DYNAMICS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/989,566 filed March 13, 2020 and U.S. Provisional Application No. 63/073,212 filed September 1, 2020. The entirety of each of these applications is hereby incorporated by reference for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 20117PCT_ST25.txt. The text file is 100 KB, was created on March 11, 2021, and is being submitted electronically via EFS-Web.

BACKGOUND

Shear stress alters certain biological signaling pathways including those involved with growth, coagulation, inflammation, and extracellular matrix deposition. For example, when shear stress is applied to cultured endothelial cells, they will re-arrange their cytoskeleton to align with the direction of flow in a matter of hours. Such changes have implications for human health, as the shear stresses induced from disturbed flow conditions may lead to life threatening conditions such as atherosclerosis and coronary microvasculature disease. Thus, there is a need to develop improved techniques for evaluating liquid dynamic in biological contexts.

Oshinowo et al. report in vitro imaging of platelets under flow. Platelets, 2020, 31(5): 570- 579. Liu et al. report molecular tension probes for imaging forces at the cell surface. Acc Chem Res, 2017, 50(12): 2915-2924. See also WO 2013/049444. Ma et al. report DNA probes that store mechanical information reveal transient piconewton forces applied by T cells. Proc Natl Acad Sci USA, 2019, 116(34): 16949-16954. See also U.S. Patent Application No. 16/913,187.

References cited herein are not an admission of prior art. SUMMARY

It is an object of this disclosure to provide systems, devices, and methods for the direct use of fluorescent reporters that measure multiaxial and dynamic shear flows that occur in vitro or in vivo across a surface of interest, where shear flows can be measured, quantified and/or correlated to physiological changes in cells or tissues in real time. In certain embodiments, this disclosure contemplates imaging or visualizing the shear field applied to a surface, e.g., a surface of cells or inner lining of a blood vessel, the lumen of pumping lymphatics, within the bile duct, vessels with significant leakage, inflamed endothelium, tumor vasculature, or other systems.

In certain embodiments, this disclosure relates to a molecular arm comprising an anchor on one end, a force indicator (optical force transducer), a tether, and a shear flow resistor (mechanical amplifier) on the other end, wherein the shear flow resistor causes the force indicator to expand providing an optical signal if exposed to a liquid that flows past the molecular arm in a stationary position at or above a critical velocity. In certain embodiment, the shear flow resistor causes the force indicator to expand providing an optical signal if the liquid flows past the arm in a stationary position or through the channel at or above a velocity of 0.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 dynes/cm ² . In certain embodiments, the molecular arm or any segment thereof, e.g., shear flow resistor further contains a label, e.g., fluorescent label.

In certain embodiments, this disclosure relates to a molecular arm comprising a specific binding agent at one end, a nucleic acid force indicator comprising multiple hairpin domains, a nucleic acid tether, and a shear flow resistor on the other end wherein the shear flow resistor causes the hairpin domains in the force indicator to expand providing an optical signal if exposed to a liquid that flows past the arm in a stationary position at or above a critical velocity.

In certain embodiments, this disclosure relates to an optical shear flow system comprising: a) a channel comprising a surface; b) a molecular arm comprising an anchor, a force indicator, a tether, and a shear flow resistor; and c) a liquid in the channel; wherein the anchor is attached to the surface; and wherein the shear flow resistor causes the force indicator to expand providing an optical signal if the liquid flows through the channel at or above a critical velocity.

In certain embodiments, it is contemplated that the channel has a cross-sectional area of less than 100, 50, 10, or 5 cm ² . In certain embodiments, it is contemplated that the surface is glass, metal, polymer, protein, cell, group of cells, or combinations thereof. In certain embodiments, it is contemplated that the channel is a vascular channel, blood vessel, artery, capillary, inside a tissue or organ.

In certain embodiments, it is contemplated that the anchor is an antibody, agent, specific binding agent, ligand or receptor and the surface comprises an antigen, specific binding agent, agent, receptor or a ligand, respectively. In certain embodiments, it is contemplated that the anchor is an antibody such as an antibody or binding fragment thereof to CD31, VCAM, CD43, or a4b1 or other specific binding agent, ligand, or receptor to CD31, VCAM, CD43, or a4b1.

In certain embodiments, it is contemplated that the tether and/or the shear flow resistor comprises nucleic acid sequences or amino acid sequences. In certain embodiments, it is contemplated that the tether and/or the shear flow resistor comprises nucleic acid sequences with a G and C content of greater than 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 50% of the total nucleotide bases. In certain embodiments, it is contemplated that the tether and/or the shear flow resistor comprises nucleic acid sequences with a G and C content of between 20-25%, 20-30%, 20-35%, 10-25%, 15-25%, 15-30%, 15-35%, of the total nucleotide bases.

In certain embodiments, it is contemplated that the force indicator and tether comprise nucleic acid sequences, and the force indicator spontaneously forms multiple hairpin domains.

In certain embodiments, it is contemplated that the hairpin domains or nearby segments contain a quencher and fluorophore in sufficiently close proximity to prevent an optical signal and the optical signal is a result of the hairpin domains dehybridizing separating the quencher from the fluorophore.

In certain embodiments, it is contemplated that the optical signal is a result of hairpin domains dehybridizing forming single stranded segments and the optical signal is a result of fluorescent probes in the liquid hybridizing the single stranded segments.

In certain embodiments, it is contemplated that the shear flow resistor is a bead attached through the tether. In certain embodiments, it is contemplated that the shear flow resistor comprises branched nucleic acids attached through the tether.

In certain embodiments, it is contemplated that the branched nucleic acids have 2, 3, 4, 5, 10, 25, 50, 100, or 150 or more primary branch points providing primary nucleic acid branches from a linear or circular nucleic acid. In certain embodiments, it is contemplated that the primary nucleic acid branches have secondary branch points providing second nucleic acid branches. In certain embodiments, it is contemplated that the secondary nucleic acid branches have tertiary branch points providing tertiary nucleic acid branches. In certain embodiments, it is contemplated that the tertiary nucleic acid branches have quaternary branch points providing quaternary nucleic acid branches.

In certain embodiments, this disclosure relates to methods of imaging, detecting, measuring, or quantifying shear flow in a channel comprising providing an optical shear flow system disclosed herein and imaging the channel or detecting, measuring, or quantifying an optical signal in the channel.

In certain embodiments, it is contemplated that imaging includes imaging the optical signal produced when the liquid flows through the channel at or above a critical velocity causing the force indicator to expand. In certain embodiments, it is contemplated that an image is recorded on computer readable medium.

In certain embodiments, this disclosure relates to in vivo methods of diagnosing shear flow of a bodily fluid such as shear flow associated with blood flow in a subject comprising administering into the circulatory system, e.g., intravenously, a molecular arm disclosed herein to a subject, wherein the molecular arm anchors to a surface or wall of a vascular channel, e.g., blood vessel, artery, capillary, or heart, wherein the shear flow resistor causes the force indicator to expand providing an optical signal if the bodily fluid flows past the surface or through the channel at or above a critical velocity, and imaging, detecting, measuring, or quantifying the optical signal, and wherein the optical signal indicates that the subject has bodily fluid flow above a calibrated value associated with the molecular arm, e.g., high blood flow at a certain location, or wherein a lack of an optical signal indicates the subject does not have a bodily fluid flow above a calibrated value associated with the molecular arm.

In certain embodiments, it is contemplated that an image, measurement, or diagnosis is recorded on computer readable medium. In certain embodiments, it is contemplated that an image, measurement, or diagnosis is communicated or transmitted to a medical professional.

In certain embodiments, this disclosure relates to any nucleic acid sequence disclosed herein (e.g., SEQ ID NO: 1-406) optionally conjugated to a label, fluorescent dye, or quencher. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figure 1 A DNA based optical reporter of shear stress. One creates a reporter that directly measures shear stress useful for shear based in vivo sensors and therapeutics and testing hypotheses related to molecular biophysics. Each bionanomechanical reporter is similar to a kite and contains: 1) an antibody-based anchor targeted to a ligand of interest, 2) a DNA or protein- based optical force transducer that fluoresces when unfolded at a threshold force, and 3) a mechanical amplifier (kite) that increases the total force on the transducer. Data indicates that a bead (kite) generates tensile force as a function of fluidic drag force and bead size. As the tensile force increases, an optical force reporter, which consists of a series of DNA hairpins with fluor- quencher pairs, unfolds and fluoresces. By creating large numbers of multiplexed reporters with varied shear force sensitivities and anchors attaching to ligands of interest, one can visualize complex, multiaxial, in vivo force fields. For example, in vitro physiologically relevant shear stresses were measured from 0.5 dynes/cm ² to 25 dynes/cm ² by changing the bead size.

Figure IB illustrates an antibody-based anchor to a cell surface and a dendrimer based mechanical amplifier.

Figure 1C illustrates the fluorescent signal (black circle) using DNA hairpins designed to unfold at a critical tension leading to fluorescence. As the bead experiences drag, which is proportional to the fluid velocity, the DNA strand tethering the bead to the wall experiences tension. This tension is reported optically by multiple added hairpins that each have fluorophore- quencher pairs which fluoresce when unfolded. In flow conditions, fluorescence occurs whenever the applied fluidic shear force exceeds a critical value which can be modified by changing the nanoreporter design. Changing the number and type of base pairs enables us to create DNA hairpins that unfold at a specified mechanical force. When combined with beads of different sizes and fluorophores of different emission spectra, one can create a multiplexed system that measures the localized shear stress.

Figure ID illustrates force probes with ten hairpins in series with a double stranded tether modified from a ml 3 bacteriophage genome (circular single-stranded DNA, 8064 bases in length) linearized with restriction enzyme BsaAI.

Figure IE illustrates alternative DNA force transducer designs e.g. contains a continuous long DNA strands as a backbone. Figure 2 shows calculations indicating a sigmoid curve of fluorescence intensity in response to increasing applied shear.

Figure 3A illustrates a DNA amplifier containing a megadalton dendrimer composed of five unique DNA strands. Each subsequent layer of the dendrimer has three times as many components as the last which can be modified to be fluorescent with Alexa 647. A single dendrimer did not generate enough force to open the hairpins. Multiple dendrimers were used to create a DNA drogue. Dendrimers (192) were added onto the tether by incorporating a dendrimer capturing extension on every short oligo used to make the linearized p8064 ml 3 double stranded.

Figure 3B illustrates a DNA drogue shear nanoreporter construct described in Figure 3A that produces a signal at shear rates of around 100 dynes/cm ² .

Figure 3C illustrates multivalently attachment of several DNA drogues onto a single hairpin chain.

Figure 4 illustrates using fluorescent probes that hybridize with de-hybridized segments as a result of force extension on hairpins which can be used alone or in combination with fluorescent dyes and quenchers.

Figure 5 illustrates that a high concentrations of double stranded tether DNA results in a supervalent bead with heavily restricted movement. Lower valency allows for beads with increased mobility which float above the glass and are not easily visible with reflection interference contrast microscopy (RICM). With applied shear, supervalent beads have restricted movement of only about 1 micron displacement: compare that to 2.5 microns of movement for of low valency beads.

Figure 6 illustrates the conjugation of DNA based fluorescent reporters using anti-CD41 antibodies for specific platelet targeting and shear flow mediated activation of nanoreporters on platelet surfaces.

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

"Consisting essentially of or "consists of or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

The term "specific binding agent" refers to a molecule, such as a proteinaceous molecule, that binds a target molecule with a greater affinity than other random molecules or proteins. Examples of specific binding agents include antibodies that bind an epitope of an antigen or a receptor which binds a ligand. "Specifically binds" refers to the ability of a specific binding agent (such as an ligand, receptor, enzyme, antibody or binding region/fragment thereof) to recognize and bind a target molecule or polypeptide, such that its affinity (as determined by, e.g., affinity ELISA or other assays) is at least 10 times as great, but optionally 50 times as great, 100, 250 or 500 times as great, or even at least 1000 times as great as the affinity of the same for any other or other random molecule or polypeptide.

In certain contexts, an “antibody” refers to a protein based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e. recognized by the animal to be a foreign molecule and an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen, and thereby targeting the antigen for elimination or degradation. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, as used herein the term “antibody” is intended to include natural antibodies, monoclonal antibody, or non-naturally produced synthetic antibodies, and binding fragments, such as single chain binding fragments. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

From a structural standpoint, an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. The heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains have the same amino acid sequence. Each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigen-binding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity determining regions, or CDRs."

As used herein, the term “ligand” refers to an organic molecule, i.e., substantially comprised of carbon, hydrogen, and oxygen, that binds a “receptor.” Receptors are organic molecules typically found on the surface of a cell. Through binding a ligand to a receptor, the cell has a signal of the extra cellular environment which may cause changes inside the cell. As a convention, a ligand is usually used to refer to the smaller of the binding partners from a size standpoint, and a receptor is usually used to refer to a molecule that spatially surrounds the ligand or portion thereof. However as used herein, the terms can be used interchangeably as they generally refer to molecules that are specific binding partners. For example, a glycan may be expressed on a cell surface glycoprotein and a lectin may bind the glycan. As the glycan is typically smaller and surrounded by the lectin during binding, it may be considered a ligand even though it is a receptor of the lectin binding signal on the cell surface. In another example, a double stranded oligonucleotide sequence contains two complimentary nucleic acid sequences. Either of the single stranded sequences may be consider the ligand or receptor of the other. In certain embodiments, a ligand is contemplated to be a small molecule. In certain embodiments, a receptor is contemplated to be a compound that has a molecular weight of greater than 2,000 or 5,000. In any of the embodiments disclosed herein the position of a ligand and a receptor may be switched.

