominant factor for transcriptional inhibition. Our results suggest that other factors, such as the directionality of PA binding or the nature of distortion of the DNA duplex imparted by the PIP could play an influential role In the potency of inhibiting transcriptionai elongation when T7 RNAP is used. Thus, while the binding affinity of a PIP for its target dsDNA sequence provides a qualitative guide, the STi method could be used to more accurately quantify a direct consequence of sequence-selective dsDNA binding with one of its primary mechanisms of gene modulation (i.e., inhibition of transcriptional elongation).

CONCLUSION

Herein, we have shown that the STI assay can be used to identify sequence-selective inhibitors of transcription. The assay is facile (45 min incubation), robust (n=3) and is performed in a plate reader format. This assay therefore has the potential to be automated into a high-throughput assay and could be used more generally as an initial screening platform to identify inhibitors of T7 RNAP or possibly RNAPII-mediated transcription. Our results also show that the effect of PIP binding to DNA sequences on transcriptional elongation is quantifiable, which could act as a complementary tool to establish structure-activity profiles for this family of gene regulatory compounds.

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