Methods and systems for analyzing molecules based on nucleic acid displacement reactions

Abstract

Methods for and systems for analyzing a target molecule are described. The methods may comprise, for example, contacting the target molecule with a first molecule and a second molecule, to form a complex; displacing an eluting nucleic acid molecule with the complex, thereby forming a reacted complex and releasing a product nucleic acid molecule from the eluting nucleic acid molecule; purifying the product nucleic acid molecule; and measuring an amount of the product nucleic acid molecule.

Description

METHODS AND SYSTEMS FOR ANALYZING MOLECULES BASED ON NUCLEICACID DISPLACEMENT REACTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 633,429, filed April 12, 2024, which is incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure relates generally to methods and systems for analyzing target molecules, and more specifically to methods and systems of approximating amounts of the target molecules by measuring the amount of product from a nucleic acid displacement reaction.BACKGROUND

[0003] The study of non-nucleic acid molecules, such as proteins, are not natively amenable to nucleic acid sequencing techniques. Accordingly, the advantages of nucleic acid sequencing techniques, such as an ease of multiplexing and scaling and a compatibility with popular sequencing platforms, are not natively available to methods of detecting and measuring non- nucleic acid molecules, like proteins. Methods that convert the analyzing of non-nucleic acid molecules into a sequencing-based workflow would improve the study of non-nucleic acid molecules by making such study available to the multiplexing and scaling benefits native to most sequencing methods. The present disclosure describes methods that provide these improvements.BRIEF SUMMARY

[0004] Disclosed herein are methods and systems for detecting non-nucleic acid molecules, such as protein molecules, based on nucleic acid displacement reactions. Existing methods for detecting and measuring non-nucleic acid molecules, e.g., protein molecules, cannot be easily multiplexed and or analyzed on a large scale. In contrast, nucleic acid molecules are routinely analyzed via easily scalable and massively parallel sequencing technologies. The methods and systems described herein incorporate nucleic acid sequencing techniques into a method foranalyzing non-nucleic acid molecules, such as proteins. In doing so, the methods and systems described herein extend the benefits native to sequencing techniques, such as an ease of multiplexing and scalability, to a method for analyzing non-nucleic acid molecules, such as proteins. By measuring the amount of a product nucleic acid molecule released from nucleic acid displacement reactions, the methods and systems described herein can approximate the amount of a target molecule.

[0005] In some aspects, disclosed herein is a protein-to-DNA signal conversion assay method that uses a molecular construct based on a proximity-assisted strand displacement (PSD) reaction at a three-way DNA junction. The initial molecular construct can consist of a first molecule and a second molecule: two matched, and single-stranded DNA-conjugated affinity ligands specific to orthogonal epitopes on a protein-of-interest. Target detection in solution can bring the two DNA-conjugated affinity ligands in close proximity to each other and can allow for the formation of a three-way DNA junction. A subsequent strand displacement reaction can result in a unique DNA barcode for each protein-of-interest which can be read out using quantitative polymerase chain reaction (qPCR), next generation sequencing (NGS), or other approaches following barcode amplification. The molecular construct may be immobilized on a solid support prior to the assay reaction to mitigate the effects of reagent cross -reactivity and allow for highly- multiplexed assays.

[0006] In some aspects, the methods and systems based on PSD reactions described herein convert a signal from a non-nucleic acid molecule, e.g., a protein molecule, to a signal from a nucleic acid molecule. In doing so, the methods and systems described herein provide high sensitivity and high specificity for detecting, in parallel, at least one to thousands of proteins in the human proteome. The molecular complex for the PSD reaction can be performed in solution as standalone hybrid constructs or on a support, e.g., solid surfaces that can be amorphous or structured DNA nanostructures, or inorganic nanoparticles, gel matrices, semiconductor or nonsemiconductor chips, etc.. The described supports can be functionalized to harvest distinct advantages suiting multiple applications. Samples can be obtained from complex biological matrices ranging from single cells to organ systems. The solid surfaces can be thermo- andenzyme-stable and / or monofunctional or have other properties suitable for a variety of human and non-human proteomic applications. The readout can be a unique DNA barcode strand for every captured analyte that can be quantified using low-to-mid throughput qPCR or mid-to-high- throughput NGS systems with traditional enzyme-based or enzyme-free amplification.

[0007] In some aspects, disclosed herein is a method for analyzing a target molecule from a sample, comprising: a) contacting the target molecule with a first molecule and a second molecule, to form a complex, the first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR, the second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR, and the PR of the first molecule hybridizing to the C-PR of the second molecule; b) displacing an eluting nucleic acid molecule with the complex, thereby forming a reacted complex and releasing a product nucleic acid molecule from the eluting nucleic acid molecule, the eluting nucleic acid molecule comprising a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR, the product nucleic acid molecule comprising the D-SDR, the ALR, and the BR, and the reacted complex comprising the TR and the SDR of the complex hybridized to the C-TR and the C-SDR from a portion of the eluting nucleic acid molecule; c) purifying the product nucleic acid molecule; and d) measuring an amount of the product nucleic acid molecule.

[0008] In some aspects, disclosed herein is a method for generating a complex, comprising: a) contacting a target molecule with a first molecule and a second molecule, the first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR, and the second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR; and b) hybridizing the PR of the first molecule to the C-PR of the second molecule, to generate thecomplex. In some embodiments, the methods can further comprise: hybridizing the complex to a support, the first nucleic acid molecule comprising a first support-binding region SBR1, the support comprising a first complementary binding region C-SBR1, and the C-SBR1 hybridizing to the SBR1, thereby hybridizing the complex to the support.

[0009] In some aspects, disclosed herein is a plurality of molecules comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR; a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR; and the PR of the first molecule hybridized to the C-PR of the second molecule.