As used herein, the term “small molecule” refers to any variety of covalently bound molecules with a molecular weight of less than 900 or 1000. Typically, the majority of atoms include carbon, hydrogen, oxygen, nitrogen, and to a lesser extent sulfur and/or a halogen. Examples include steroids, short peptides, mono or polycyclic aromatic or non-aromatic, heterocyclic compounds.

As used herein, the term “surface” refers to the outside part of an object. Examples of contemplated surfaces are on a particle, bead, wafer, array, well, microscope slide, transparent or opaque glass, polymer, or metal, or in vitro or in vivo cell, or group of cells.

A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a peptide "label " refers to incorporation of a peptide, wherein the sequence can be identified by a specific binding agent, antibody, or bind to a metal such as nickel/ nitrilotriacetic acid, e.g., a poly-histidine sequence. Specific binding agents and metals can be conjugated to solid surfaces to facilitate isolation and purification methods. A label contemplates the covalent attachment of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling nucleic acids, polypeptides and glycoproteins are known in the art and may be used. Examples of labels include, but are not limited to, the following: radioisotopes or radionucleotides (such as ³⁵ S or ^m I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels may be attached by spacer arms of various lengths to reduce potential steric hindrance.

As used herein, the term "nucleic acid" is meant to include ribonucleic or deoxyribonucleic acid, nucleobase polymers, or mixtures thereof. A nucleic acid can include native or non-native bases. In this regard, a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. It will be understood that a deoxyribonucleic acid used in the methods or compositions set forth herein can include uracil bases and a ribonucleic acid can include a thymine base.

With regard to the nucleobases, it is contemplated that the term encompasses isobases, otherwise known as modified bases, e.g., are isoelectronic or have other substitutes configured to mimic naturally occurring hydrogen bonding base-pairs, e.g., within any of the sequences herein U may be substituted for T, or T may be substituted for U. Examples of nucleotides with modified adenosine or guanosine include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine. Examples of nucleotides with modified cytidine, thymidine, or uridine include 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine. Contemplated isobases include 2'-deoxy-5- methylisocytidine (iC) and 2'-deoxy-isoguanosine (iG) (see U.S. Pat. No. 6,001,983; No. 6,037,120; No. 6,617,106; and No. 6,977,161).

The term "nucleobase polymer" refers to nucleic acids and chemically modified forms with nucleobase monomers. In certain embodiments, methods and compositions disclosed herein may be implemented with a nucleobase polymers comprising units of a ribose, 2’deoxyribose, locked nucleic acids (l-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol), 2'-0-methyl groups, a 3'- 3 '-inverted thymidine, phosphorothioate linkages, or combinations thereof. In certain embodiments, the nucleobase polymer may be less than 100, 50, or 35 nucleotides or nucleobases. Nucleobase polymers may be chemically modified, e.g., within the sugar backbone or on the 5’ or 3’ ends. As such, in certain embodiments, nucleobase polymers disclosed herein may contain monomers of phosphodiester, phosphorothioate, methylphosphonate, phosphorodiamidate, piperazine phosphorodiamidate, ribose, 2'-0-methylribose, 2'-0-methoxyethyl ribose, 2'- fluororibose, deoxyribose, l-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol, P-(2- (hydroxymethyl)morpholino)-N,N-dimethylphosphonamidate, morpholin-2-ylmethanol, (2- (hydroxymethyl)morpholino) (piperazin-l-yl)phosphinate, or peptide nucleic acids or combinations thereof. In certain embodiments, the nucleobase polymer can be modified to contain a phosphodiester bond, methylphosphonate bond or phosphorothioate bond. The nucleobase polymers can be modified, for example, 2'-amino, 2'-fluoro, 2'-0-methyl, 2'-H of the ribose ring. Constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography and re-suspended in water. In certain embodiments, nucleobase polymers include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA "locked nucleic acid" nucleotides such as a 2',4'-C methylene bicyclo nucleotide (see for example U.S. Patent No. 6,639,059, U.S. Patent No. 6,670,461, U.S. Patent No. 7,053,207). In one embodiment, the disclosure features modified nucleobase polymers, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.

As used herein, the term “conjugated” refers to linking molecular entities through covalent bonds, or by other specific binding interactions, such as due to hydrogen bonding or other van der Walls forces. The force to break a covalent bond is high, e.g., about 1500 pN for a carbon to carbon bond. The force to break a combination of strong protein interactions is typically a magnitude less, e.g., biotin to streptavidin is about 150 pN. Thus, a skilled artisan would understand that conjugation must be strong enough to restrict the breaking of bonds in order to implement the intended results. In certain embodiments, the term conjugated is intended to include linking molecular entities that do not break unless exposed to a force of about greater than about 5, 10, 25, 50, 75, 100, 125, or 150 pN depending on the context.

As used herein, "subject" refers to any animal, preferably a human patient, livestock, or domestic pet.

Unless stated otherwise as apparent from the following discussion, it will be appreciated that terms such as “detecting,” “receiving,” “quantifying,” “mapping,” “generating,” “registering,” “determining,” “obtaining,” “processing,” “computing,” “deriving,” “estimating,” “calculating,” “inferring” or the like may refer to the actions and processes of a computer system, or similar electronic computing device, that manipulates and transforms data represented as physical (e.g., electronic) quantities within the computer system's registers and memories into other data similarly represented as physical quantities within the computer system memories or registers or other such information storage, transmission or display devices. Embodiments of the methods described herein may be implemented using computer software. If written in a programming language conforming to a recognized standard, sequences of instructions designed to implement the methods may be compiled for execution on a variety of hardware platforms and for interface to a variety of operating systems. In addition, embodiments are not described with reference to any particular programming language. It will be appreciated that a variety of programming languages may be used to implement embodiments of the disclosure.

In some embodiments, the disclosed methods may be implemented using software applications that are stored in a memory and executed by a processor (e.g., CPU) provided on the system. In some embodiments, the disclosed methods may be implanted using software applications that are stored in memories and executed by CPUs distributed across the system. As such, the modules of the system may be a general purpose computer system that becomes a specific purpose computer system when executing the routine of the disclosure. The modules of the system may also include an operating system and micro instruction code. The various processes and functions described herein may either be part of the micro instruction code or part of the application program or routine (or combination thereof) that is executed via the operating system.

It is to be understood that the embodiments of the disclosure may be implemented in various forms of hardware, software, firmware, special purpose processes, or a combination thereof. In one embodiment, the disclosure may be implemented in software as an application program tangible embodied on a computer readable program storage device. The application program may be uploaded to, and executed by, a machine comprising any suitable architecture. The system and/or method of the disclosure may be implemented in the form of a software application running on a computer system, for example, a mainframe, personal computer (PC), handheld computer, server, etc. The software application may be stored on a recording media locally accessible by the computer system and accessible via a hard wired or wireless connection to a network, for example, a local area network, or the Internet.

It is to be further understood that, because some of the constituent system components and method steps depicted in the accompanying figures may be implemented in software, the actual connections between the systems components (or the process steps) may differ depending upon the manner in which the disclosure is programmed. Given the teachings of the disclosure provided herein, one of ordinary skill in the related art will be able to contemplate these and similar implementations or configurations of the disclosure. Fluidic Shear Sensor

A tool capable of measuring changes in the spatial fluid velocity directly at a vessel wall is useful for scientific research. This disclosure relates to bionanomechanical reporters that are similar to a kite and contains: 1) an antibody-based anchor targeted to a ligand of interest, 2) a optical force transducer (e.g., DNA or protein-based) that fluoresces when unfolded at a threshold force, and 3) a mechanical amplifier (kite) that increases the total force on the transducer (Fig. 1 A). A bead (kite) generates tensile force as a function of fluidic drag force and bead size. As the tensile force increases, the optical force reporter, which consists of a series of DNA hairpins with fluorophore-quencher pairs, unfolds and fluoresces.

DNA nanomechanics can be used to: 1) spatially constrain an object to be within a specified distance of a surface and 2) measure the forces coupled into a DNA strand assembly. DNA nanostructures were created that constrain the movement of a bead near a wall (Fig. IB). The position of a bead in relation to the wall depends on lift forces generated near the wall surface as well as drag forces. As the bead experiences drag, which is proportional to the fluid velocity, the DNA strand tethering the bead to the wall experiences tension. This tension is reported optically by multiple (e.g., ten) added hairpins that each have fluorophore-quencher pairs, which fluoresce when unfolded (Fig. 1C). In flow conditions, the system will fluoresce whenever the applied fluidic shear force exceeds a critical value. Changing the number and type of base pairs enables us to create DNA hairpins that unfold at a specified mechanical force.

A DNA bead-based structure are created to report the wall shear stress applied by a fluid to an interface. This structure may be: 1) designed to measure a range of shear stresses; 2) used en masse; and 3) have consistent static and dynamic performance. The fluorescence signal generated from a single fluorophore quencher is typically undetectable using confocal microscopy. About 10-12 fluorophores contained within the point spread function (about 250 nm) are sufficient to create a signal. A nanoreporter featuring 10 serially connected hairpins was created.

The sequence of the hairpin relates to the force required for the hairpin to open. GC base pairing utilizes 3 hydrogen bonds compared to the 2 or AT base pairs. Thus, less GC pairs require less force to separate; however, 0% GC hairpins could open spontaneously at room temperature. Preliminary calculations suggested that a hairpin sequence of 22% GC would be a good place to start. A functional unit was designed that could be repeated as desired to increase the number of force-sensing hairpins in series (Figure ID). This single functional unit needed five domains, two (one at either end) for connecting to neighboring units, and three in the middle for assembling the hairpin and its fluorophore and quencher components. In order to reduce unit-to-unit variability and promote full signal of the serial hairpin assembly in the smallest flow range possible, the three domains were conserved for hairpin-fluorophore-quencher assembly across all hairpin units. Contrarily, the two sticky end domains flanking the hairpin component are unique for all hairpin units. At one end of the entire hairpin chain, DNA strand is modified with digoxigenin allowing for selective attachment of the strand to a surface bound protein that specifically binds digoxigenin. At the other end of the chain, a strand connects the hairpin assembly to a double stranded DNA tether.

The double stranded tether (dsTether) was generated by modifying ml3 bacteriophage genome (circular single-stranded DNA, 8064 bases in length). Exposure to a restriction enzyme BsaAI resulted in linear form. Bacteriophage genome p8064 has many restriction sites for the BsaAI enzyme. Thus, to enhance single location cleavage a single short oligo was hybridized to the ml 3 that made one restriction site double stranded allowing for controlled cleavage at the desired site. The BsaAI enzyme was inactivated by heating.

Linearized sequence (SEQ ID NO: 406)

CACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAA

GCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTA

GCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCC CG

TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT C

GACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAG

ACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTC C

AAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTT T

GCCGATTTCGGAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGT

GGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGC

CCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCT

CCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAA

AGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCC

AGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAAC AATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGG

GGATCCTCAACTGTGAGGAGGCTCACGGACGCGAAGAACAGGCACGCGTGCTGGCA

GAAACCCCCGGTATGACCGTGAAAACGGCCCGCCGCATTCTGGCCGCAGCACCACA

GAGTGCACAGGCGCGCAGTGACACTGCGCTGGATCGTCTGATGCAGGGGGCACCGG

CACCGCTGGCTGCAGGTAACCCGGCATCTGATGCCGTTAACGATTTGCTGAACACAC

CAGTGTAAGGGATGTTTATGACGAGCAAAGAAACCTTTACCCATTACCAGCCGCAG

GGCAACAGTGACCCGGCTCATACCGCAACCGCGCCCGGCGGATTGAGTGCGAAAGC

GCCTGCAATGACCCCGCTGATGCTGGACACCTCCAGCCGTAAGCTGGTTGCGTGGGA

TGGCACCACCGACGGTGCTGCCGTTGGCATTCTTGCGGTTGCTGCTGACCAGACCAG

CACCACGCTGACGTTCTACAAGTCCGGCACGTTCCGTTATGAGGATGTGCTCTGGCC

GGAGGCTGCCAGCGACGAGACGAAAAAACGGACCGCGTTTGCCGGAACGGCAATC

AGCATCGTTTAACTTTACCCTTCATCACTAAAGGCCGCCTGTGCGGCTTTTTTTACG G

GATTTTTTTATGTCGATGTACACAACCGCCCAACTGCTGGCGGCAAATGAGCAGAAA

TTTAAGTTTGATCCGCTGTTTCTGCGTCTCTTTTTCCGTGAGAGCTATCCCTTCACC AC

GGAGAAAGTCTATCTCTCACAAATTCCGGGACTGGTAAACATGGCGCTGTACGTTTC

GCCGATTGTTTCCGGTGAGGTTATCCGTTCCCGTGGCGGCTCCACCTCTGAAAGCTT

GGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT

TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCG

CACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTG

GTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGG

CCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCT

ACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATC

CGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCC

AGACGCGAATTATTTTTGATGGCGTTCCTATTGGTTAAAAAATGAGCTGATTTAACA

AAAATTTAATGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATATTTGCTTA T

ACAATCTTCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGAC A

TGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCA A

TGACCTGATAGCCTTTGTAGATCTCTCAAAAATAGCTACCCTCTCCGGCATTAATTT A

TCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCT C

ACCCTTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGG G

TTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACA GGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTT

AATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTAATGCTACTACT AT

TAGTAGAATTGATGCCACCTTTTCAGCTCGCGCCCCAAATGAAAATATAGCTAAACA

GGTTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTTCGCA

GAATTGGGAATCAACTGTTATATGGAATGAAACTTCCAGACACCGTACTTTAGTTGC

ATATTTAAAACATGTTGAGCTACAGCATTATATTCAGCAATTAAGCTCTAAGCCATC

CGCAAAAATGACCTCTTATCAAAAGGAGCAATTAAAGGTACTCTCTAATCCTGACCT

GTTGGAGTTTGCTTCCGGTCTGGTTCGCTTTGAAGCTCGAATTAAAACGCGATATTT G

AAGTCTTTCGGGCTTCCTCTTAATCTTTTTGATGCAATCCGCTTTGCTTCTGACTAT A

ATAGTCAGGGTAAAGACCTGATTTTTGATTTATGGTCATTCTCGTTTTCTGAACTGT T

T A A AGC ATTT GAGGGGG ATT C A AT GA AT ATTT AT GAC GATTCC GC AGT ATT GGAC GC

TATCCAGTCTAAACATTTTACTATTACCCCCTCTGGCAAAACTTCTTTTGCAAAAGC C

TCTCGCTATTTTGGTTTTTATCGTCGTCTGGTAAACGAGGGTTATGATAGTGTTGCT C

TTACTATGCCTCGTAATTCCTTTTGGCGTTATGTATCTGCATTAGTTGAATGTGGTA T

TCCTAAATCTCAACTGATGAATCTTTCTACCTGTAATAATGTTGTTCCGTTAGTTCG T

TTTATTAACGTAGATTTTTCTTCCCAACGTCCTGACTGGTATAATGAGCCAGTTCTT A

AAATCGCATAAGGTAATTCACAATGATTAAAGTTGAAATTAAACCATCTCAAGCCCA

ATTTACTACTCGTTCTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATGAGCA G

CTTTGTTACGTTGATTTGGGTAATGAATATCCGGTTCTTGTCAAGATTACTCTTGAT G

AAGGTCAGCCAGCCTATGCGCCTGGTCTGTACACCGTTCATCTGTCCTCTTTCAAAG T

TGGTCAGTTCGGTTCCCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACA T

GGAGCAGGTCGCGGATTTCGACACAATTTATCAGGCGATGATACAAATCTCCGTTGT

ACTTT GTTTCGCGCTT GGT AT AATCGCTGGGGGT C AAAGAT GAGT GTTTT AGT GT ATT

CTTTTGCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCC GT

TTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTG

CTACCCTCGTTCCGATGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCAAAAGCGG

CCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGG

TTGTTGTCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGA

AAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTGGAG

ATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTTAGTTGTTCCTTTCTATTCT CA

CTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAATCCCATACAGAAAATTCATTTAC TAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCT

GTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGTTACGGTAC

ATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGG

CGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGA

TACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGG T

ACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAAT

ACTTTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTAT

ACGGGCACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCT

GTATCATCAAAAGCCATGTATGACGCTTACTGGAACGGTAAATTCAGAGACTGCGCT

TTCCATTCTGGCTTTAATGAGGATTTATTTGTTTGTGAATATCAAGGCCAATCGTCT G

ACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCG

GCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGA

GGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAAC

GCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGC

TAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCAT T

GGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCT A

ATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCC

GTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCG C

TGGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGT C

TTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAAC AT

ACTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTG C

GTTTCCTCGGTTTCCTTCTGGTAACTTTGTTCGGCTATCTGCTTACTTTTCTTAAAA AG

GGCTTCGGTAAGATAGCTATTGCTATTTCATTGTTTCTTGCTCTTATTATTGGGCTT A

ACTCAATTCTTGTGGGTTATCTCTCTGATATTAGCGCTCAATTACCCTCTGACTTTG T

TCAGGGTGTTCAGTTAATTCTCCCGTCTAATGCGCTTCCCTGTTTTTATGTTATTCT CT

CTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAACAAAAAATCGTTTCTTATTTGG A

TT GGGAT AAAT AAT ATGGCTGTTT ATTTT GT AACTGGC AAATT AGGCTCTGGAAAGA

CGC T C GTT AGC GTT GGT A AGATT C AGGAT A A A ATTGT AGC T GGGT GCA A A AT AGC A

ACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGCTAAAACG

CCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATATCTGATTTGCTTGCTATTGGG C

GCGGTAATGATTCCTACGATGAAAATAAAAACGGCTTGCTTGTTCTCGATGAGTGCG GTACTTGGTTTAATACCCGTTCTTGGAATGATAAGGAAAGACAGCCGATTATTGATT

GGTTTCTACATGCTCGTAAATTAGGATGGGATATTATTTTTCTTGTTCAGGACTTAT C

TATTGTTGATAAACAGGCGCGTTCTGCATTAGCTGAACATGTTGTTTATTGTCGTCG T

CTGGACAGAATTACTTTACCTTTTGTCGGTACTTTATATTCTCTTATTACTGGCTCG A

AAATGCCTCTGCCTAAATTACATGTTGGCGTTGTTAAATATGGCGATTCTCAATTAA

GCCCTACTGTTGAGCGTTGGCTTTATACTGGTAAGAATTTGTATAACGCATATGATA

CTAAACAGGCTTTTTCTAGTAATTATGATTCCGGTGTTTATTCTTATTTAACGCCTT A

TTTATCACACGGTCGGTATTTCAAACCATTAAATTTAGGTCAGAAGATGAAATTAAC

TAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGCGATTGGATTTGCATC A

GCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTTAAAAAGGTAGTCTCT

CAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAATCTAAGC T

ATCGCT AT GTTTT C AAGGATTCT AAGGGAAA ATT AATT AAT AGCGACGATTT AC AGA

AGCAAGGTTATTCACTCACATATATTGATTTATGTACTGTTTCCATTAAAAAAGGTA

ATTCAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCA TC

TTCTTTTGCTCAGGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTTGTAACTTG G

TATTCAAAGCAATCAGGCGAATCCGTTATTGTTTCTCCCGATGTAAAAGGTACTGTT

ACTGTATATTCATCTGACGTTAAACCTGAAAATCTACGCAATTTCTTTATTTCTGTT T

TACGTGCAAATAATTTTGATATGGTAGGTTCTAACCCTTCCATTATTCAGAAGTATA

ATCCAAACAATCAGGATTATATTGATGAATTGCCATCATCTGATAATCAGGAATATG

ATGATAATTCCGCTCCTTCTGGTGGTTTCTTTGTTCCGCAAAATGATAATGTTACTC A

AACTTTTAAAATTAATAACGTTCGGGCAAAGGATTTAATACGAGTTGTCGAATTGTT

TGTAAAGTCTAATACTTCTAAATCCTCAAATGTATTATCTATTGACGGCTCTAATCT A

TTAGTTGTTAGTGCTCCTAAAGATATTTTAGATAACCTTCCTCAATTCCTTTCAACT G

TTGATTTGCCAACTGACCAGATATTGATTGAGGGTTTGATATTTGAGGTTCAGCAAG

GTGATGCTTTAGATTTTTCATTTGCTGCTGGCTCTCAGCGTGGCACTGTTGCAGGCG G

TGTTAATACTGACCGCCTCACCTCTGTTTTATCTTCTGCTGGTGGTTCGTTCGGTAT TT

TTAATGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATTCAA

AAATATTGTCTGTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATCTCTG T

TGGCCAGAATGTCCCTTTTATTACTGGTCGTGTGACTGGTGAATCTGCCAATGTAAA

TAATCCATTTCAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCC T

GTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTG AGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCTACAACG

GTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAAC

ACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTG T

TTAGCTCCCGCTCTGATTCTAACGAGGAAAG

A batch of 192 short DNA oligos of equal length were added to make the long, linearized tether double stranded except for a short region on the end opposite the hairpin chain. This single stranded region hybridizes with a connecting strand that captures a biotinylated DNA strand, which binds streptavidin-coated mechanical amplifiers. To assemble the sensors, the hairpin sequences, sequences with a fluorophore, sequences with a quencher, sequences with digoxigenin strand, connecting sandwich strands, cut ml 3, 192 short ml3 complimentary oligos, and biotin strand were mixed together with salted TE buffer. This mixture self-assembles during an overnight annealing protocol in a thermocycler. The samples are electrophoresed in a 1% agarose gel and purified by band excision.

Before adding the mechanical transducer component, the ability of the sensor to fluoresce and quench (by adding complementary DNA strands) were verified with total internal reflection fluorescence (TIRF) microscopy. An oligo complimentary to the entire hairpin was added to one of the samples, which effectively forces all hairpins in each sensor to adopt the “open” configuration. Samples purified from the gel with this added opening strand exhibited fluorescence while samples without the opening strand could thus adopt the “closed” configuration and fluorescence signal was quenched. It is also worth noting that the linearized double strand tether with added open or closed hairpin chains exhibits gel mobility indistinguishable from the linearized dsTether with no hairpins added. This is because the hairpin chain adds only a few hundred base pairs to the 8064 bp long tether. Additionally, the excess hairpins can be visualized in the gel. However, as the samples are purified based on the mobility of the tether band, visible fluorescence in the open hairpin sample indicates that the hairpins are attaching to the dsTether. Hairpin attachment to the glass depends on the designed digoxigenin/anti-digoxigenin chemistry as very few points of fluorescence were visible in the open sensor sample incubated on glass without prior anti-digoxigenin treatment. In such a scenario, any present fluorescence was deemed to be nonspecific attachment to the glass. Hairpin(sensor) Strands tctactaaaactctatcacaCCGGAGCGCCTCCGTGTATAAATGTTTTCATTTATACgcg tcaatgtacac gtcttggcaggcatca, SEQ ID NO: 1 tgacgccaagttcgaCCGGAGCGCCTCCGTGTATAAATGTTTTCATTTATACgcgtcaat gtacacgtga tgcctgccaaga, SEQ ID NO: 2 tcgaacttggcgtcaCCGGAGCGCCTCCGT GT AT AAATGTTTT C ATTT AT ACgcgtcaatgtacacgcag cgttattcgcga, SEQ ID NO: 3 caatttcgaggaccgCCGGAGCGCCTCCGTGTATAAATGTTTTCATTTATACgcgtcaat gtacacgtcg cgaataacgctg, SEQ ID NO: 4 cggtcctcgaaattgCCGGAGCGCCTCCGT GT AT AAATGTTTT C ATTT AT ACgcgtcaatgtacacgggc tctcagcttaag, SEQ ID NO: 5 gtcgtcaccagagatCCGGAGCGCCTCCGTGT AT A AAT GTTTT C ATTT AT ACgcgtcaatgtacacgctta agctgagagcc, SEQ ID NO: 6 atctctggtgacgacCCGGAGCGCCTCCGT GT AT AAATGTTTT C ATTT AT ACgcgtcaatgtacacggcc aagtcgtcattg, SEQ ID NO: 7 aagctacctgcgatgCCGGAGCGCCTCCGTGTATAAATGTTTTCATTTATACgcgtcaat gtacacgcaa tgacgacttggc, SEQ ID NO: 8 catcgcaggtagcttCCGGAGCGCCTCCGT GT AT AAATGTTTT C ATTT AT ACgcgtcaatgtacacggac gcacgctttgta, SEQ ID NO: 9 tcctccatcccttccCCGGAGCGCCTCCGTGTATAAATGTTTTCATTTATACgcgtcaat gtacacgtaca aagcgtgcgtc, SEQ ID NO: 10

Sensor to tether connector tgtgatagagttttagtagaCTTTCCTCGTTAGAATCAGAG, SEQ ID NO: 11

Tether to biotin connector

GGGCGCGTACTATGGTTGCTTttaggagtgtgggaa, SEQ ID NO: 12 biotin connector

/5biosg/ttcccacactcctaa, SEQ ID NO: 13 Sensor to digoxigenin ggaagggatggaggatt/3Dig_N/, SEQ ID NO: 14

