Compositions and methods for detecting binding interactions under equilibrium or non-equilibrium conditions

Abstract

Provided are methods of detecting analytes. In some configurations, the methods can employ analytes attached to a solid support or particle and affinity reagents that are attached to the solid support or particle via a flexible linker. In some configurations, the methods can employ analytes attached to a solid support or particle and solution-phase affinity reagents can be attracted to the analytes via application of a stimulus.

Description

COMPOSITIONS AND METHODS FOR DETECTING BINDING INTERACTIONS UNDER EQUILIBRIUM OR NON-EQUILIBRIUM CONDITIONSCROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63 / 578,624, filed on Aug. 24, 2023, and U.S. Provisional Application No. 63 / 640,664, filed on Apr. 30, 2024, each of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 21, 2024, is named SL_50109_4023WO.xml and is 1,780 bytes in size.BACKGROUND

[0003] Affinity reagents include a broad class of reagents that form detectable interactions with analytes and other molecules. Affinity reagents can be configured to form reversible bound complexes with analytes. The observation of binding between an affinity reagent and analyte can provide useful information for characterizing the structure and properties of the analyte based on known recognition properties of the affinity reagent. Substantial resources and effort are typically invested into producing affinity reagents that bind to analytes of interest with sufficient specificity to distinguish the analytes in complex mixtures, such as biopsy samples, which typically contain a large number and variety of proteins, nucleic acids and other biochemical analytes. The specificity and strength of binding between an affinity reagent and analyte can be heavily influenced by small changes in conditions used for the assay such as changes in temperature, ionic strength, pH, and concentration of affinity reagent and analyte. This combination of factors can constrain the design of multiplex assays in which a large number of different analytes are to be evaluated, in parallel, for binding to a given affinity reagent. The present disclosure provides compositions and methods that improve binding assays and provide advantages that extend to multiplexed formats. Other advantages are provided as well.SUMMARY

[0004] The present disclosure provides a method of detecting analytes. The method can include steps of (a) providing a mixture of analytes with affinity reagents, wherein the analytes are immobilized on a surface and the affinity reagents are in fluid phase; (b) applying a stimulus to attract the affinity reagents to the surface; and (c) detecting binding of affinity reagents to analytes on the surface. For ease of explanation, several configurations of the method will be exemplified herein in the context of using immobilized analytes and fluid-phase affinity reagents. However, it will be understood that the method can be carried out using immobilized affinity reagents and fluid-phase analytes. Accordingly, the method can include steps of (a) providing a mixture of analytes with affinity reagents, wherein the affinity reagents are immobilized on a surface and the analytes are in fluid phase; (b) applying a stimulus to attract the analytes to the surface; and (c) detecting binding of analytes to affinity reagents on the surface.

[0005] In some configurations, a method of detecting analytes on an array can include steps of (a) contacting an array with affinity reagents in fluid phase, wherein analytes are attached at addresses in the array; (b) allowing affinity reagents in the fluid phase to bind to analytes attached at addresses in the array; (c) removing a plurality of the affinity reagents from contact with the array, thereby retaining a fraction of the affinity reagents in contact with the array; (d) applying a stimulus to attract the fraction of affinity reagents to the array; and (e) detecting binding of affinity reagents in the fraction to analytes attached at addresses in the array. Again, the method will be exemplified herein in the context of using immobilized analytes and fluidphase affinity reagents. However, the method can be carried out using immobilized affinity reagents and fluid-phase analytes. Accordingly, the method can include steps of (a) contacting an array with analytes in fluid phase, wherein affinity reagents are attached at addresses in the array; (b) allowing analytes in the fluid phase to bind to affinity reagents attached at addresses in the array; (c) removing a plurality of the analytes from contact with the array, thereby retaining a fraction of the analytes in contact with the array; (d) applying a stimulus to attract the fraction of analytes to the array; and (e) detecting binding of analytes in the fraction to affinity reagents attached at addresses in the array.