[0010] In some aspects, disclosed herein is an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D- SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

[0011] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR; a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR; the PR of the first molecule hybridized to the C-PR of the second molecule; and an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

[0012] In some aspects, disclosed herein is an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, acomplementary strand displacement region C-SDR, a duplicate strand displacement region D- SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

[0013] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR; a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR; the PR of the first molecule hybridized to the C-PR of the second molecule; and an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

[0014] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a reacted complex comprising a toehold region TR and a strand displacement region SDR of the complex hybridized to a complementary toehold region C-TR and a complementary strand displacement region C-SDR from a portion of the eluting nucleic acid molecule, and; a product nucleic acid molecule comprising a duplicate strand displacement region D-SDR, an anti-leak region ALR, and a barcode region BR.

[0015] In some aspects, disclosed herein is an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D- SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

[0016] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR; asecond molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR; the PR of the first molecule hybridized to the C-PR of the second molecule; and an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

[0017] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a reacted complex comprising a toehold region TR and a strand displacement region SDR of the complex hybridized to a complementary toehold region C-TR and a complementary strand displacement region C-SDR from a portion of the eluting nucleic acid molecule, and; a product nucleic acid molecule comprising a duplicate strand displacement region D-SDR, an anti-leak region ALR, and a barcode region BR.

[0018] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, a toehold region TR, and a first support-binding region SBR1; a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, a strand displacement region SDR, and a second support-binding region SBR2; a support comprising a first complementary support-binding region C-SBR1 or a second complementary support-binding region C-SBR2; the PR of the first molecule hybridized to the C-PR of the second molecule, and the SBR1 of the first molecule or the SBR2 of the second molecule hybridized to the C-SBR1 of the support or the C-SBR2 of the support; and an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary antileak region C-ALR, and a barcode region BR.

[0019] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a reacted complex comprising a toehold region TR, a support-binding region SBR, and a strand displacement region SDR of the complex hybridized to a complementary toehold region C-TR, a complementary support-binding region C-SBR, and a complementary strand displacement region C-SDR from a portion of the eluting nucleic acid molecule; and a product nucleic acid molecule comprising a duplicate strand displacement region D-SDR, an anti-leak region ALR, and a barcode region BR.

[0020] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, a toehold region TR, and a first loop-binding region LBR1; a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, a strand displacement region SDR, and a second loop-binding region LBR2; a nucleic acid loop comprising a first complementary loop-binding region C-LBR1 or a second complementary loop-binding region C-LBR2; the PR of the first molecule hybridized to the C-PR of the second molecule, and the LBR1 of the first molecule or the LBR2 of the second molecule hybridized to the C-LBR1 of the nucleic acid loop or the C-LBR2 of the nucleic acid loop; and an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR. In some embodiments, the methods disclosed herein further comprise a support comprising a sequence complementary to the nucleic acid loop.

[0021] In some aspects, disclosed herein is a system for analyzing a target molecule from a sample, comprising: a reacted complex comprising a toehold region TR, a support-binding region SBR, and a strand displacement region SDR of the complex hybridized to a complementary toehold region C-TR, a complementary loop-binding region C-BR, and a complementary strand displacement region C-SDR from a portion of the eluting nucleic acidmolecule; and a product nucleic acid molecule comprising a duplicate strand displacement region D-SDR, an anti-leak region ALR, and a barcode region BR.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Various aspects of the disclosed methods, devices, and systems are set forth with particularity in the appended claims. A better understanding of the features and advantages of the disclosed methods, devices, and systems will be obtained by reference to the following detailed description of illustrative embodiments and the accompanying drawings.

[0023] FIG. 1 provides exemplary methods for analyzing a target molecule by using a nucleic acid displacement reaction to measure an amount of a product nucleic acid molecule.

[0024] FIG. 2 provides exemplary methods for analyzing a target molecule by using a nucleic acid displacement reaction immobilized on a support to measure an amount of a product nucleic acid molecule.

[0025] FIG. 3 provides exemplary methods for analyzing a target molecule by using a nucleic acid displacement reaction immobilized on a nucleic acid loop to measure an amount of a product nucleic acid molecule.

[0026] FIG. 4 provides exemplary methods for establishing a proximity-assisted strand displacement reaction.

[0027] FIG. 5 provides exemplary methods for establishing a proximity-assisted strand displacement reaction on a support.

[0028] FIG. 6 provides exemplary methods for establishing a proximity-assisted strand displacement reaction on a nucleic acid loop.

[0029] FIG. 7A provides a schematic of a complex for a proximity-assisted strand displacement reaction. FIG. 7B provides a schematic of an eluting nucleic acid molecule for a proximity- assisted strand displacement reaction.

[0030] FIG. 8A provides a schematic of a reacted complex from a proximity-assisted strand displacement reaction. FIG. 8B provides a schematic of a product nucleic acid molecule from a proximity-assisted strand displacement reaction.

[0031] FIG. 9 provides a schematic of a complex reacting with an eluting nucleic acid molecule via a nucleic acid displacement reaction to generate a reacted complex and a product nucleic acid molecule.

[0032] FIG. 10 provides a schematic for establishing a proximity-assisted strand displacement reaction in solution, on a support, or a nucleic acid loop.

[0033] FIG. 11 provides a schematic for an experimental workflow for a proximity-assisted strand displacement reaction.

[0034] FIGS. 12A-12F provide a schematic for a proximity-assisted strand displacement reaction on a support.

[0035] FIGS. 13A-13F provide a schematic for a proximity-assisted strand displacement reaction on a nucleic acid loop on a support.DETAILED DESCRIPTION

[0036] Methods and systems for analyzing a target molecule from a sample are described. First, the target molecule can be contacted with a first molecule and a second molecule, to form a complex. The first molecule can include a first binder molecule and a first nucleic acid molecule. The first nucleic acid molecule can include a first spacer region SRI, a priming region PR, and a toehold region TR. The second molecule can include a second binder molecule and a second nucleic acid molecule. The second nucleic acid molecule can include a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR. The first molecule’s PR can hybridize to the second molecule’s C-PR. Then, the complex can be displaced by an eluting nucleic acid molecule, to form a reacted complex. In doing so, the product nucleic acid molecule can be released from the eluting nucleic acid molecule. The eluting nucleic acid molecule can include a tag, a third spacer region SR3, a complementarytoehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR. The product nucleic acid molecule can include the D-SDR, the ALR, and the BR. The reacted complex can include the TR and the SDR of the complex hybridized to the C-TR and the C-SDR from a part of the eluting nucleic acid molecule. The product nucleic acid molecule can then be purified and measured.