Fluorophore

/5Alex488N/ACGGAGGCGCTCCGG, SEQ ID NO: 15

Quencher cgtgtacattgacgc/3BHQ_l/, SEQ ID NO: 16

Tether complimentary

CGGGAGCTAAACAGGAGGCCGATTAAAGGGATTTTAGACAGG, SEQ ID NO: 17 AACGGTACGCCAGAATCCTGAGAAGTGTTTTTATAATCAGTG, SEQ ID NO: 18 AGGCCACCGAGTAAAAGAGTCTGTCCATCACGCAAATTAACC, SEQ ID NO: 19 GTTGTAGCAATACTTCTTTGATTAGTAATAACATCACTTGCC, SEQ ID NO: 20 TGAGTAGAAGAACTCAAACTATCGGCCTTGCTGGTAATATCC, SEQ ID NO: 21 AGAACAATATTACCGCCAGCCATTGCAACAGGAAAAACGCTC, SEQ ID NO: 22 ATGGAAATACCTACATTTTGACGCTCAATCGTCTGAAATGGA, SEQ ID NO: 23 TTATTTACATTGGCAGATTCACCAGTCACACGACCAGTAATA, SEQ ID NO: 24 AAAGGGACATTCTGGCCAACAGAGATAGAACCCTTCTGACCT, SEQ ID NO: 25 GAAAGCGT AAGAAT ACGT GGC AC AGAC AAT ATTTTT GAAT GG, SEQ ID NO: 26 CTATTAGTCTTTAATGCGCGAACTGATAGCCCTAAAACATCG, SEQ ID NO: 27 CCATTAAAAATACCGAACGAACCACCAGCAGAAGATAAAACA, SEQ ID NO: 28 GAGGT GAGGC GGT C AGT ATT A AC AC CGC CTGC A AC AGT GC C A, SEQ ID NO: 29 CGCTGAGAGCCAGCAGCAAATGAAAAATCTAAAGCATCACCT, SEQ ID NO: 30 TGCTGAACCTCAAATATCAAACCCTCAATCAATATCTGGTCA, SEQ ID NO: 31 GTTGGCAAATCAACAGTTGAAAGGAATTGAGGAAGGTTATCT, SEQ ID NO: 32 AAAAT ATCTTT AGGAGC ACT A AC AACT AAT AGATT AGAGCCG, SEQ ID NO: 33 TCAATAGATAATACATTTGAGGATTTAGAAGTATTAGACTTT, SEQ ID NO: 34 ACAAACAATTCGACAACTCGTATTAAATCCTTTGCCCGAACG, SEQ ID NO: 35 TTATTAATTTTAAAAGTTTGAGTAACATTATCATTTTGCGGA, SEQ ID NO: 36 ACAAAGAAACCACCAGAAGGAGCGGAATTATCATCATATTCC, SEQ ID NO: 37 TGATTATCAGATGATGGCAATTCATCAATATAATCCTGATTG, SEQ ID NO: 38 TTTGGATTATACTTCTGAATAATGGAAGGGTTAGAACCTACC, SEQ ID NO: 39 AT ATC AAAATT ATTTGC ACGT AAAAC AGAAAT AAAGAAATT G, SEQ ID NO: 40 CGTAGATTTTCAGGTTTAACGTCAGATGAATATACAGTAACA, SEQ ID NO: 41 GTACCTTTTACATCGGGAGAAACAATAACGGATTCGCCTGAT, SEQ ID NO: 42 T GCTTTGAAT ACC AAGTT AC AAAATCGCGC AGAGGCGAATT A, SEQ ID NO: 43 TTCATTTCAATTACCTGAGCAAAAGAAGATGATGAAACAAAC, SEQ ID NO: 44 ATCAAGAAAACAAAATTAATTACATTTAACAATTTCATTTGA, SEQ ID NO: 45 ATTACCTTTTTTAATGGAAACAGTACATAAATCAATATATGT, SEQ ID NO: 46 GAGTGAATAACCTTGCTTCTGTAAATCGTCGCTATTAATTAA, SEQ ID NO: 47 TTTTCCCTTAGAATCCTTGAAAACATAGCGATAGCTTAGATT, SEQ ID NO: 48 AAGACGCTGAGAAGAGT C AAT AGT GAATTT ATC AAAAT CAT A, SEQ ID NO: 49 GGTCTGAGAGACTACCTTTTTAACCTCCGGCTTAGGTTGGGT, SEQ ID NO: 50 T AT AT AACT AT ATGT AAAT GCTGAT GC AAATCC A ATCGC AAG, SEQ ID NO: 51 ACAAAGAACGCGAGAAAACTTTTTCAAATATATTTTAGTTAA, SEQ ID NO: 52 TTTCATCTTCTGACCTAAATTTAATGGTTTGAAATACCGACC, SEQ ID NO: 53 GT GT GAT AAAT A AGGC GT T AAAT A AG A AT A A AC AC C GG A AT C , SEQ ID NO: 54 ATAATTACTAGAAAAAGCCTGTTTAGTATCATATGCGTTATA, SEQ ID NO: 55 CAAATTCTTACCAGTATAAAGCCAACGCTCAACAGTAGGGCT, SEQ ID NO: 56 T AATT GAGAATCGCC AT ATTT AAC AACGCC AAC AT GT AATTT, SEQ ID NO: 57 AGGC AGAGGCATTTTCGAGCC AGT AAT AAGAGAAT AT AAAGT, SEQ ID NO: 58 ACCGACAAAAGGTAAAGTAATTCTGTCCAGACGACGACAATA, SEQ ID NO: 59 AACAACATGTTCAGCTAATGCAGAACGCGCCTGTTTATCAAC, SEQ ID NO: 60 AATAGATAAGTCCTGAACAAGAAAAATAATATCCCATCCTAA, SEQ ID NO: 61 TTTACGAGCATGTAGAAACCAATCAATAATCGGCTGTCTTTC, SEQ ID NO: 62 CTTATCATTCCAAGAACGGGTATTAAACCAAGTACCGCACTC, SEQ ID NO: 63 ATCGAGAACAAGCAAGCCGTTTTTATTTTCATCGTAGGAATC, SEQ ID NO: 64 ATTACCGCGCCCAATAGCAAGCAAATCAGATATAGAAGGCTT, SEQ ID NO: 65 ATCCGGTATTCTAAGAACGCGAGGCGTTTTAGCGAACCTCCC, SEQ ID NO: 66 GACTT GCGGGAGGTTTT GAAGCCTT AAAT C AAGATT AGTT GC, SEQ ID NO: 67 TATTTTGCACCCAGCTACAATTTTATCCTGAATCTTACCAAC, SEQ ID NO: 68 GCTAACGAGCGTCTTTCCAGAGCCTAATTTGCCAGTTACAAA, SEQ ID NO: 69 AT AAAC AGCC AT ATT ATTT ATCCC AATCC A AAT AAGAAACGA, SEQ ID NO: 70 TTTTTTGTTTAACGTCAAAAATGAAAATAGCAGCCTTTACAG, SEQ ID NO: 71 AG AG A AT A AC AT A A A A AC AGGG A AGC GC AT T AG AC GGG AG A A, SEQ ID NO: 72 TTAACTGAACACCCTGAACAAAGTCAGAGGGTAATTGAGCGC, SEQ ID NO: 73 TAATATCAGAGAGATAACCCACAAGAATTGAGTTAAGCCCAA, SEQ ID NO: 74 T AAT AAGAGC AAG AAAC AAT GAAAT AGC AAT AGCT ATCTT AC, SEQ ID NO: 75 CGAAGCCCTTTTTAAGAAAAGTAAGCAGATAGCCGAACAAAG, SEQ ID NO: 76 TTACCAGAAGGAAACCGAGGAAACGCAATAATAACGGAATAC, SEQ ID NO: 77 CCAAAAGAACTGGCATGATTAAGACTCCTTATTACGCAGTAT, SEQ ID NO: 78 GTT AGC AAAC GT AGA A A AT AC AT AC AT A A AGGT GGC A AC AT A, SEQ ID NO: 79 TAAAAGAAACGCAAAGACACCACGGAATAAGTTTATTTTGTC, SEQ ID NO: 80 ACAATCAATAGAAAATTCATATGGTTTACCAGCGCCAAAGAC, SEQ ID NO: 81 AAAAGGGCGAC ATT C AACCGATTGAGGGAGGGAAGGT AAAT A, SEQ ID NO: 82 TTGACGGAAATTATTCATTAAAGGTGAATTATCACCGTCACC, SEQ ID NO: 83 GACTTGAGCCATTTGGGAATTAGAGCCAGCAAAATCACCAGT, SEQ ID NO: 84 AGCACCATTACCATTAGCAAGGCCGGAAACGTCACCAATGAA, SEQ ID NO: 85 ACCATCGATAGCAGCACCGTAATCAGTAGCGACAGAATCAAG, SEQ ID NO: 86 TTTGCCTTTAGCGTCAGACTGTAGCGCGTTTTCATCGGCATT, SEQ ID NO: 87 TTCGGTCATAGCCCCCTTATTAGCGTTTGCCATCTTTTCATA, SEQ ID NO: 88 ATCAAAATCACCGGAACCAGAGCCACCACCGGAACCGCCTCC, SEQ ID NO: 89 CTCAGAGCCGCCACCCTCAGAACCGCCACCCTCAGAGCCACC, SEQ ID NO: 90 ACCCTCAGAGCCGCCACCAGAACCACCACCAGAGCCGCCGCC, SEQ ID NO: 91 AGC ATT GAC AGGAGGTT GAGGC AGGT C AGACGATTGGCCTTG, SEQ ID NO: 92 AT ATT C AC AAAC AAAT AAATCCTC ATT AAAGCC AGAAT GGAA, SEQ ID NO: 93 AGCGCAGTCTCTGAATTTACCGTTCCAGTAAGCGTCATACAT, SEQ ID NO: 94 GGC TTTT GAT GAT AC AGGAGT GT ACTGGT AAT A AGTTTT A AC , SEQ ID NO: 95 GGGGT C AGT GC C TT G AGT A AC AGT GC C C GT AT A A AC AGT T A A, SEQ ID NO: 96 TGCCCCCTGCCTATTTCGGAACCTATTATTCTGAAACATGAA, SEQ ID NO: 97 AGT ATT A AGAGGCTGAGAC T C CTC A AGAGA AGGATT AGGATT, SEQ ID NO: 98 AGCGGGGTTTTGCTCAGTACCAGGCGGATAAGTGCCGTCGAG, SEQ ID NO: 99 AGGGTTGATATAAGTATAGCCCGGAATAGGTGTATCACCGTA, SEQ ID NO: 100 CTCAGGAGGTTTAGTACCGCCACCCTCAGAACCGCCACCCTC, SEQ ID NO: 101 AGAACCGCCACCCTCAGAGCCACCACCCTCATTTTCAGGGAT, SEQ ID NO: 102 AGC AAGCCC AAT AGGAACCC AT GT ACCGT AAC ACTGAGTTTC, SEQ ID NO: 103 GTCACCAGTACAAACTACAACGCCTGTAGCATTCCACAGACA, SEQ ID NO: 104 GCCCTCATAGTTAGCGTAACGATCTAAAGTTTTGTCGTCTTT, SEQ ID NO: 105 CCAGACGTTAGTAAATGAATTTTCTGTATGGGATTTTGCTAA, SEQ ID NO: 106 AC AACTTTC AAC AGTTTC AGCGGAGT GAG AAT AGA AAGGAAC, SEQ ID NO: 107 AACTAAAGGAATTGCGAATAATAATTTTTTCACGTTGAAAAT, SEQ ID NO: 108 CTCCAAAAAAAAGGCTCCAAAAGGAGCCTTTAATTGTATCGG, SEQ ID NO: 109 TTTATCAGCTTGCTTTCGAGGTGAATTTCTTAAACAGCTTGA, SEQ ID NO: 110 TACCGATAGTTGCGCCGACAATGACAACAACCATCGCCCACG, SEQ ID NO: 111 CAT AACCGAT AT ATTCGGTCGCTGAGGCTT GC AGGGAGTT AA, SEQ ID NO: 112 AGGCCGCTTTTGCGGGATCGTCACCCTCAGCAGCGAAAGACA, SEQ ID NO: 113 GCATCGGAACGAGGGTAGCAACGGCTACAGAGGCTTTGAGGA, SEQ ID NO: 114 CTAAAGACTTTTTCATGAGGAAGTTTCCATTAAACGGGTAAA, SEQ ID NO: 115 ATACGTAATGCCACTACGAAGGCACCAACCTAAAACGAAAGA, SEQ ID NO: 116 GGCAAAAGAATACACTAAAACACTCATCTTTGACCCCCAGCG, SEQ ID NO: 117 ATTATACCAAGCGCGAAACAAAGTACAACGGAGATTTGTATC, SEQ ID NO: 118 ATCGCCTGATAAATTGTGTCGAAATCCGCGACCTGCTCCATG, SEQ ID NO: 119 TT ACTT AGCCGGAACGAGGCGC AGACGGT C AAT CAT AAGGGA, SEQ ID NO: 120 ACCGAACTGACC AACTTT GAA AGAGGAC AGAT GAACGGT GT A, SEQ ID NO: 121 CAGACCAGGCGCATAGGCTGGCTGACCTTCATCAAGAGTAAT, SEQ ID NO: 122 CTTGACAAGAACCGGATATTCATTACCCAAATCAACGTAACA, SEQ ID NO: 123 AAGCTGCTCATTCAGTGAATAAGGCTTGCCCTGACGAGAAAC, SEQ ID NO: 124 ACCAGAACGAGTAGTAAATTGGGCTTGAGATGGTTTAATTTC, SEQ ID NO: 125 AACTTTAATCATTGTGAATTACCTTATGCGATTTTAAGAACT, SEQ ID NO: 126 GGC TC ATT AT AC C AGT C AGGACGTT GGGA AGA A A A ATCT ACG, SEQ ID NO: 127 TT AAT AAAACGAACT AACGGA AC AAC ATT ATT AC AGGT AGA A, SEQ ID NO: 128 AGATTCATCAGTTGAGATTTAGGAATACCACATTCAACTAAT, SEQ ID NO: 129 GC AG AT AC AT AAC GC C A A A AGG A AT T AC G AGGC AT AGT A AG A, SEQ ID NO: 130 GCAACACTATCATAACCCTCGTTTACCAGACGACGATAAAAA, SEQ ID NO: 131 CC AAAAT AGCGAGAGGCTTTTGC AAAAGAAGTTTT GCC AGAG, SEQ ID NO: 132 GGGGT A AT AGT A A A AT GT TT AG AC T GG AT AGC GT C C A AT ACT, SEQ ID NO: 133 GCGGAATCGTCATAAATATTCATTGAATCCCCCTCAAATGCT, SEQ ID NO: 134 TT AAAC AGTT C AGA AAACGAGA ATGACC AT AAAT C AAAAATC, SEQ ID NO: 135 AGGTCTTTACCCTGACTATTATAGTCAGAAGCAAAGCGGATT, SEQ ID NO: 136 GCATCAAAAAGATTAAGAGGAAGCCCGAAAGACTTCAAATAT, SEQ ID NO: 137 CGCGTTTTAATTCGAGCTTCAAAGCGAACCAGACCGGAAGCA, SEQ ID NO: 138 AACTCCAACAGGTCAGGATTAGAGAGTACCTTTAATTGCTCC, SEQ ID NO: 139 TTTTGATAAGAGGTCATTTTTGCGGATGGCTTAGAGCTTAAT, SEQ ID NO: 140 TGCTGAATATAATGCTGTAGCTCAACATGTTTTAAATATGCA, SEQ ID NO: 141 ACT AAAGT ACGGTGTCTGGAAGTTT C ATTCC AT AT AAC AGTT, SEQ ID NO: 142 GATTCCCAATTCTGCGAACGAGTAGATTTAGTTTGACCATTA, SEQ ID NO: 143 GATACATTTCGCAAATGGTCAATAACCTGTTTAGCTATATTT, SEQ ID NO: 144 TCATTTGGGGCGCGAGCTGAAAAGGTGGCATCAATTCTACTA, SEQ ID NO: 145 ATAGTAGTAGCATTAACATCCAATAAATCATACAGGCAAGGC, SEQ ID NO: 146 AAAGAATT AGC AAAATT AAGC AAT AAAGCCTC AGAGC AT AAA, SEQ ID NO: 147 GCTAAATCGGTTGTACCAAAAACATTATGACCCTGTAATACT, SEQ ID NO: 148 TTTGCGGGAGAAGCCTTTATTTCAACGCAAGGATAAAAATTT, SEQ ID NO: 149 TT AGA AC CC T CAT AT ATTTT AAAT GCA AT GCC T GAGT AAT GT, SEQ ID NO: 150 GT AGGT AAAGATT C AAAAGGGT GAGAAAGGCCGGAGAC AGTC, SEQ ID NO: 151 AAATCACCATCAATATGATATTCAACCGTTCTAGCTGATAAA, SEQ ID NO: 152 TT AAT GCCGGAGAGGGT AGCT ATTTTT GAGAGATCT AC AAAG, SEQ ID NO: 153 GCT AT C AGGT C ATTGC CTG AGAGT C T GG AGC A A AC A AG AG A A, SEQ ID NO: 154 TCGAT GAACGGT AATCGT AAAACT AGC AT GT C AAT CAT ATGT, SEQ ID NO: 155 ACCCCGGTTGATAATCAGAAAAGCCCCAAAAACAGGAAGATT, SEQ ID NO: 156 GTATAAGCAAATATTTAAATTGTAAACGTTAATATTTTGTTA, SEQ ID NO: 157 AAATTCGCATTAAATTTTTGTTAAATCAGCTCATTTTTTAAC, SEQ ID NO: 158 CAATAGGAACGCCATCAAAAATAATTCGCGTCTGGCCTTCCT, SEQ ID NO: 159 GT AGCC AGCTTT CAT C AAC ATT AAATGT GAGCGAGT A AC AAC, SEQ ID NO: 160 CCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAA, SEQ ID NO: 161 T GGG AT AGGT C AC GTT GGT GT AG AT GGGC GC ATC GT A AC C GT , SEQ ID NO: 162 GCATCTGCCAGTTTGAGGGGACGACGACAGTATCGGCCTCAG, SEQ ID NO: 163 GAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTG, SEQ ID NO: 164 CCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGCTGCGCA, SEQ ID NO: 165 ACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTAC, SEQ ID NO: 166 GCC AGCTGGCGAA AGGGGGATGT GCTGC AAGGCGATT AAGTT, SEQ ID NO: 167 GGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA, SEQ ID NO: 168 CGGCCAGTGCCAAGCTTTCAGAGGTGGAGCCGCCACGGGAAC, SEQ ID NO: 169 GGATAACCTCACCGGAAACAATCGGCGAAACGTACAGCGCCA, SEQ ID NO: 170 TGTTTACCAGTCCCGGAATTTGTGAGAGATAGACTTTCTCCG, SEQ ID NO: 171 T GGT GA AGGGAT AGC TC T C ACGGA A A A AGAGACGC AGA A AC A, SEQ ID NO: 172 GCGGATCAAACTTAAATTTCTGCTCATTTGCCGCCAGCAGTT, SEQ ID NO: 173 GGGC GGTT GT GT AC ATC G AC AT A A A A A A AT C C C GT A A A A A A A, SEQ ID NO: 174 GCCGCACAGGCGGCCTTTAGTGATGAAGGGTAAAGTTAAACG, SEQ ID NO: 175 ATGCTGATTGCCGTTCCGGCAAACGCGGTCCGTTTTTTCGTC, SEQ ID NO: 176 TCGTCGCTGGCAGCCTCCGGCCAGAGCACATCCTCATAACGG, SEQ ID NO: 177 A AC GT GC C GG AC TT GT AG A AC GT C AGC GT GGT GC T GGT C T GG, SEQ ID NO: 178 TCAGCAGCAACCGCAAGAATGCCAACGGCAGCACCGTCGGTG, SEQ ID NO: 179 GTGCCATCCCACGCAACCAGCTTACGGCTGGAGGTGTCCAGC, SEQ ID NO: 180 ATCAGCGGGGTCATTGCAGGCGCTTTCGCACTCAATCCGCCG, SEQ ID NO: 181 GGCGCGGTTGCGGTATGAGCCGGGTCACTGTTGCCCTGCGGC, SEQ ID NO: 182 TGGTAATGGGTAAAGGTTTCTTTGCTCGTCATAAACATCCCT, SEQ ID NO: 183 T AC ACTGGTGTGTTC AGC AA ATCGTT AACGGC AT C AGAT GCC, SEQ ID NO: 184 GGGTTACCTGCAGCCAGCGGTGCCGGTGCCCCCTGCATCAGA, SEQ ID NO: 185 CGATCCAGCGCAGTGTCACTGCGCGCCTGTGCACTCTGTGGT, SEQ ID NO: 186 GCTGC GGCCAGAATGCGGCGGGCCGTTTTCACGGTCATACCG, SEQ ID NO: 187 GGGGTTTCTGCCAGCACGCGTGCCTGTTCTTCGCGTCCGTGA, SEQ ID NO: 188 GCCTCCTCACAGTTGAGGATCCCCGGGTACCGAGCTCGAATT, SEQ ID NO: 189 CGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATC, SEQ ID NO: 190 CGC TC AC A ATTC C AC AC A AC AT AC GAGCC GGA AGC AT A A AGT, SEQ ID NO: 191 GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAA, SEQ ID NO: 192 TTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT, SEQ ID NO: 193 CGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAG, SEQ ID NO: 194 GCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACC, SEQ ID NO: 195 AGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCC, SEQ ID NO: 196 TGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGC, SEQ ID NO: 197 AGGCGAAAATCCTGTTTGATGGTGGTTCCGAAATCGGCAAAA, SEQ ID NO: 198 TCCCTT AT AAAT C AAAAGAAT AGCCCGAGAT AGGGTTGAGTG, SEQ ID NO: 199 TTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGG, SEQ ID NO: 200 ACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATG, SEQ ID NO: 201 GCCCACTACGTGAACCATCACCCAAATCAAGTTTTTTGGGGT, SEQ ID NO: 202 CGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCC, SEQ ID NO: 203 CCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGA, SEQ ID NO: 204 GAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGC, SEQ ID NO: 205 TGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCG, SEQ ID NO: 206 CCGCGCTTAATGCGCCGCTACA, SEQ ID NO: 207