[0006] The present disclosure further provides a method of detecting an analyte, including steps of (a) providing an analyte attached to a solid support or particle; (b) attaching an affinity reagent to the solid support or particle via a flexible linker; and (c) detecting binding between the affinity reagent and the analyte on the solid support or particle.

[0007] Also provided is a method of identifying an analyte in an array. The method can include steps of (a) providing an array of individually resolved analytes, wherein analytes are attached at addresses in the array, and wherein individual addresses in the array are each attached to a single analyte; (b) attaching affinity reagents to the addresses in the array via flexible linkers, whereby individual addresses in the array each have a single, attached affinity reagent and a single, attached analyte; (c) detecting a plurality of addresses in the array, thereby distinguishing a higher level of binding between an attached affinity reagent and an attached analyte at a first address in the array from a lower level of binding between an attached affinity reagent and an attached analyte at a second address in the array; and (d) identifying an analyte detected at an address in the array in step (c).

[0008] Also provided is a method of detecting a first reaction, comprising: (a) providing immobilized on a solid support: (i) an analyte; and (ii) a first reactant, the first reactant being immobilized on the support within a first distance from the analyte; (b) contacting the immobilized analyte with a probe, the probe comprising an affinity reagent and a second reactant, the affinity reagent having binding specificity for the analyte, and the second reactant being capable of a second reaction with the first reactant when within a second distance from the first reactant; (c) forming a first reaction between the analyte and the affinity reagent, thereby bringing the second reactant within the second distance of the first reactant; (d) after forming the first reaction, forming the second reaction between the first reactant and the second reactant; and (e) detecting the first reaction.

[0009] Also provided is a method, comprising: (a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, wherein the equilibration period is sufficient to form a binding equilibrium between affinity reagents of the plurality of affinity reagents and analytes of the plurality of analytes; and (b) after the equilibration period, detecting complexes comprising an affinity reagent bound to an analyte, wherein the quantity of detected complexes is greater than a steady-state quantity of complexes that exists at the binding equilibrium.

[0010] Also provided is a method, comprising: (a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, wherein each analyte of the plurality of analytes is individually paired to a unique identifier; (b) during the equilibration period, recording detection proxies at unique identifiers containing an affinity reagent bound to an analyte; and (c) detecting signals from detection proxies at a subset of the unique identifiers, thereby identifying analytes bound by affinity reagents during the equilibration period.

[0011] Also provided is a method, comprising: (a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, thereby coupling affinity reagents of the plurality of affinity reagents to analytes of the plurality of analytes, wherein each analyte of the plurality of analytes is paired to a unique identifier; (b) forming a detectable reaction for analytes bound by an affinity reagent during the equilibration period; and (c) detecting presence of the detectable reaction at a subset of unique identifiers, thereby detecting the subset of analytes of the plurality of analytes bound by an affinity reagent of the plurality of affinity reagents.

[0012] Also provided is a method, comprising: (a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, wherein the equilibration period is sufficient to form a binding equilibrium between affinity reagents of the plurality of affinity reagents and analytes of the plurality of analytes; and (b) detecting during the equilibration period complexes comprising an affinity reagent bound to an analyte, wherein the quantity of detected complexes is greater than an steady-state quantity of complexes that exists at the binding equilibrium.

[0013] Also provided is a method, comprising: (a) binding an affinity reagent to an analyte at a fixed spatial address; (b) coupling a kinetically-controlled detection agent to the fixed spatial address; and (c) after coupling the kinetically-controlled detection agent to the fixed spatial address, detecting a signal at the fixed spatial address.