[0037] Existing methods for detecting and measuring non-nucleic acid molecules, e.g., protein molecules, cannot be easily multiplexed and or analyzed on a large scale. In contrast, nucleic acid molecules are routinely analyzed via easily scalable and massively parallel sequencing technologies. The methods and systems described herein incorporate nucleic acid sequencing techniques into a method for analyzing non-nucleic acid molecules, such as proteins. In doing so, the methods and systems described herein extend the benefits native to sequencing techniques, such as an ease of multiplexing and scalability, to a method for analyzing non-nucleic acid molecules, such as proteins. By measuring the amount of a product nucleic acid molecule released from nucleic acid displacement reactions, the methods and systems described herein can approximate the amount of a target molecule.

[0038] The detecting and measuring of protein molecules especially have longstanding shortcomings. For example, conventional immunoassays face limitations in their multiplexing ability, because cross -reactive binding of antibodies can contaminate the signal readout. This issue becomes more pronounced as the degree of multiplexing increases. In contrast, the methods and systems based on proximity-assisted strand displacement (PSD), as described herein, overcome such limitations by requiring the dual recognition of correctly matched first and second molecules, e.g., PSD probes (DNA-labeled affinity binders). Furthermore, the DNA sequence-specific protein-to-DNA conversion underlying the PSD reaction reduces the probability of signal due to cross-reactivity. The PSD reaction can be immobilized on a support, to potentially further improve the signal-to-noise ratio. The methods and systems described herein provides a highly scalable, programmable method to increase the sensitivity and specificity of detecting non-nucleic acid molecules, such as proteins.

[0039] The requirements for protein biomarker analysis can vary greatly across different applications, ranging from targeted investigations that require ultra-high sensitivity measurement to broad studies aimed at screening large numbers of samples against many different proteins simultaneously. The PSD-based methods and systems described herein are highly multiplexed and scalable, and accordingly, provides robust, highly sensitive, and reproducible measurements of one to one thousand or more protein species, simultaneously, in a single experiment. Furthermore, the PSD-based techniques described herein allow for mid-to-high throughput readout by NGS or low-to-mid throughput readout using qPCR. Both throughputs significantly expand the protein library and provide a more cost-efficient solution for large studies. The methods and systems described herein provide a highly sensitive, high-throughput, and highly specific approach for detecting low-abundance proteins that are difficult to measure using traditional immunoassays. In addition, the PSD reaction operates based on favorable thermodynamics, such that an enzyme is not necessary to drive the reactants to react and generate products. Given that PSD reactions do not require enzymes, the methods and systems described herein are robust, and cannot fail due to enzyme denaturation that may arise during improper transport or storage — enzymes can irreversibly denature at room temperature. The robustness, high-sensitivity, and multiplexing capacity of the PSD reaction-based methods and systems described herein constitute numerous advantages over traditional methods of measuring non-nucleic acid molecules, such as protein molecules.

[0040] The described systems and methods are highly sensitive in detecting the target molecule, e.g., protein, while providing high signal-to-noise ratios for the measured signals that indicate the target molecule amounts. The systems and methods described herein take advantage of the exponential amplification properties of enzyme-based or enzyme-free techniques to achieve a strong readout signal, which provides assay sensitivity on par or better than traditional enzyme- linked immunosorbent assays (ELIS As). Furthermore, the systems and methods described herein use extremely small sample volumes to measure large numbers of proteins simultaneously, making them highly beneficial in cases where precious samples are in limited supply, such as in studies that use human samples from clinical cohorts or biobank material. The systems and methods described herein are expected to use only 5-10 uL of sample for its assays, regardless ofthe scale required. Additionally, low sample volumes reduce the concentration of potentially interfering substances, and specifically tailored blocking reagents in the methods described herein can further minimize sample matrix interference.

[0041] The PSD reaction-based methods and systems described herein are suitable for a broad range of applications, ranging from highly targeted, hypothesis-driven studies, to the largest screening projects. For largescale projects, next generation sequencing (NGS) readouts are suitable, for example, for conducting high-throughput studies with large numbers of human serum or plasma samples against the complete library of proteins. This technology can provide a highly specific and sensitive approach for detecting low-abundance proteins, which can allow researchers to search for actionable protein signatures within a low-abundant proteome. For small- and medium-scale projects, e.g., highly targeted, hypothesis-driven studies, qPCR readouts can be used. Doing so provides a high-quality flexible solution for more targeted investigations, by using one or several panels most relevant to the subject of study, or when sample matrices other than serum or plasma are measured. The systems and methods described herein can provide a highly specific and scalable approach to detect and measure the expression levels of multiple non-nucleic acid molecules, e.g., proteins, simultaneously. Enzyme-free isothermal approaches such as hybridization chain reaction, rolling circle amplification, or other approaches, can also be used as a readout for the systems and methods described herein.

[0042] The PSD reaction-based systems and methods described herein can be used by clinicians for liquid biopsy investigations to improve disease detection, aid more personalized healthcare, and allow a better understanding of real-time human biology. The systems and methods described herein comprise a combination of immunoassay and functional DNA nanotechnology, to provide a powerful technique for proteomics within research and clinical settings. The systems and methods described herein merge an affinity binder-based immunoassay with highly efficient DNA strand displacement combined with the ubiquity of efficient amplification reactions. Furthermore, the described systems and methods offer readout options using either quantitative real-time PCR (qPCR) or next generation sequencing (NGS). The flexibility of the readout thatcan be used results in a highly specific, scalable, and multiplexed method for simultaneously quantifying one to thousands of protein biomarkers.