Dendrimer prebackbone aatcctccatcccttccttaatcctccatcccttccttaatcctccatcccttccttTAG TGGAGATAATGGATTGG, SEQ ID NO: 208

Backbone ggaagggatggaggattgatctactatagcactgcttgatctactatagcactgcttgat ctactatagcactgc, SEQ ID NO: 209

Layer 1 tacgtgcttttacaggtgtttacgtgcttttacaggtgtttacgtgcttttacaggtgtt GC AGT GCT AT AGT AGATC, SEQ ID NO: 210

Layer 2

C ACCTGT AAAAGC ACGT Attgagcctacttagttgtacttgagcctacttagttgtacttgagcctacttagttgta c, SEQ ID NO: 211 Layer 3 tctatgctactgactaggtttctatgctactgactaggtttctatgctactgactaggtt GT AC A AC T A AGT AGGC T C , SEQ ID NO: 212

Layer 4f

CCTAGTCAGTAGCATAGAttCCAATCCATTATCTCCACTACCAATCCATTATCTCCA CT ACC AATCCATTATCTCC ACTA, SEQ ID NO: 213

Tether dendrimer grabber

CGGGAGCT AAAC AGGAGGCCGATT AAAGGGATTTT AGAC AGGttCC AATCC ATT ATC TCCACTA, SEQ ID NO: 214

AACGGTACGCCAGAATCCTGAGAAGTGTTTTTATAATCAGTGttCCAATCCATTATC T CCACTA, SEQ ID NO: 215

AGGCCACCGAGTAAAAGAGTCTGTCCATCACGCAAATTAACCttCCAATCCATTATC T CCACTA, SEQ ID NO: 216

GTTGTAGCAATACTTCTTTGATTAGTAATAACATCACTTGCCttCCAATCCATTATC TC CACTA, SEQ ID NO: 217

TGAGTAGAAGAACTCAAACTATCGGCCTTGCTGGTAATATCCttCCAATCCATTATC T CCACTA, SEQ ID NO: 218

AGAACAATATTACCGCCAGCCATTGCAACAGGAAAAACGCTCttCCAATCCATTATC T CCACTA, SEQ ID NO: 219

ATGGAAATACCTACATTTTGACGCTCAATCGTCTGAAATGGAttCCAATCCATTATC T CCACTA, SEQ ID NO: 220

TT ATTT AC ATTGGC AGATTC ACC AGT C AC ACGACC AGT AATAttCC AATCC ATT ATCTC CACTA, SEQ ID NO: 221

AAAGGGACATTCTGGCCAACAGAGATAGAACCCTTCTGACCTttCCAATCCATTATC T CCACTA, SEQ ID NO: 222

GAAAGCGTAAGAATACGTGGCACAGACAATATTTTTGAATGGttCCAATCCATTATC T CCACTA, SEQ ID NO: 223

CTATTAGTCTTTAATGCGCGAACTGATAGCCCTAAAACATCGttCCAATCCATT ATCTC CACTA, SEQ ID NO: 224 CCATTAAAAATACCGAACGAACCACCAGCAGAAGATAAAACAttCCAATCCATTATC TCCACTA, SEQ ID NO: 225

GAGGTGAGGCGGTCAGTATTAACACCGCCTGCAACAGTGCCAttCCAATCCATTATC T CCACTA, SEQ ID NO: 226

CGCTGAGAGCCAGCAGCAAATGAAAAATCTAAAGCATCACCTttCCAATCCATTATC T CCACTA, SEQ ID NO: 227

TGCTGAACCTCAAATATCAAACCCTCAATCAATATCTGGTCAttCCAATCCATTATC TC CACTA, SEQ ID NO: 228

GTTGGCAAATCAACAGTTGAAAGGAATTGAGGAAGGTTATCTttCCAATCCATTATC T CCACTA, SEQ ID NO: 229

AAAATATCTTTAGGAGCACTAACAACTAATAGATTAGAGCCGttCCAATCCATTATC T CCACTA, SEQ ID NO: 230

TCAATAGATAATACATTTGAGGATTTAGAAGTATTAGACTTTttCCAATCCATTATC TC CACTA, SEQ ID NO: 231

ACAAACAATTCGACAACTCGTATTAAATCCTTTGCCCGAACGttCCAATCCATTATC T CCACTA, SEQ ID NO: 232

TTATTAATTTTAAAAGTTTGAGTAACATTATCATTTTGCGGAttCCAATCCATTATC TC CACTA, SEQ ID NO: 233

ACAAAGAAACCACCAGAAGGAGCGGAATTATCATCATATTCCttCCAATCCATTATC T CCACTA, SEQ ID NO: 234

TGATTATCAGATGATGGCAATTCATCAATATAATCCTGATTGttCCAATCCATTATC TC CACTA, SEQ ID NO: 235

TTTGGATTATACTTCTGAATAATGGAAGGGTTAGAACCTACCttCCAATCCATTATC TC CACTA, SEQ ID NO: 236

AT ATC AAAATT ATTTGC ACGT AAAAC AGAAAT AAAGAAATTGttCC AATCC ATT ATCT CCACTA, SEQ ID NO: 237

CGTAGATTTTCAGGTTTAACGTCAGATGAATATACAGTAACAttCCAATCCATTATC T CCACTA, SEQ ID NO: 238

GTACCTTTTACATCGGGAGAAACAATAACGGATTCGCCTGATttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 239 TGCTTTGAATACCAAGTTACAAAATCGCGCAGAGGCGAATTAttCCAATCCATTATCT CCACTA, SEQ ID NO: 240