[0014] Also provided is a system, comprising: (a) a solid support comprising a plurality of sites, wherein each site of the plurality of sites is optically resolvable from any other site of the plurality of sites; and (b) a plurality of analytes immobilized on the solid support, wherein each site of the plurality of sites is attached to one and only one analyte of the quantity of analytes; wherein, in a first configuration, the system further comprises a plurality of affinity reagents, wherein a fraction of the affinity reagents is bound to analytes of the plurality of analytes, wherein a quantity of the fraction of affinity reagents is determined by a binding equilibrium of the affinity reagents for the analytes; and wherein, in a second configuration, the system issubstantially devoid of affinity reagents, wherein each individual site of a fraction of sites of the plurality of sites comprises a detectable label, wherein a quantity of the fraction of sites is greater than a quantity of the second fraction of affinity reagents.

[0015] Also provided is a system, comprising: (a) a solid support comprising a plurality of sites, wherein each site of the plurality of sites is optically resolvable from any other site of the plurality of sites; (b) a plurality of analytes immobilized on the solid support, wherein each site of the plurality of sites is attached to one and only one analyte of the quantity of analytes; and (c) a plurality of affinity reagents bound analytes of the plurality of analytes at sites of the plurality of sites, wherein the affinity reagents have a known binding equilibrium for binding to analytes of the plurality of analytes, and wherein a quantity of the sites of the plurality of sites is greater than a quantity of analytes bound to affinity reagents based upon the known binding equilibrium.

[0016] Also provided is a method of detecting a first reaction, comprising: (a) providing immobilized on a solid support: (i) an analyte; and (ii) a first reactant, the first reactant being immobilized on the support within a first distance from the analyte; (b) contacting the immobilized analyte with a probe, the probe comprising an affinity reagent and a second reactant, the affinity reagent having binding specificity for the analyte, and the second reactant being capable of a second reaction with the first reactant when within a second distance from the first reactant; (c) forming a first reaction between the analyte and the affinity reagent, and forming a second reaction between the first reactant and the second reactant; and (d) detecting the first reaction.

[0017] Also provided is a composition, comprising: (a) an affinity reagent having a binding specificity for at least a first analyte; (b) a first reactant, wherein the first reactant has reactivity with a second reactant; and (c) a linker, wherein the linker couples the affinity reagent to the first reactant, wherein the linker permits the first reactant to react with the second reactant when the affinity reagent is bound to the analyte.INCORPORATION BY REFERENCE

[0018] All publications, items of information available on the internet, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications, items of informationavailable on the internet, patents, or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF DRAWINGS

[0019] FIGs. 1 A and IB provide a diagrammatic representation of a binding assay method wherein an applied stimulus is used to attract affinity reagents to proteins in an array, in accordance with some embodiments.

[0020] FIG. 2 shows a cross section of a region of a flow cell having two protein analytes (white globules) immobilized on the lower surface and a fluid phase containing affinity reagents (Y shapes), wherein the affinity reagents are attached to a first luminophore (open circle) and the protein analytes are attached to a second luminophore (closed circle), in accordance with some embodiments.

[0021] FIG. 3 A shows a diagram of a protein (white globule) attached to a surface, wherein the protein is attached to a first luminophore (closed circle) via a nucleic acid docker, and an affinity reagent (Y-shape) that is bound to the protein, wherein the affinity reagent is attached to a second luminophore (open circle) via a nucleic acid tether, and wherein the docker and tether hybridize to position the luminophores for proximity-based detection (e.g. FRET), in accordance with some embodiments.

[0022] FIG. 3B shows a diagram of a protein (white globule) attached to a surface, wherein the protein is attached to a first luminophore (closed circle) via a nucleic acid docker, and an affinity reagent (Y-shape) that is bound to the protein, wherein the affinity reagent is attached to a second luminophore (open circle) via a nucleic acid tether, and wherein the docker and tether hybridize to a splint oligonucleotide to position the luminophores for proximity-based detection (e g. FRET), in accordance with some embodiments.

[0023] FIGs. 4A and 4B provide a diagrammatic representation of a binding assay method wherein analytes are attached to addresses on a surface and affinity reagents are linked to the addresses, in accordance with some embodiments.