[0043] The complex at the core of the PSD reaction-based methods comprises a first and second molecule that can assume the form of two matched, DNA-conjugated affinity binder pairs — e.g., NHS-amine, DBCO-azide, or other approaches for monofunctional affinity binder-DNA complexes — with partial complementarity over their DNA domains. Two such affinity binders can be used to detect any given protein analyte of interest. While floating freely in solution, these reagents may interact with other affinity reagents. Still, the DNA domains are designed specifically to keep any one pair as orthogonal from the others as possible. In addition, the designed orthogonality can be tuned to not limit the workflow and operations of the methods and systems based on PSD reactions.

[0044] When analyzing, e.g., detecting or measuring, a target molecule such as a non-nucleic acid molecule, e.g., protein molecule, the freely diffusing matched pairs (i.e., the first and second molecules) can find their binding domains on the target molecule and form a two-lever arm complex based on the proximity of the DNA domains. In the absence of the target molecule, the first and second molecules would not form a stable complex at room temperature. The forming of the complex is followed by the introduction of a single population or multiple sets of “eluting strands” containing multiple domains. The domains of the eluting strand and / or the complex can comprise the following components: a) Biotin or other tags: The tag, e.g., biotin, pulls down the strand-displaced three-way junction away from the released barcode. b) Spacer: The spacer region (e.g., SRI, SR2, SR3, or SR4) can be a Poly-T, PEG, or other such domain. c) Toehold: The toehold region (e.g., TR) induces the displacement and release of the barcode region by interacting and hybridizing with the immunocomplex formed after the introduction of the target analyte. d) Strand displacement region: The encroachment of the toehold onto the two-lever molecular construct begins the process of unraveling the strand displacement region (e.g., SDR) fromits original position, with its complementary strand (associated with the toehold region) strand hopping to form a three-way junction on the previously two-junction immunocomplex construct and resulting in the release of the unique barcode associated with the target molecule. e) Anti-leak region: The anti-leak region allows for the displacement of the two-way junction at a later timepoint and is an indirect way of determining the concentration of unique elution strands consumed during the reaction, thereby quantifying the target analyte. f) Barcode region: The barcode region (e.g., BR) is uniquely assigned to each target molecule. Therefore, amplifying and sequencing the barcode region allows for the determining of the target molecule’s identity. Given that the barcode sequence is a nucleic acid sequence, potentially unlimited permutations of the sequence space are available for scaling up the technology, so that multiplexed studies can be performed at arbitrarily large or small levels. The barcode region is read using qPCR, NGS, or other enzyme-free methods to identify the sequence associated with every construct. The barcode region allows for the transduction of a protein signal into a DNA signal. The PSD reactions can be interpreted as comprising a highly efficient biosensor and transducer.

[0045] The components described above can then operate according to the following: A) complex formation, B) binder proximity and nucleic acid strand displacement, and C) barcode release and amplification. The target molecule, e.g., protein, can be bound by the first molecule and the second molecule, in order to generate the complex. The formed complex can be stable either independently in solution or on a support. The complex can then be subject to strand displacement, such that a stable three-way junction is formed due to the proximity of the affinity binder pairs (e.g., the first binder molecule and the second binder molecule) and the partial complementarity in the DNA extensions. The strand displacement reaction can result in barcode release, and the barcode can be isolated from the construct for amplification using enzyme-based or enzyme-free approaches via specifically designed primers, dNTPs, and polymerases via thermal cycling or under isothermal reaction conditions. Depending on a target molecule’s expected or known concentration, a dilution factor may be introduced, or amplification may not be necessary. The DNA barcode can then be read out using qPCR or NGS approaches, depending on the number of unique barcodes generated through the assay.Definitions

[0046] Unless otherwise defined, all of the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field to which this disclosure belongs.

[0047] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0048] ‘ ‘About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0049] As used herein, the terms "comprising" (and any form or variant of comprising, such as "comprise" and "comprises"), "having" (and any form or variant of having, such as "have" and "has"), "including" (and any form or variant of including, such as "includes" and "include"), or "containing" (and any form or variant of containing, such as "contains" and "contain"), are inclusive or open-ended and do not exclude additional, un-recited additives, components, integers, elements, or method steps.

[0050] As used herein, ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0051] As used herein, the term “region” refers to a portion of a nucleic acid molecule that can be either single-stranded or double-stranded. A double-stranded region of a nucleic acid molecule can refer to two single-stranded nucleic acid molecules that are hybridized to each other. A region, e.g., a toehold region, can at one point over the course of a method, be a singlestranded region, and at another point over the course of the method, be a double- stranded region, and the region, e.g., toehold region, can still be referred to as the region, e.g., the toehold region, although further clarification regarding the strandedness of the region, e.g., the toehold region, can be provided. A double-stranded region of a nucleic acid molecule can be referred to by its constituent single- single-stranded regions. For example, the strand displacement region, which can be double- stranded, can be referred to by its constituent single- stranded strand displacement region SDR, or its constituent single-stranded strand displacement region SDR’, and SDR and SDR’ can be strands that have hybridized to each other.

[0052] As used herein, the prefix “C-” refers to a sequence that is complementary to another sequence. Accordingly, as an example, “C-A” refers to the complementary sequence to “A”.

[0053] As used herein, the prefix “D” refers to a sequence that is a duplicate of another sequence. Accordingly, as an example, “D-A” refers to the duplicate sequence of “A”.

[0054] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. The description is presented to enable one of ordinary skill in the art to make and use the invention and is provided in the context of a patent application and its requirements. Various modifications to the described embodiments will be readily apparent to those persons skilled in the art and the generic principles herein may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiment shown but is to be accorded the widest scope consistent with the principles and features described herein.