TTCATTTCAATTACCTGAGCAAAAGAAGATGATGAAACAAACttCCAATCCATTATC T CCACTA, SEQ ID NO: 241

ATCAAGAAAACAAAATTAATTACATTTAACAATTTCATTTGAttCCAATCCATTATC TC CACTA, SEQ ID NO: 242

ATTACCTTTTTTAATGGAAACAGTACATAAATCAATATATGTttCCAATCCATTATC TC CACTA, SEQ ID NO: 243

GAGTGAATAACCTTGCTTCTGTAAATCGTCGCTATTAATTAAttCCAATCCATTATC TC CACTA, SEQ ID NO: 244

TTTTCCCTTAGAATCCTTGAAAACATAGCGATAGCTTAGATTttCCAATCCATTATC TC CACTA, SEQ ID NO: 245

AAGACGCTGAGAAGAGTCAATAGTGAATTTATCAAAATCATAttCCAATCCATTATC T CCACTA, SEQ ID NO: 246

GGTCTGAGAGACTACCTTTTTAACCTCCGGCTTAGGTTGGGTttCCAATCCATTATC TC CACTA, SEQ ID NO: 247

TATATAACTATATGTAAATGCTGATGCAAATCCAATCGCAAGttCCAATCCATTATC T CCACTA, SEQ ID NO: 248

ACAAAGAACGCGAGAAAACTTTTTCAAATATATTTTAGTTAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 249

TTTCATCTTCTGACCTAAATTTAATGGTTTGAAATACCGACCttCCAATCCATTATC TC CACTA, SEQ ID NO: 250

GTGTGATAAATAAGGCGTTAAATAAGAATAAACACCGGAATCttCCAATCCATTATC T CCACTA, SEQ ID NO: 251

ATAATTACTAGAAAAAGCCTGTTTAGTATCATATGCGTTATAttCCAATCCATTATC TC CACTA, SEQ ID NO: 252

CAAATTCTTACCAGTATAAAGCCAACGCTCAACAGTAGGGCTttCCAATCCATTATC T CCACTA, SEQ ID NO: 253

TAATTGAGAATCGCCATATTTAACAACGCCAACATGTAATTTttCCAATCCATTATC TC CACTA, SEQ ID NO: 254 AGGCAGAGGCATTTTCGAGCCAGTAATAAGAGAATATAAAGTttCCAATCCATTATCT CCACTA, SEQ ID NO: 255

ACCGACAAAAGGTAAAGTAATTCTGTCCAGACGACGACAATAttCCAATCCATTATC T CCACTA, SEQ ID NO: 256

AACAACATGTTCAGCTAATGCAGAACGCGCCTGTTTATCAACttCCAATCCATTATC T CCACTA, SEQ ID NO: 257

AAT AGAT A AGTCCTGAAC AAGAAAAAT AAT ATCCC ATCCT AAttCC AATCC ATT ATCT CCACTA, SEQ ID NO: 258

TTTACGAGCATGTAGAAACCAATCAATAATCGGCTGTCTTTCttCCAATCCATTATC TC CACTA, SEQ ID NO: 259

CTTATCATTCCAAGAACGGGTATTAAACCAAGTACCGCACTCttCCAATCCATTATC T CCACTA, SEQ ID NO: 260

ATCGAGAACAAGCAAGCCGTTTTTATTTTCATCGTAGGAATCttCCAATCCATTATC TC CACTA, SEQ ID NO: 261

ATTACCGCGCCCAATAGCAAGCAAATCAGATATAGAAGGCTTttCCAATCCATTATC T CCACTA, SEQ ID NO: 262

ATCCGGTATTCTAAGAACGCGAGGCGTTTTAGCGAACCTCCCttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 263

GACTT GCGGGAGGTTTT GAAGCCTT AAAT C AAGATT AGTT GCttCC AATCC ATT ATCT CCACTA, SEQ ID NO: 264

TATTTTGCACCCAGCTACAATTTTATCCTGAATCTTACCAACttCCAATCCATTATC TC CACTA, SEQ ID NO: 265

GCTAACGAGCGTCTTTCCAGAGCCTAATTTGCCAGTTACAAAttCCAATCCATTATC TC CACTA, SEQ ID NO: 266

ATAAACAGCCATATTATTTATCCCAATCC AAAT AAGAAACGAttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 267

TTTTTTGTTTAACGTCAAAAATGAAAATAGCAGCCTTTACAGttCCAATCCATTATC TC CACTA, SEQ ID NO: 268

AGAGAATAACATAAAAACAGGGAAGCGCATTAGACGGGAGAAttCCAATCCATTATC TCCACTA, SEQ ID NO: 269 TTAACTGAACACCCTGAACAAAGTCAGAGGGTAATTGAGCGCttCCAATCCATTATCT CCACTA, SEQ ID NO: 270

TAATATCAGAGAGATAACCCACAAGAATTGAGTTAAGCCCAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 271

TAATAAGAGCAAGAAACAATGAAATAGCAATAGCTATCTTACttCCAATCCATTATC T CCACTA, SEQ ID NO: 272

CGAAGCCCTTTTTAAGAAAAGTAAGCAGATAGCCGAACAAAGttCCAATCCATTATC T CCACTA, SEQ ID NO: 273

TTACCAGAAGGAAACCGAGGAAACGCAATAATAACGGAATACttCCAATCCATTATC TCCACTA, SEQ ID NO: 274

CCAAAAGAACTGGCATGATTAAGACTCCTTATTACGCAGTATttCCAATCCATTATC T CCACTA, SEQ ID NO: 275

GTT AGC AAACGT AGAAAAT AC AT AC AT A AAGGTGGC AAC AT AttCC AATCC ATT ATCT CCACTA, SEQ ID NO: 276

TAAAAGAAACGCAAAGAC ACC ACGGAATAAGTTTATTTTGTCttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 277

ACAATCAATAGAAAATTCATATGGTTTACCAGCGCCAAAGACttCCAATCCATTATC T CCACTA, SEQ ID NO: 278

AAAAGGGCGAC ATTC AACCGATT GAGGGAGGGAAGGT AAAT AttCC AATCC ATT AT C TCCACTA, SEQ ID NO: 279

TTGACGGAAATTATTCATTAAAGGTGAATTATCACCGTCACCttCCAATCCATTATC TC CACTA, SEQ ID NO: 280

GACTTGAGCCATTTGGGAATTAGAGCC AGC AAAATCACCAGTttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 281

AGC ACCATT ACC ATT AGC AAGGCCGGAAACGTCACCAATGAAttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 282

ACCATCGATAGCAGCACCGTAATCAGTAGCGACAGAATCAAGttCCAATCCATTATC T CCACTA, SEQ ID NO: 283

TTTGCCTTTAGCGTCAGACTGTAGCGCGTTTTCATCGGCATTttCCAATCCATTATC TC CACTA, SEQ ID NO: 284 TTCGGTCATAGCCCCCTTATTAGCGTTTGCCATCTTTTCATAttCCAATCCATTATCTCC ACTA, SEQ ID NO: 285

ATCAAAATCACCGGAACCAGAGCCACCACCGGAACCGCCTCCttCCAATCCATTATC T CCACTA, SEQ ID NO: 286

CTCAGAGCCGCCACCCTCAGAACCGCCACCCTCAGAGCCACCttCCAATCCATTATC T CCACTA, SEQ ID NO: 287

ACCCTCAGAGCCGCCACCAGAACCACCACCAGAGCCGCCGCCttCCAATCCATTATC T CCACTA, SEQ ID NO: 288

AGCATTGACAGGAGGTTGAGGCAGGTCAGACGATTGGCCTTGttCCAATCCATTATC T CCACTA, SEQ ID NO: 289

ATATTCACAAACAAATAAATCCTCATTAAAGCCAGAATGGAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 290

AGCGCAGTCTCTGAATTTACCGTTCCAGTAAGCGTCATACATttCCAATCCATTATC TC CACTA, SEQ ID NO: 291

GGCTTTTGATGATACAGGAGTGTACTGGTAATAAGTTTTAACttCCAATCCATTATC TC CACTA, SEQ ID NO: 292

GGGGTCAGTGCCTTGAGTAACAGTGCCCGTATAAACAGTTAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 293

TGCCCCCTGCCTATTTCGGAACCTATTATTCTGAAACATGAAttCCAATCCATTATC TC CACTA, SEQ ID NO: 294

AGTATTAAGAGGCTGAGACTCCTCAAGAGAAGGATTAGGATTttCCAATCCATTATC T CCACTA, SEQ ID NO: 295

AGCGGGGTTTTGCTCAGTACCAGGCGGATAAGTGCCGTCGAGttCCAATCCATTATC T CCACTA, SEQ ID NO: 296

AGGGTTGATATAAGTATAGCCCGGAATAGGTGTATCACCGTAttCCAATCCATTATC T CCACTA, SEQ ID NO: 297

CTCAGGAGGTTTAGTACCGCCACCCTCAGAACCGCCACCCTCttCCAATCCATTATC T CCACTA, SEQ ID NO: 298

AGAACCGCCACCCTCAGAGCCACCACCCTCATTTTCAGGGATttCCAATCCATTATC T CCACTA, SEQ ID NO: 299 AGCAAGCCCAATAGGAACCCATGTACCGTAACACTGAGTTTCttCCAATCCATTATCT CCACTA, SEQ ID NO: 300

GTCACCAGTACAAACTACAACGCCTGTAGCATTCCACAGACAttCCAATCCATTATC T CCACTA, SEQ ID NO: 301

GCCCTCATAGTTAGCGTAACGATCTAAAGTTTTGTCGTCTTTttCCAATCCATTATC TC CACTA, SEQ ID NO: 302

CCAGACGTTAGTAAATGAATTTTCTGTATGGGATTTTGCTAAttCCAATCCATTATC TC CACTA, SEQ ID NO: 303

AC AACTTTC AAC AGTTTC AGCGGAGT GAGAAT AGA AAGGAACttCC AATCC ATT ATCT CCACTA, SEQ ID NO: 304

AACTAAAGGAATTGCGAATAATAATTTTTTCACGTTGAAAATttCCAATCCATTATC T CCACTA, SEQ ID NO: 305

CTCCAAAAAAAAGGCTCCAAAAGGAGCCTTTAATTGTATCGGttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 306

TTTATCAGCTTGCTTTCGAGGTGAATTTCTTAAACAGCTTGAttCCAATCCATTATC TC CACTA, SEQ ID NO: 307

TACCGATAGTTGCGCCGACAATGACAACAACCATCGCCCACGttCCAATCCATTATC T CCACTA, SEQ ID NO: 308

CAT AACCGATATATTCGGTCGCTGAGGCTTGCAGGGAGTTAAttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 309

AGGCCGCTTTTGCGGGATCGTCACCCTCAGCAGCGAAAGACAttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 310

GCATCGGAACGAGGGTAGCAACGGCTACAGAGGCTTTGAGGAttCCAATCCATTATC TCCACTA, SEQ ID NO: 311

CTAAAGACTTTTTCATGAGGAAGTTTCCATTAAACGGGTAAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 312

ATACGTAATGCCACTACGAAGGCACCAACCTAAAACGAAAGAttCCAATCCATTATC TCCACTA, SEQ ID NO: 313

GGCAAAAGAATACACTAAAACACTCATCTTTGACCCCCAGCGttCCAATCCATTATC T CCACTA, SEQ ID NO: 314 ATTATACCAAGCGCGAAACAAAGTACAACGGAGATTTGTATCttCCAATCCATTATCT CCACTA, SEQ ID NO: 315