[0024] FIG. 5A shows protein analytes (white globules) attached to addresses of an array and an affinity reagents (Y-shapes) attached to each address via a nucleic acid linker, wherein FRET occurs between luminophores (12 point stars) when the affinity reagent is bound to the protein(lefthand address) and FRET is not observed when the affinity reagent is dissociated from the protein (righthand address), in accordance with some embodiments.

[0025] FIG. 5B shows a configuration similar to FIG. 5A except the second fluorophore (closed 12 point star) is attached to the linker via a complementary oligonucleotide, in accordance with some embodiments.

[0026] FIG. 5C shows an exemplary configuration in which analytes (white globules) are attached to addresses of an array and an affinity reagent (Y-shape) is attached to each address via a nucleic acid linker. A first fluorophore (open 12 point star) is attached to the affinity reagent via a nucleic acid tether and a second fluorophore (closed 12 point star) is attached to the linker via a nucleic acid docker. At the address on the left (“+”), the affinity reagent is bound to the analyte and the tether is hybridized to the docker. A nucleotide sequence of the tether is complementary to a nucleotide sequence of the docker.

[0027] FIG. 6 shows protein analytes (white globules) attached to zero mode waveguides (ZMWs) and an affinity reagents (Y-shapes) attached to each ZMW via a nucleic acid linker, wherein emission from a luminophores (12 point star) is detectable when the affinity reagent is bound to the protein (lefthand ZMW) and emission is not observed when the affinity reagent is dissociated from the protein (righthand ZMW), in accordance with some embodiments.

[0028] FIGs. 7A and 7B show cross-sections of respective substrates that are capable of generating an electrophoretic force to attract affinity reagents, in accordance with some embodiments.

[0029] FIGs. 8A, 8B, and 8C illustrate methods of indirectly recording a binding interaction by transferring a transferrable moiety from an affinity reagent that is bound to an analyte to a unique identifier co-localized with the analyte, in accordance with some embodiments.

[0030] FIGs. 9A, 9B, and 9C illustrate a method of indirectly recording a binding interaction by catalytically modifying a unique identifier co-localized with an analyte, in accordance with some embodiments.

[0031] FIG. 10A depicts a method of directly recording a binding interaction between an analyte and an affinity reagent utilizing a bridging moiety that immobilizes the affinity reagent at the unique identifier containing the analyte, in accordance with some embodiments.

[0032] FIG. 10B depicts a method of dissociating an affinity reagent from an analyte after recording a binding interaction, in accordance with some embodiments.

[0033] FIG. 11 depicts oligonucleotide systems that utilize secondary structure of the oligonucleotides to control the kinetic on-rate of binding between the oligonucleotides, in accordance with some embodiments.

[0034] FIG. 12 illustrates a system for directly recording a binding interaction between an affinity reagent and an analyte that utilizes a luminescent enzyme, in accordance with some embodiments.

[0035] FIGs. 13A, 13B, 13C, and 13D display various systems for recording binding interactions that utilize a gap between docker and tether strands, in accordance with some embodiments.

[0036] FIGs. 14A, 14B, and 14C depict various configurations of an affinity reagent attached to a unique identifier by a linker, in which the configurations vary with respect to reaction for recording the binding interaction, in accordance with some embodiments.

[0037] FIG. 1 shows a diagram of a binding reaction in which a protein (cloud shape), which is attached to a first target nucleic acid (open, black line arrow) via a retaining component (black rectangle), binds to an affinity reagent (Y shape), which is attached to a second target nucleic acid (hatched, black line arrow). The target nucleic acids are hybridized with first hairpin oligonucleotides (labeled “1”) and second hairpin oligonucleotides (labeled “2”) to produce a nicked double helix. The arrow heads indicate 3’ ends of the nucleic acids and oligonucleotides, in accordance with some embodiments.