[0055] The figures illustrate processes according to various embodiments. In the exemplary processes, some blocks are, optionally, combined, the order of some blocks is, optionally, changed, and some blocks are, optionally, omitted. In some examples, additional steps may beperformed in combination with the exemplary processes. Accordingly, the operations as illustrated (and described in greater detail below) are exemplary by nature, and, as such, should not be viewed as limiting.Methods for analyzing molecules based on nucleic acid displacement reactions

[0056] The study of non-nucleic acid molecules, such as proteins, is an important area of focus in real-time human biology, following the advances in genomics. Identifying protein biomarkers through discovery allows for detecting signatures with pathophysiological significance and connects the gap between genomes and phenotypes. Progress in this field, however, has been hindered by a shortage of technologies that can provide high specificity, sensitivity, precision, dynamic range, and throughput, which are all essential for reliable results.

[0057] Conventional immunoassays face limitations in their multiplexing ability, e.g., the number of proteins or other target molecules that can be detected in a single assay, because cross-reactive binding of affinity ligands floating freely in the solution can contaminate the signal readout. This issue becomes more pronounced as the degree of multiplexing increases. In contrast, the methods and systems described herein differentiate itself by using strand displacement reactions. It provides a high signal-to-noise ratio by requiring the dual and simultaneous recognition of correctly matched proximity-assisted strand displacement (PSD) reaction probes (e.g., a first molecule and a second molecule, such as DNA-labeled affinity ligands) to the target molecule, e.g., protein, to result in a signal. Further, both probes can be immobilized in close proximity on solid phase supports either before or after complex formation so that they are likely to not interact with other PSD probes, thereby further reducing crosstalk. The combination of both these approaches reduces the probability of contaminated signal due to cross-reactivity. The result is a highly scalable, programmable method to increase specificity and aid multiplexed assays.

[0058] FIG. 1 shows an exemplary schematic showing a general process 100 for analyzing a target molecule from a sample. The method can include: contacting the target molecule with a first molecule and a second molecule, to form a complex, the first molecule comprising a firstbinder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR, the second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR, and the PR of the first molecule hybridizing to the C-PR of the second molecule (102); displacing an eluting nucleic acid molecule with the complex, thereby forming a reacted complex and releasing a product nucleic acid molecule from the eluting nucleic acid molecule, the eluting nucleic acid molecule comprising a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C- ALR, and a barcode region BR, the product nucleic acid molecule comprising the D-SDR, the ALR, and the BR, and the reacted complex comprising the TR and the SDR of the complex hybridized to the C-TR and the C-SDR from a portion of the eluting nucleic acid molecule (104); purifying the product nucleic acid molecule (106); and measuring an amount of the product nucleic acid molecule (108).

[0059] At 102 in FIG. 1, the target molecule is contacted with a first molecule and a second molecule, to form a complex. The first molecule can comprise a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR, the second molecule can comprise a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR, and the PR of the first molecule can hybridize to the kc- PR of the second molecule.

[0060] At 104 in FIG. 1, an eluting nucleic acid molecule is displaced with the complex, thereby forming a reacted complex and releasing a product nucleic acid molecule from the eluting nucleic acid molecule. The eluting nucleic acid molecule can comprise a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D- SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode regionBR, the product nucleic acid molecule comprising the D-SDR, the ALR, and the BR, and the reacted complex comprising the TR and the SDR of the complex hybridized to the C-TR and the C-SDR from a portion of the eluting nucleic acid molecule.

[0061] At 106 in FIG. 1, the product nucleic acid molecule is purified.

[0062] At 108 in FIG. 1, an amount of the product nucleic acid molecule is measured.Surface immobilization

[0063] The complex, e.g., the two-lever arm molecular construct, can be hosted on a variety of solid surfaces to allow for the molecular manipulation through external fields such as magnetic, electric, electrochemical, or other types of fields. The solid surfaces can range from several square nanometers to several square millimeters in their surface areas. Some examples of potential surfaces can be amorphous, such as DNA scaffolds, and others can comprise structured DNA nanostructures, such as DNA origami. Other examples of potential surfaces can comprise inorganic nanoparticles (e.g., magnetically active, fluorescently labeled nanoparticles), gel matrices, or functionalized or unfunctionalized semiconductor chips. The two arms of the complex, e.g., the first nucleic acid molecule and the second nucleic acid molecule of the complex, can be attached to the surfaces using DNA, PEG-DNA, NHS-amine, azide-DBCO, maleimide-thiol, or other chemistries. The surfaces can additionally have linker moieties such as biotin, PEG, poly-l-lysine, amines, azides, DBCO, or photocleavable ligands (photocleavable- PEG-biotin-NHS or other such chemistries). The two arms, e.g., the first and second nucleic acid molecules of the complex, can be immobilized on the solid phase surfaces prior to or after the introduction of the protein analyte of interest for operational reasons. For example, the immobilization on the solid phase surface may mitigate the cross -reactivity observed between free-floating affinity binders when the sample is introduced into the solution. Some of the features of the solid surfaces can comprise:1. Shape and material flexibility2. Several orders of magnitude surface area: few sq. nanometers to few sq. millimeters3. Fluorescence, autofluorescence, or light scattering4. Enzyme- and thermal-stability5. Electrically conductive or non-conductive6. Magnetically active7. Mono- or poly-functional with control over the number and pattern of ligands bound8. Compatibility with existing or novel flow cells (patterned or un-pattemed), microfluidic devices, and well plates (96-well or 384-well).