ATCGCCTGATAAATTGTGTCGAAATCCGCGACCTGCTCCATGttCCAATCCATTATC TC CACTA, SEQ ID NO: 316

TTACTTAGCCGGAACGAGGCGCAGACGGTCAATCATAAGGGAttCCAATCCATTATC T CCACTA, SEQ ID NO: 317

ACCGAACTGACCAACTTTGAAAGAGGACAGATGAACGGTGTAttCCAATCCATTATC T CCACTA, SEQ ID NO: 318

CAGACCAGGCGCATAGGCTGGCTGACCTTCATCAAGAGTAATttCCAATCCATTATC T CCACTA, SEQ ID NO: 319

CTTGACAAGAACCGGATATTCATTACCCAAATCAACGTAACAttCCAATCCATTATC T CCACTA, SEQ ID NO: 320

AAGCTGCTCATTCAGTGAATAAGGCTTGCCCTGACGAGAAACttCCAATCCATTATC T CCACTA, SEQ ID NO: 321

ACCAGAACGAGTAGTAAATTGGGCTTGAGATGGTTTAATTTCttCCAATCCATTATC T CCACTA, SEQ ID NO: 322

AACTTTAATCATTGTGAATTACCTTATGCGATTTTAAGAACTttCCAATCCATTATC TC CACTA, SEQ ID NO: 323

GGCTCATTATACCAGTCAGGACGTTGGGAAGAAAAATCTACGttCCAATCCATTATC T CCACTA, SEQ ID NO: 324

TTAATAAAACGAACTAACGGAACAACATTATTACAGGTAGAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 325

AGATTCATCAGTTGAGATTTAGGAATACCACATTCAACTAATttCCAATCCATTATC TC CACTA, SEQ ID NO: 326

GCAGATACATAACGCCAAAAGGAATTACGAGGCATAGTAAGAttCCAATCCATTATC TCCACTA, SEQ ID NO: 327

GCAACACTATCATAACCCTCGTTTACCAGACGACGATAAAAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 328

CCAAAATAGCGAGAGGCTTTTGCAAAAGAAGTTTTGCCAGAGttCCAATCCATTATC T CCACTA, SEQ ID NO: 329 GGGGTAATAGTAAAATGTTTAGACTGGATAGCGTCCAATACTttCCAATCCATTATCT CCACTA, SEQ ID NO: 330

GCGGAATCGTCATAAATATTCATTGAATCCCCCTCAAATGCTttCCAATCCATTATC TC CACTA, SEQ ID NO: 331

TTAAACAGTTCAGAAAACGAGAATGACCATAAATCAAAAATCttCCAATCCATTATC T CCACTA, SEQ ID NO: 332

AGGTCTTTACCCTGACTATTATAGTCAGAAGCAAAGCGGATTttCCAATCCATTATC T CCACTA, SEQ ID NO: 333

GC AT C A AAAAGATT AAGAGGA AGCCCGAAAGACTT C AAAT ATttCC AATCC ATT ATCT CCACTA, SEQ ID NO: 334

CGCGTTTTAATTCGAGCTTCAAAGCGAACCAGACCGGAAGCAttCCAATCCATTATC T CCACTA, SEQ ID NO: 335

AACTCCAACAGGTCAGGATTAGAGAGTACCTTTAATTGCTCCttCCAATCC ATT ATCT CCACTA, SEQ ID NO: 336

TTTTGATAAGAGGTCATTTTTGCGGATGGCTTAGAGCTTAATttCCAATCCATTATC TC CACTA, SEQ ID NO: 337

TGCTGAATATAATGCTGTAGCTCAACATGTTTTAAATATGCAttCCAATCCATTATC TC CACTA, SEQ ID NO: 338

ACTAAAGTACGGTGTCTGGAAGTTTCATTCCATATAACAGTTttCCAATCCATTATC TC CACTA, SEQ ID NO: 339

GATTCCCAATTCTGCGAACGAGTAGATTTAGTTTGACCATTAttCCAATCCATTATC TC CACTA, SEQ ID NO: 340

GATACATTTCGCAAATGGTCAATAACCTGTTTAGCTATATTTttCCAATCCATTATC TC CACTA, SEQ ID NO: 341

TCATTTGGGGCGCGAGCTGAAAAGGTGGCATCAATTCTACTAttCCAATCCATTATC T CCACTA, SEQ ID NO: 342

AT AGT AGT AGC ATT A AC ATCC AAT AAAT CAT AC AGGCAAGGCttCC AATCC ATT ATCT CCACTA, SEQ ID NO: 343

AAAGAATTAGCAAAATTAAGCAATAAAGCCTCAGAGCATAAAttCCAATCCATTATC TCCACTA, SEQ ID NO: 344 GCTAAATCGGTTGTACCAAAAACATTATGACCCTGTAATACTttCCAATCCATTATCTC CACTA, SEQ ID NO: 345

TTTGCGGGAGAAGCCTTTATTTCAACGCAAGGATAAAAATTTttCCAATCCATTATC T CCACTA, SEQ ID NO: 346

TTAGAACCCTCATATATTTTAAATGCAATGCCTGAGTAATGTttCCAATCCATTATC TC CACTA, SEQ ID NO: 347

GTAGGTAAAGATTCAAAAGGGTGAGAAAGGCCGGAGACAGTCttCCAATCCATTATC TCCACTA, SEQ ID NO: 348

AAATCACCATCAATATGATATTCAACCGTTCTAGCTGATAAAttCCAATCCATTATC TC CACTA, SEQ ID NO: 349

TTAATGCCGGAGAGGGTAGCTATTTTTGAGAGATCTACAAAGttCCAATCCATTATC T CCACTA, SEQ ID NO: 350

GCTATCAGGTCATTGCCTGAGAGTCTGGAGCAAACAAGAGAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 351

TCGATGAACGGTAATCGTAAAACTAGCATGTCAATCATATGTttCCAATCCATTATC T CCACTA, SEQ ID NO: 352

ACCCCGGTTGATAATCAGAAAAGCCCCAAAAACAGGAAGATTttCCAATCCATTATC T CCACTA, SEQ ID NO: 353

GTATAAGCAAATATTTAAATTGTAAACGTTAATATTTTGTTAttCCAATCCATTATC TC CACTA, SEQ ID NO: 354

AAATTCGCATTAAATTTTTGTTAAATCAGCTCATTTTTTAACttCCAATCCATTATC TC CACTA, SEQ ID NO: 355

CAATAGGAACGCCATCAAAAATAATTCGCGTCTGGCCTTCCTttCCAATCCATTATC T CCACTA, SEQ ID NO: 356

GTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAACttCCAATCCATTATC T CCACTA, SEQ ID NO: 357

CCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 358

TGGGATAGGTCACGTTGGTGTAGATGGGCGCATCGTAACCGTttCCAATCCATTATC T CCACTA, SEQ ID NO: 359 GCATCTGCCAGTTTGAGGGGACGACGACAGTATCGGCCTCAGttCCAATCCATTATCT CCACTA, SEQ ID NO: 360

GAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTGttCCAATCCATTATC T CCACTA, SEQ ID NO: 361

CCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGCTGCGCAttCCAATCCATTATC T CCACTA, SEQ ID NO: 362

ACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACttCCAATCCATTATC T CCACTA, SEQ ID NO: 363

GCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTttCCAATCCATTATC T CCACTA, SEQ ID NO: 364

GGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGAttCCAATCCATTATC T CCACTA, SEQ ID NO: 365

CGGCCAGTGCCAAGCTTTCAGAGGTGGAGCCGCCACGGGAACttCCAATCCATTATC T CCACTA, SEQ ID NO: 366

GGATAACCTCACCGGAAACAATCGGCGAAACGTACAGCGCCAttCCAATCCATTATC TCCACTA, SEQ ID NO: 367

TGTTTACCAGTCCCGGAATTTGTGAGAGATAGACTTTCTCCGttCCAATCCATTATC TC CACTA, SEQ ID NO: 368

TGGTGAAGGGATAGCTCTCACGGAAAAAGAGACGCAGAAACAttCCAATCCATTATC TCCACTA, SEQ ID NO: 369

GCGGATCAAACTTAAATTTCTGCTCATTTGCCGCCAGCAGTTttCCAATCCATTATC TC CACTA, SEQ ID NO: 370

GGGCGGTTGTGTACATCGACATAAAAAAATCCCGTAAAAAAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 371

GCCGCACAGGCGGCCTTTAGTGATGAAGGGTAAAGTTAAACGttCCAATCCATTATC T CCACTA, SEQ ID NO: 372

ATGCTGATTGCCGTTCCGGCAAACGCGGTCCGTTTTTTCGTCttCCAATCCATTATC TC CACTA, SEQ ID NO: 373

TCGTCGCTGGCAGCCTCCGGCCAGAGCACATCCTCATAACGGttCCAATCCATTATC T CCACTA, SEQ ID NO: 374 AACGTGCCGGACTTGTAGAACGTCAGCGTGGTGCTGGTCTGGttCCAATCCATTATCT CCACTA, SEQ ID NO: 375

TCAGCAGCAACCGCAAGAATGCCAACGGCAGCACCGTCGGTGttCCAATCCATTATC T CCACTA, SEQ ID NO: 376

GTGCCATCCCACGCAACCAGCTTACGGCTGGAGGTGTCCAGCttCCAATCCATTATC T CCACTA, SEQ ID NO: 377

ATCAGCGGGGTCATTGCAGGCGCTTTCGCACTCAATCCGCCGttCCAATCCATTATC T CCACTA, SEQ ID NO: 378

GGCGCGGTTGCGGTATGAGCCGGGTCACTGTTGCCCTGCGGCttCCAATCCATTATC T CCACTA, SEQ ID NO: 379

TGGTAATGGGTAAAGGTTTCTTTGCTCGTCATAAACATCCCTttCCAATCCATTATC TC CACTA, SEQ ID NO: 380

TACACTGGTGTGTTCAGCAAATCGTTAACGGCATCAGATGCCttCCAATCCATTATC T CCACTA, SEQ ID NO: 381

GGGTTACCTGCAGCCAGCGGTGCCGGTGCCCCCTGCATCAGAttCCAATCCATTATC T CCACTA, SEQ ID NO: 382

CGATCCAGCGCAGTGTCACTGCGCGCCTGTGCACTCTGTGGTttCCAATCCATTATC TC CACTA, SEQ ID NO: 383

GCTGCGGCCAGAATGCGGCGGGCCGTTTTCACGGTCATACCGttCCAATCCATTATC T CCACTA, SEQ ID NO: 384

GGGGTTTCTGCCAGCACGCGTGCCTGTTCTTCGCGTCCGTGAttCCAATCCATTATC TC CACTA, SEQ ID NO: 385

GCCTCCTCACAGTTGAGGATCCCCGGGTACCGAGCTCGAATTttCCAATCCATTATC T CCACTA, SEQ ID NO: 386

CGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCttCCAATCCATTATC TC CACTA, SEQ ID NO: 387

CGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTttCCAATCCATTATC T CCACTA, SEQ ID NO: 388

GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 389 TTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTttCCAATCCATTATCTC CACTA, SEQ ID NO: 390

CGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGttCCAATCCATTATC T CCACTA, SEQ ID NO: 391

GCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCttCCAATCCATTATC TC CACTA, SEQ ID NO: 392

AGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCttCCAATCCATTATC T CCACTA, SEQ ID NO: 393

TGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCttCCAATCCATTATC T CCACTA, SEQ ID NO: 394

AGGCGAAAATCCTGTTTGATGGTGGTTCCGAAATCGGCAAAAttCCAATCCATTATC T CCACTA, SEQ ID NO: 395

TCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTGttCCAATCCATTATC T CCACTA, SEQ ID NO: 396

TTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGttCCAATCCATTATC T CCACTA, SEQ ID NO: 397

ACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGttCCAATCCATTATC T CCACTA, SEQ ID NO: 398

GCCCACTACGTGAACCATCACCCAAATCAAGTTTTTTGGGGTttCCAATCCATTATC TC CACTA, SEQ ID NO: 399

CGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCttCCAATCCATTATC TCCACTA, SEQ ID NO: 400

CCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAttCCAATCCATTATC T CCACTA, SEQ ID NO: 401

GAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCttCCAATCCATTAT CTCCACTA, SEQ ID NO: 402

TGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGttCCAATCCATTATC T CCACTA, SEQ ID NO: 403

CCGCGCTTAATGCGCCGCTACAttCCAATCCATTATCTCCACTA, SEQ ID NO: 404 GGGCGCGTACTATGGTTGCTTtgacgagcac, SEQ ID NO: 405 Testing of Nanoreporter

Following adequate demonstration that the hairpins and tethers were assembling and anchoring to the glass as designed, to assemble the full nanoreporter with the mechanical amplifier was attempted. Addition of the mechanical amplifier would allow for generation of tension in the dsTether and hairpin chain to assess if the hairpins could be opened by addition of shear flow. To test this, a simple microfluidic device was used, consisting of 2mm x 25mm x lOOpm PDMS channels attached to a #1.5 cover glass. The microfluidics were incubated with 50 pg/mL anti- digoxigenin for 3 minutes, then washed and blocked with a 1% BSA PBS buffer with Tween-20. The purified hairpin-tethers were incubated for one hour with streptavidin coated silica microbeads washed 3x with 1% BSA PBS buffer + Tween-20. Silica proved to be an optimal bead material due to its low autofluorescence as compared to magnetic or polystyrene beads. Following a 30- minute incubation of the prepared hairpin-tether-bead nanoreporters, the microfluidics were ready for flow and imaging. A syringe pump with PBS was hooked up and connected to the microfluidic with friction fit tubing. By applying gradually increasing shear, the hairpins began opening at 15 dynes/cm ² and gradually increased in fluorescence intensity until about 25 dynes/cm ² was applied for a bead size of 1 micron in diameter. Further increase in applied shear did not result in increased fluorescence, indicating that all 10 hairpins were opened in equilibrium. Following removal of shear, fluorescence signal likewise promptly disappeared. This process was repeated many tens of times, or until the fluorophores bleached.