[0038] FIG. 16A shows a hairpin oligonucleotide hybridizing to a pair of target nucleic acids via annealing of the 3’ overhang (shown as the open section of the strand) of the oligonucleotide to a first target nucleic acid and annealing of a first of two hairpin sequences (shown as the grey hatched section of the strand) of the oligonucleotide to a second target nucleic acid, in accordance with some embodiments.

[0039] FIG. 16B shows a hairpin oligonucleotide hybridizing to a pair of target nucleic acids via annealing of the 3’ overhang (shown as the open section of the strand) of the oligonucleotide to a portion of the first target nucleic acid and annealing of a first of two hairpin sequences (shown as the grey hatched section of the strand) of the oligonucleotide to portions of both the first and second target nucleic acids, in accordance with some embodiments.

[0040] FIG. 17 shows a diagram of a reaction in which two species of oligonucleotides (black and grey arrows) hybridize to target nucleic acids of a protein-affinity reagent complex to form a nicked double helix. The oligonucleotides form hairpins and include labels (asterisks) which arein a quenched state in the hairpin conformation. The labels are competent for detection when hybridized in the nicked double helix, in accordance with some embodiments.

[0041] FIG. 18 illustrates differences in observation of binding equilibrium between a plurality of affinity reagents and a plurality of analytes when the binding interactions are not recorded (upper) or are recorded (lower), in accordance with some embodiments.

[0042] FIG. 19 displays a plot of effective affinity reagent concentration as a function of linker length when the linker co-localizes the affinity reagent with an analyte on a solid support, in accordance with some embodiments.

[0043] FIG. 20 displays a plot of equilibrium binding curves for an affinity reagent to a higher affinity target (superordinate) and a lower affinity target (subordinate), in accordance with some embodiments.

[0044] FIG. 21 displays detection rates for recording of binding interactions of affinity reagents with target peptides and non-target peptides via a biotin ligase-facilitated recording method.DETAILED DESCRIPTION

[0045] A fundamental challenge to the design and implementation of binding assays is to detect binding of an affinity reagent to an analyte of interest in the presence of an excess of non-bound affinity reagent and / or non-bound analyte. Typically, a binding assay includes an incubation period wherein at least one reactant (e.g., affinity reagent and / or analyte) is present at a relatively high concentration in solution. During this incubation period, an equilibrium is typically established between three species: non-bound affinity reagent, non-bound analyte and analyte: affinity reagent complex. For many assays, the complex is detected via a label that is attached to the affinity reagent or analyte. However, one or both of the non-bound species are typically present in a large excess compared to the complex, and this can result in a level of background signal from non-bound species that overwhelms detection of signal from the complexes. One approach to reducing unwanted background signal is to separate non-bound species from complexes, for example, by removal of the non-bound species from the assay, prior to detection. However, removal of the non-bound species results in a disequilibrium condition wherein, depending upon the kinetics of the binding reaction, the complexes may begin to dissociate. Substantial degrees of dissociation prior to or during the timeframe of detection create a risk of anomalous results.

[0046] Binding equilibrium represents a dynamic balance between association and dissociation of molecules. For example, in considering the binding equilibrium between a first species and a second species, observation of complexed molecules and unbound molecules will provide a steady-state measurement of the equilibrium, but over a long enough time, virtually all molecules of the first species and the second species will at least briefly form a complex with the other species. Observation of binding at equilibrium between molecules may provide incomplete information due to not observing all binding interactions that occurred.

[0047] The present disclosure provides binding assays that are used to form a complex between an affinity reagent and analyte, optionally with at least one of the affinity reagent and analyte labeled in the non-bound state, and detecting the complex via the detection of the label. Alternatively, some binding assay may introduce a third molecule that binds to the complex formed by the affinity reagent and analyte, in which the third molecule optionally contains a label. In some configurations, the binding assays allow detection of the labeled complex in the presence of non-bound species that also contain the label. In some cases, non-bound species are in substantial excess compared to the concentration or amount of complex present during a detection step. A binding assay of the present disclosure can be configured to detect complexes under an equilibrium condition, a disequilibrium condition, or in a simulated equilibrium condition.