[0064] The immobilizing of the complex before or after strand displacement onto DNA or other solid phase supports may depend on the number of analytes to be detected and the orthogonality between the various binders involved. The immobilizing surface may be monofunctional, thermostable, both, or have unique material or functional properties. In some cases, an intermediate step of transferring strand-displaced complexes to other solid phase surfaces may be introduced for the purposes of separation, purification, signal-to-noise improvement, or similar.Surface composition

[0065] The solid surfaces could be composed of functional moieties arranged in a patterned or unpattemed fashion using lithographic or other techniques. The functional moieties can be responsible for linkage to the complex’s lever arms (e.g., the first and second nucleic acid molecules) or other solid surfaces. The functional moieties can be interspersed with passivating moieties of various shapes, sizes, charges, to boost the signal to noise ratio (and thereby sensitivity) and may include, but not be limited to, any one of the following in singular or in combination:1. DNA spacers such as Poly T with or without other functional moieties;2. PEG with or without DNA or other linkers ranging from a few hundred to a few thousand repeating units;3. Silicon or Silica-based chemistries;4. Zwitterionic molecules;5. Bovine Serum Albumin (BSA);6. Sheared Salmon Sperm DNA.EXAMPLES

[0066] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.Example 1

[0067] This section provides an example protocol that can be used for analyzing a target molecule in accordance with the systems and methods described herein.Materials for protocol• Two matched affinity binders, e.g., antibodies, specific to orthogonal epitopes on the target molecule, e.g., protein. o IL-6■ Capture - M AB 206■ Non-biotinylated poly - AF-206-NA o TNF-alpha■ Capture - M AB 61 OR■ Non-biotinylated poly - AF-210-NA o PSA■ Capture - MAB 13442■ Non-biotinylated poly - AF 1344Amine-modified DNA oligonucleotides with partial complementarity l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) N-Hydroxysuccinimide (NHS)• Target protein• Eluting strand containing a biotin tag, spacer, toehold, strand displacement region, and a unique barcode• Streptavidin-coated magnetic beads• PCR reagents (primers, dNTPs, DNA polymerase, buffer)• qPCR instrument or NGS platformProtocol1. Conjugate the two matched affinity binders to their respective amine-modified DNA oligonucleotides using EDC / NHS chemistry: a. Activate the carboxyl groups on the antibodies by mixing them with EDC (2 mM) and NHS (5 mM) in MES buffer (pH 6.0) for 15 minutes at room temperature. b. Add the amine-modified DNA oligonucleotides (10 pM) to the activated antibodies and incubate for 2 hours at room temperature. c. Quench the reaction by adding ethanolamine or hydroxylamine to a final concentration of 10 mM and incubate for 10 minutes at room temperature. d. Purify the conjugates using size-exclusion chromatography or desalting columns to remove unconjugated DNA and ensure monofunctional coupling. This step may take 1-2 hours, depending on the purification method.2. Mix the two affinity binder-DNA conjugates (50 nM each) in a buffer suitable for antigen binding (e.g., PBS with 0.1% BSA). Incubate for 30 minutes at room temperature to allow the formation of the molecular construct.3. Add the target protein to the mixture at a concentration within the dynamic range of the assay (e.g., 1 pM to 10 nM). Incubate for 1 hour at room temperature to allow the formation of the immunocomplex.4. Introduce the elution strand (100 nM) containing the biotin tag, spacer, toehold, strand displacement region, and unique barcode. Incubate for 30 minutes at room temperature to allow strand displacement and barcode release.5. Capture the biotin-tagged three-way junction complex using streptavidin-coated magnetic beads. Incubate for 15 minutes at room temperature.6. Separate the magnetic beads using a magnet and collect the supernatant containing the released barcode. This step should take about 5 minutes.7. Quantify the barcodes using either qPCR or next generation sequencing (NGS): a. For qPCR: i. Prepare the qPCR reaction mix with the appropriate primers, dNTPs, DNA polymerase, and buffer. Use the following thermal cycling conditions:1. Initial denaturation: 95°C for 5 minutes.2. 40 cycles of: a. Denaturation: 95°C for 15 seconds b. Annealing / Extension: 60°C for 1 minute ii. Analyze the qPCR data to determine the concentration of the target protein in the sample based on the quantification of the barcode. iii. The qPCR step should take around 1.5 to 2 hours. b. For NGS: i. Prepare the sequencing library by adding platform- specific adapters to the amplified barcodes following the manufacturer's protocol. This step may take 2-4 hours, depending on the library preparation method. ii. Perform sequencing on the chosen NGS platform (e.g., Illumina). Sequencing time varies based on the platform and read length, but generally takes 1-2 days. iii. Analyze the sequencing data to identify and quantify the unique barcodes associated with the target protein. Data analysis time depends on the complexity of the data and the bioinformatics pipeline used.

[0068] It should be understood from the foregoing that, while particular implementations of the disclosed methods and systems have been illustrated and described, various modifications can be made thereto and are contemplated herein. It is also not intended that the invention be limited bythe specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the preferable embodiments herein are not meant to be construed in a limiting sense. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a person skilled in the art. It is therefore contemplated that the invention shall also cover any such modifications, variations and equivalents.

Claims

CLAIMSWhat is claimed is:

1. A method for analyzing a target molecule from a sample, comprising: a) contacting the target molecule with a first molecule and a second molecule, to form a complex, the first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR, the second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR, and the PR of the first molecule hybridizing to the C-PR of the second molecule; b) displacing an eluting nucleic acid molecule with the complex, thereby forming a reacted complex and releasing a product nucleic acid molecule from the eluting nucleic acid molecule, the eluting nucleic acid molecule comprising a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR, the product nucleic acid molecule comprising the D-SDR, the ALR, and the BR, andthe reacted complex comprising the TR and the SDR of the complex hybridized to the C-TR and the C-SDR from a portion of the eluting nucleic acid molecule; c) purifying the product nucleic acid molecule; and d) measuring an amount of the product nucleic acid molecule.

2. The method of claim 1, further comprising contacting the first molecule with a support or contacting the second molecule with the support.

3. The method of claim 1, further comprising contacting the first molecule with a nucleic acid loop or contacting the second molecule with the nucleic acid loop.

4. The method of claim 3, wherein the nucleic acid loop contacts the support and the complex.

5. The method of any of claims 1-4, wherein the method is catalyst- free.

6. The method of any of claims 1-4, wherein the method comprises a catalyst.

7. The method of claim 6, wherein the catalyst is an enzyme.

8. The method of claim 7, wherein the catalyst is a poly (l-lysine)-graft dextran polymer.

9. The method of any of claims 1-8, wherein the method comprises contacting the target molecule with a competitor molecule.