Furthermore, when viewed with brightfield or RICM imaging, the beads can be seen moving around via Brownian motion in a zero shear environment. Upon application of shear flow, the beads move in direction of the applied shear then stop after having displaced around 2.7 microns, which is the length of the tether. Since the beads are not stationary when no shear is applied, measuring the exact displacement is difficult. This controlled displacement indicates that the beads are tethered to the surface and is helpful for identifying active nanoreporters as beads nonspecifically bound to the glass do not move when shear is applied.

Another important point pertaining to the ability of these nanoreporters to directly measure shear is the exact vertical location of the bead. Given that the flow velocity profile near the wall may be linear, a bead anywhere within this linear region would technically experience the same shear. However, within this region, a bead further away from the wall will experience greater flow velocities and thus generate more drag. As such, function of the nanoreporter is inexorably tied to flow velocity, and thus the vertical position of the bead within the flow profile. For the nanoreporter to be called a shear sensor, and not a flow sensor, its bead must be in approximately the same y position in all samples and testing conditions. During our preliminary experiments, this is exactly what was observe. Tethered beads move freely in static conditions, and often are barely visible in RICM imaging as they float around over a micron away from the glass. But in flow conditions, even just a few dynes/cm ² , the beads will come down to the glass. This tells us that the nanoreporter beads are sensing flow velocity conditions consistently with the mechanical amplifier in the same y location, which is right up against the glass.

Improved Nanoreporter Yield

Initial experiments revealed consistent and repeatable nanoreporter function, but with few sensors per unit area compared to how many beads were being added to the chamber. Experiments were performed to determine if the yield of active sensors on glass surface could be dependent on the duration of the hairpin-tether with microbead incubation. Instead of 1 hour, an overnight incubation on a rotator increased active sensors per unit area over 50-fold. Additionally, blocking both the glass surface and the beads with a PBS + Tween-20 + 1% BSA solution showed improvements. Otherwise, the entire glass surface would be covered with nonspecifically bound beads. It was discovered that large nonspecifically bound clumps of beads could be removed by a brief sonication of the beads after washing and before incubation with the dsTethers. Other areas of optimization that are contemplated are anti-digoxigenin concentration on glass, bead-tether- hairpin incubation on glass, and different blocking buffers.

Multi-valent Mono-active Tether Attachment

As the protocols used to create the nanoreporters would logically result in beads with more than one dsTether/hairpin chain attached to it (10 x excess molar incubation of dsTether onto bead), it is likely the beads might be multivalent yet mono-active sensors. This means in a given flow direction, only one of the multiple tethers to a bead would experience tension and therefore produce fluorescent signal. This was confirmed this by subjecting the same region of interest with different directions of flow. Some of the fluorescing hairpin chains remained in the same location regardless of which direction of flow - thus suggesting the specific nanoreporter was truly monovalent. Other beads displayed disappearance of one hairpin chain but the appearance of another one upstream of the new flow direction relative to the first hairpin chain signal. Furthermore, reverting the flow direction results in a return of the initial hairpin chain location.

Multi-valent Multi-active Tether Attachment

While holding bead concentration and incubation times constant, the number of tethered beads on the glass surface is proportional to the concentration of purified hairpin-tethers incubated with the beads. As the tether concentration is increased to about 200 pM, the active 1 -micron diameter beads per 100 square microns peaks at about 8. Further increase of tether concentration instead produces a proportional increasing prevalence of a second population of beads that are connected to more than one active hairpin-tether. The phenotype of this multi-active nanoreporter is two or more fluorescent spots near each other which are relatively perpendicular to the direction of flow, and visibly associated with a single bead. An increased flow rate is necessary to elicit full hairpin opening in the multi-tethered nanoreporter suggesting that the drag force is being shared in parallel across the two hairpin chains. At 200 pM dsTether, the occurrence of this multi active nanoreporter is less than 1%, but at 500 pM dsTether, beads with 3 or even 4 active hairpin chains are commonplace.

The concept of multivalency was taken to the extreme by using a very high tether concentration of 1.4 nanomolar. In order to produce a tether concentration this high, the hairpin chain and tether assembly processes were separated. The surface was saturated with 15 nM of purified hairpin chains, while the beads were incubated with varying concentrations of double stranded linearized ml3. The high concentration of dsTether resulted in beads with highly restricted movement. Even in static conditions, the beads appeared to be tied down to the glass, demonstrating less than half of the usual displacement under flow (See figure 5).

Expanded nanoreporter functionality

The molecular shear sensitive nanoreporter described above consists of a microbead, DNA tether, and fluorescence force transducer. To fully explore the design space of this approach, nanoreporters with different features were synthesize. This disclosure contemplates modifications such as: 1) the DNA hairpin sequence, which determines the threshold force for the opening of the hairpin; 2) the number of hairpins in the fluorescence force transducer; 3) the size and material of the microbead, and 4) the length of the DNA tether. Each one of these design parameters affects the behaviors of the nanoreporter. The DNA tether can be prepared by using m 13 DNA, or longer DNA tethers can be produced by using lambda DNA, or by hierarchically assembling multiple ml3 DNA strands. Shorter tethers can be prepared by cutting the current ml3 scaffolds into approximate desired lengths with restriction enzymes.

GC 22% Hairpin Fl/2 modeling

The hairpin chain opens over a narrow range of applied shear stress. After a base flow rate is reached, the fluorescent signal increases with flow rate until a maximum where all hairpins in the sensor assembly are open. Quantification of this fluorescence yields a sigmoid curve of the hairpin assembly’s active range (Figure 2). This means that at a given flow rate within the range of this sigmoid curve, an equilibrium number of ten hairpins in the chain are open.

Scaffolded Nanoreporter

Scaffolded versions are contemplated where force sensitive components are included on the linearized tether strand. This way, tension is bore on the continuous tether, and not across unligated sticky ends. Preliminary exploration suggests non-specific interactions between the scaffold loops. Adding short staples into the loop are contemplated to reduce secondary structure in such a way that does not add tension to the hybridization between fluorophore and quencher strands (Fig. IE).

DNA-based branched kite components

It is contemplated that a shear nanoreporter could also be created using DNA-based organic components (i.e. no bead). Shear nanoreporters are contemplated using an organic structure to generate drag forces. Drag is induced on linear structures, and the total applied force is proportional to the square root of the length of the polymer. Polymers of sufficient length can be used to measure the applied shear stress if a reporter is incorporated into the structure. A completely biomolecule- based structure is contemplated to be biodegradable improving in vivo compatibility. Assembly of nanoreporters driven by DNA hybridization streamlines the process and is contemplated to improves the yield and stability of the nanoreporter. Different geometries (e.g. dendrimers) enables incorporations of different shapes. The nanoreporters can be adapted to constricted anatomical locations. DNA dendrimer-type construct are contemplated where each layer consists of three times more DNA strands than the previous layer. By first employing a single fluorescent dendrimer in place of the bead (Fig. 3A). An interesting side effect of attaching the fluorescent dendrimer to the dsTether was that they became easily distinguishable from dendrimers nonspecifically attached the glass. The active dendrimers appeared in the exposure as a fuzzy cloud of fluorescence, as they experience Brownian motion but are ultimately constrained by the dsTether. Contrarily, nonspecifically attached dendrimers appear as sharp points of fluorescence as they are stationary.

In static conditions, the fluorescent dendrimer can be seen co-localized on top of constitutively open hairpins. After application of shear, the dendrimer fluorescence displaces from the hairpin chain fluorescence in the direction of flow. With increasing applied shear, the displacement of the dendrimer from the hairpin chain increases. The dsTether could be stretched and displacement of the dendrimer from the hairpins did not exceed 2.8 microns, which is the designed length of the dsTether. The experiment was repeated with closed hairpins and increase the size of the dendrimer.

The open hairpin experiment was repeated with three different dendrimers: 2L (300 kD), 3L (1 MD), and 4L (3.2 MD). At a spread of different applied shears, the measured amount of tether extension was almost the exact same for all three dendrimer sizes. The only noticeable difference was at very high shear rates for the microfluidic, where the larger dendrimers produced greater extension than the smallest one. The 4L dendrimer with 3.2 MD mass failed to produce any hairpin signal even with shear increased to almost 300 dynes/cm ² approaching the limit of the friction fitted microfluidic system This suggested to us that the dendrimers were barely contributing to the dsTethers.

While the dendrimer enabled extension of the tether was confirmed in flow, a single dendrimer had limited force to open the hairpins. Multiple dendrimers (192) were added onto the tether by incorporating a dendrimer capturing extension on short oligos used to make the linearized p8064 ml3 double stranded (Fig. 3 A). These capturing extensions hybridize the backbone strand of each dendrimer, or the strand that every other strand branches off of. This way a controlled number of dendrimers were program to hybridize to the long tether. DNA drogues comprised of 3L, 4L, and 5L dendrimers were compared respectively. During flow testing, the 5L dendrimer managed to produce fluorescent signal. The 5L dendrimer DNA drogue has a total designed mass of 2.2 billion Daltons. Although this is an incredibly large DNA structure, it still runs into the agarose gel with limited aggregation in the wells. This DNA drogue shear nanoreporter could produce signal at high shear rates of 100 dynes/cm ² . TEM imaging after agarose gel purification revealed a long and snakelike electron-dense megastructure.

Larger DNA drogues are contemplated by (1) adding more layers per dendrimer or (2) using multiple long DNA drogues with a single hairpin chain. It is contemplated that a 1 :3 layer n-1 to layer n ratio to 1 :2 or 1 : 1 can be created. It is also contemplated that one can use multiple long scaffold DNA to create an even larger structure, specifically using an intermediate size circular p3015 ml3 DNA to simultaneously grab many fully formed DNA drogues (Fig. 3C).

Targeting of Shear Nanoreporter to Cells

Shear may affect numerous cell types in various anatomical locations. A key aspect of measuring the shear stress on these cells will be attaching a nanoreporter directly to the cell surface. This can be accomplished by conjugating molecules or proteins to the nanoreporter that will facilitate cell binding. Targeting specific antigens on the cell surface confers the additional advantage of targeting specific cell types and even cellular states. For example, vascular cell adhesion molecule-1 (VCAM-1) is expressed on activated endothelial cells and is a key marker of pro-atherosclerotic conditions. Hence, a shear nanoreporter targeting VCAM-1 will identify when pro-atherosclerotic conditions are present and report on the localized shear in that area. Importantly, given the versatility of DNA, numerous biomolecular conjugation techniques are available to bind targeting molecules to DNA.

Experiments were performed to determine whether nanoreporters could target specific cell markers. Initial experiments were directed to platelets. A DNA oligo with sequence was conjugated to anti-CD41 antibody using a commercially available kit (SoluLink® Protein-Oligo Conjugation Kit). Substitution of digoxigenin with this antibody-DNA conjugate switches the targeted binding site from anti-digoxigenin to platelet-specific integrin al Ibp3, which is abundantly expressed on the platelet surface. Proper activity of the antibody after conjugation with DNA was first verified. Platelets were isolated using standard protocols and plated in a simple microfluidic structure created from PDMS channels (40 mm x 2 mm x 100 pm) and a No. 1.5 coverslip. The platelets were coated with the FPLC purified anti-CD41-DNA then washed with tween-20-free buffer. The platelets were incubated with constitutively open 10-hairpin-chains with either free or blocked anti-CD41-DNA hybridization sites. The hairpin chains with blocked hybridization sites showed very low binding, while the hairpin chains with free binding sites demonstrated excellent binding and fluorescence. Images were taken with TIRF so only the edges of the platelets are clearly visible - thicker areas of the platelets cannot be visualized with TIRF.

Following these results, we attempted to assemble and use shear to activate the complete nanoreporter on platelets (Fig. 6). Purified hairpin-tether assemblies were incubated on beads as done previously, but without the anti-CD41-DNA. Following a washing step of the platelets using 1%BSA PBS buffer, the platelets were then incubated with a high concentration of anti-CD41- DNA and washed again. Finally, the hairpin-tether-bead assembly was incubated on the prepared platelets shortly before imaging.

One concern was that the applied forces will alter the binding affinity of the antibodies and that they will be unable to attach to the surface of the cells under flow. However, data from experiments suggest that this is not a concern for CD41 on platelets as the nanoreporter fluoresces at 25 dynes/cm ² . With an active sensor yield of at best 5%, significant non-specific binding from the streptavidin coated beads was noted, which is not unexpected given previous experience that beads stick to any biological materials present on the glass. It is contemplated that one can reduce adhesion by passivating the bead surface by saturating unbound streptavidin with biotinylated PEG at least 1 kD in size. Another option is to coat a bead in mutated streptavidin that does not contain a RYE ⁾ sequence. The RYE ⁾ sequence expressed by wild type streptavidin and mimics RGD (Arg- Gly-Asp). RGD is the universal recognition domain present in fibronectin and other adhesion- related molecules.

Nanoreporter in Endothelialized Microfluidics

A series of sensors can be designed to target various antigens starting with endothelial cell markers CD31/PEC AM, VCAM, and CD43. Both microfluidic and larger “microfluidics” can be coated in a 3D conformal layer of endothelial cells that recapitulates the essential features of a biological system. This system can be modified by conjugating various antibodies to the previously characterized endothelial targets (CD31, VCAM, CD43, a4b1). It is contemplated that testing can be performed using blood products, e.g., whole blood.