10. The method of any of claims 1-9, wherein the portion of the eluting nucleic acid molecule comprises the tag, the SR3, the C-TR, the SR4, the C-SDR, and the C-ALR.

11. The method of any of claims 1-10, wherein the first molecule comprises an antibody or an aptamer.

12. The method of any of claims 1-11, wherein the second binder molecule comprises an antibody or an aptamer.

13. The method of any of claims 1-12, wherein the first binder molecule and the first nucleic acid molecule are bound together by a N-hydroxysuccinimide (NHS)-amine bond or a dibenzocyclooctyne (DBCO)-azide bond.

14. The method of any of claims 1-13, wherein the second binder molecule and the second nucleic acid molecule are bound together by a N-hydroxysuccinimide (NHS)-amine bond or a dibenzocyclooctyne (DBCO)-azide bond.

15. The method of any of claims 1-14, wherein the tag comprises biotin, FLAG-tag, HA-tag, His-tag, Myc-tag V5-tag, HaloTag, SNAP-tag, CLIP-tag, or a combination thereof.

16. The method of any of claims 1-15, wherein the SRI, SR2, SR3, or SR4 comprises a poly- T spacer or a spacer 18 spacer.

17. The method of any of claims 1-16, wherein the TR, the C-TR, the PR, the C-PR, the SRI, the SR2, the SR3, the SR4, the C-PR, the SDR, the C-SDR, the D-SDR, the ALR, or the C-ALR is a single- stranded nucleic acid.

18. The method of any of claims 1-17, wherein the BR is a double- stranded nucleic acid.

19. The method of any of claims 1-18, wherein the TR is complementary in sequence to the C-TR, the PR is complementary in sequence to C-PR, the SDR is complementary in sequence to C-SDR, the SDR is complementary in sequence to D-SDR, or the ALR is complementary in sequence to the C-ALR.

20. The method of any of claims 1-19, wherein the D-SDR is identical in sequence to the SDR.

21. The method of any of claims 1-20, wherein the support comprises a molecular layer.

122. The method of claim 21, wherein the molecular layer comprises a PEG chain, a DNA strand, or a photocleavable group.

23. The method of any of claims 2-22, wherein the support is a particle.

24. The method of claim 23, wherein the particle is a nanoparticle or a microparticle.

25. The method of claim 24, wherein the support is a DNA nanostructure.

26. The method of claim 25, wherein the DNA nanostructure is an amorphous DNA nanostructure or a structured DNA nanostructure.

27. The method of claim 26, wherein the DNA nanostructure is a DNA origami structure.

28. The method of claim 26, wherein the amorphous DNA nanostructure is a bacteriophage DNA scaffold.

29. The method of claim 28, wherein the bacteriophage DNA scaffold is M13mpl8.

30. The method of any of claims 2-29, wherein the support is a chip.

31. The method of claim 30, wherein the chip is a semi-conductor surface or a flow cell.

32. The method of any of claims 2-31, wherein the support is a porous gel membrane.

33. The method of any of claims 2-32, wherein the support comprises a mono-functional group, a poly-functional group, or a combination thereof.

34. The method of any of claims 2-33, wherein the support comprises a fluorescent group.

35. The method of claim 34, wherein the support comprises DNA spacers, polyethylene glycol (PEG), silicon or silica-based groups, zwitterionic molecules, bovine serum albumin (BSA), sheared salmon sperm DNA, or any combination thereof.

36. The method of claim 35, wherein the DNA spacers comprise a poly-thymidine (poly-T) motif.

37. The method of claim 35, wherein the PEG comprises DNA linkers.

38. The method of any of claims 2-37, wherein the support comprises 0 to 1000 groups.

39. The method of any of claims 2-38, wherein the support is enzyme-stable.

40. The method of any of claims 2-39, wherein the support is thermal- stable.

41. The method of any of claims 2-40, wherein the support is electrically conductive.

42. The method of any of claims 2-41, wherein the support is magnetically active.

43. The method of any of claims 1-42, wherein the purifying comprises a pull-down assay.

44. The method of claim 43, wherein the pull-down assay comprises an SDS-PAGE assay.

45. The method of any of claims 1-44, wherein the measuring comprises measuring a digital signal based on the amount of the product nucleic acid molecule.

46. The method of claim 45, wherein the digital signal is a binary signal.

47. The method of any of claims 1-46, wherein the measuring comprises measuring an analog signal based on the amount of the product nucleic acid molecule.

48. The method of any of claims 1-47, wherein the measuring the amount of the product nucleic acid molecule comprises detecting the BR.

49. The method of any of claims 1-48, wherein the measuring the amount of the product nucleic acid molecule comprises detecting the ALR or detecting the BR, after the purifying the product nucleic acid molecule.

50. The method of any of claims 1-49, wherein the measuring the amount of the product nucleic acid molecule comprises fluorescence microscopy.

51. The method of any of claims 1-50, wherein the measuring the amount of the product nucleic acid molecule comprises measuring the amount of the product nucleic acid molecule with a bait set.

52. The method of claim 51, wherein the measuring the amount of the product nucleic acid molecule with the bait set comprises amplifying the product nucleic acid molecule.

53. The method of claim 52, wherein the amplifying the product nucleic acid molecule comprises quantitative polymerase chain reaction (qPCR) amplification.

54. The method of any of claims 51-53, wherein the measuring the amount of the product with the bait set comprises rolling circle amplification (RCA) or hybridization chain reaction.

55. The method of any of claims 51-54, wherein the measuring the amount of the product nucleic acid molecule with the bait set comprises sequencing.

56. The method of claim 55, wherein the sequencing comprises sequencing a portion of the BR.

57. The method of claim 56, wherein the sequenced portion of the BR corresponds to the target molecule.

58. The method of any of claims 55-57, wherein the sequencing comprises Sanger sequencing.

59. The method of any of claims 55-58, wherein the sequencing comprises next- generation sequencing.

60. The method of claim 59, wherein the next-generation sequencing comprises sequencing by synthesis, sequencing by hybridization, or sequencing by avidity.

61. The method of any of claims 51-60, wherein the measuring the amount of the product nucleic acid molecule with the bait set comprises hybridizing the product nucleic acid molecule to a probe molecule.

62. The method of claim 61, wherein the hybridizing the product nucleic acid molecule to a probe molecule comprises using a microarray.

63. The method of any of claims 1-62, wherein the sample is the product of an in vitro assay.

64. The method of claim 63, wherein the in vitro assay is an in vitro protein expression assay.

65. The method of any of claims 1-64, wherein the sample is from a subject.

66. The method of claim 65, wherein the subject is a human.

67. The method of any of claims 1-66, wherein the sample comprises a tissue biopsy sample.

68. The method of any of claims 1-67, wherein the sample comprises a liquid biopsy sample.

69. The method of claim 68, wherein the liquid biopsy sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

70. The method of any of claims 1-69, wherein the sample comprises 5 pL or less.

71. A method for generating a complex, comprising: a) contacting a target molecule with a first molecule and a second molecule, the first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR, and the second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR; andb) hybridizing the PR of the first molecule to the C-PR of the second molecule, to generate the complex.

72. The method of claim 71, further comprising: hybridizing the complex to a support, the first nucleic acid molecule comprising a first support-binding region SBR1, the support comprising a first complementary binding region C-SBR1, and the C-SBR1 hybridizing to the SBR1, thereby hybridizing the complex to the support.

73. The method of claim 71 or 72, wherein the second nucleic acid molecule comprises a second support-binding region SBR2, the support comprises a second complementary binding region C-SBR2, and the C-SBR2 hybridizes to the SBR2, thereby hybridizing the complex to the support.

74. The method of any of claims 71-73, comprising: hybridizing a nucleic acid loop to the support; and hybridizing the nucleic acid loop to the complex.

75. The method of any of claims 71-74, wherein the first binder molecule and the first nucleic acid molecule are bound together by a N-hydroxysuccinimide (NHS)-amine bond or a dibenzocyclooctyne (DBCO)-azide bond.

76. The method of any of claims 71-75, wherein the second binder molecule and the second nucleic acid molecule are bound together by a N-hydroxysuccinimide (NHS)-amine bond or a dibenzocyclooctyne (DBCO)-azide bond.

77. The method of any of claims 72-76, wherein the support is a particle.

78. The method of claim 77, wherein the particle is a nanoparticle or a microparticle.

79. A plurality of molecules comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR; a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR; and the PR of the first molecule hybridized to the C-PR of the second molecule.

80. An eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

81. A system for analyzing a target molecule from a sample, comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, and a toehold region TR;a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, and a strand displacement region SDR; the PR of the first molecule hybridized to the C-PR of the second molecule; and an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

82. A system for analyzing a target molecule from a sample, comprising: a reacted complex comprising a toehold region TR and a strand displacement region SDR of the complex hybridized to a complementary toehold region C-TR and a complementary strand displacement region C-SDR from a portion of the eluting nucleic acid molecule, and; a product nucleic acid molecule comprising a duplicate strand displacement region D-SDR, an anti-leak region ALR, and a barcode region BR.

83. A system for analyzing a target molecule from a sample, comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, a toehold region TR, and a first support-binding region SBR1; a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, a strand displacement region SDR, and a second support-binding region SBR2;a support comprising a first complementary support-binding region C-SBR1 or a second complementary support-binding region C-SBR2; the PR of the first molecule hybridized to the C-PR of the second molecule, and the SBR1 of the first molecule or the SBR2 of the second molecule hybridized to the C- SBR1 of the support or the C-SBR2 of the support; and an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

84. A system for analyzing a target molecule from a sample, comprising: a reacted complex comprising a toehold region TR, a support-binding region SBR, and a strand displacement region SDR of the complex hybridized to a complementary toehold region C-TR, a complementary support-binding region C-SBR, and a complementary strand displacement region C-SDR from a portion of the eluting nucleic acid molecule; and a product nucleic acid molecule comprising a duplicate strand displacement region D-SDR, an anti-leak region ALR, and a barcode region BR.

85. A system for analyzing a target molecule from a sample, comprising: a first molecule comprising a first binder molecule and a first nucleic acid molecule comprising a first spacer region SRI, a priming region PR, a toehold region TR, and a first loop-binding region LBR1;a second molecule comprising a second binder molecule and a second nucleic acid molecule comprising a second spacer region SR2, a complementary priming region C-PR, a strand displacement region SDR, and a second loop-binding region LBR2; a nucleic acid loop comprising a first complementary loop-binding region C- LBR1 or a second complementary loop-binding region C-LBR2; the PR of the first molecule hybridized to the C-PR of the second molecule, and the LBR1 of the first molecule or the LBR2 of the second molecule hybridized to the C- LBR1 of the nucleic acid loop or the C-LBR2 of the nucleic acid loop; and an eluting nucleic acid molecule comprising: a tag, a third spacer region SR3, a complementary toehold region C-TR, a fourth spacer region SR4, a complementary strand displacement region C-SDR, a duplicate strand displacement region D-SDR, an anti-leak region ALR, a complementary anti-leak region C-ALR, and a barcode region BR.

86. The system of claim 85, further comprising a support comprising a sequence complementary to the nucleic acid loop.

87. A system for analyzing a target molecule from a sample, comprising: a reacted complex comprising a toehold region TR, a support-binding region SBR, and a strand displacement region SDR of the complex hybridized to a complementary toehold region C-TR, a complementary loop-binding region C-BR, and a complementary strand displacement region C-SDR from a portion of the eluting nucleic acid molecule; and a product nucleic acid molecule comprising a duplicate strand displacement region D-SDR, an anti-leak region ALR, and a barcode region BR.

88. A kit comprising the first molecule and the second molecule of any of claims 1-87, and instructions for administering the first molecule and the second molecule to a sample.

89. The kit of claim 88, further comprising a blocking buffer reagent.

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