[0048] The present disclosure provides a method of detecting a first reaction, comprising: (a) providing immobilized on a solid support: (i) an analyte, and (ii) a first reactant, the first reactant being immobilized on the support within a first distance from the analyte, (b) contacting the immobilized analyte with a probe, the probe comprising an affinity reagent and a second reactant, the affinity reagent having binding specificity for the analyte, and the second reactant being capable of a second reaction with the first reactant when within a second distance from the first reactant, (c) forming a first reaction between the analyte and the affinity reagent, and forming a second reaction between the first reactant and the second reactant, and (d) detecting the first reaction. The present disclosure further provides a method of detecting a first reaction, comprising: (a) providing immobilized on a solid support: (i) an analyte, and (ii) a first reactant, the first reactant being immobilized on the support within a first distance from the analyte, (b) contacting the immobilized analyte with a probe, the probe comprising an affinity reagent and a second reactant, the affinity reagent having binding specificity for the analyte, and the secondreactant being capable of a second reaction with the first reactant when within a second distance from the first reactant, (c) forming a first reaction between the analyte and the affinity reagent, thereby bringing the second reactant within the second distance of the first reactant, (d) after forming the first reaction, forming the second reaction between the first reactant and the second reactant, and (e) detecting the first reaction.

[0049] Methods set forth herein may comprise a step of forming a reaction between a first reactant and a second reactant. The reaction may require bringing the first reactant into a sufficient proximity to the second reactant to form the reaction. Sufficient proximity between a first reactant and a second reactant can depend upon the nature of the interaction between the pair. A reaction may occur at a nanometer-scale separation distance between the first reactant and the second reactant (e.g., a photon transfer reaction between a dye pair), or may occur at an Angstrom-scale separation distance (e.g., covalent bond formation, certain electrostatic non- covalent interactions). A separation distance between a first reactant and a second reactant may be no more than about 50 nanometers (nm), 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, 1 nm, 0.5 nm, 0.1 nm, 0.05 nm, 0.01 nm, or less than 0.01 nm. Alternatively or additionally, a separation distance between a first reactant and a second reactant may be at least about 0.01 nm, 0.05 nm, 0.1 nm, 0.5 nm, 1 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, or more than 50 nm. In some cases, a separation distance between a first reactant and a second reactant may be less than a separation distance between an analyte and the first reactant, in which the analyte and first reactant are co-localized at a same address.

[0050] A first reactant that is configured to react with a second reactant may be co-located with an analyte at an address of a solid support. There may be a separation distance between the first reactant and the analyte at an address, for example as measured by the distance between their respective attachment points to the address. Alternatively, a reactive moiety of a first reactant may be attached by a linker to a solid support, thereby facilitating some diffusion of the reactive moiety adjacent to a co-located analyte. In such case, the separation distance between the first reactant and the analyte may be an average distance between the analyte and the reactive moiety of the first reactant. The separation distance between a first reactant and an analyte at an address may be chosen to balance between the selectivity of the reaction between the first reactant and the second reactant and the reactivity of the first reactant. For example, it may be preferable to attach a first reactant in close proximity to an analyte to decrease the likelihood that the firstreactant binds to a second reactant when the affinity reagent attached to the second reactant is not bound to the analyte co-located with the first reactant. Further, if a first reactant is too close to an analyte, the first reactant may become partially- or fully-occluded (e.g., sterically, electrostatically, etc.) from reacting with a second reactant. Alternatively, partial occlusion of a first reactant may facilitate kinetic control of the reaction rate of the first reactant. Accordingly, a separation distance between a first reactant and an analyte at an address may be an optically non- resolvable distance, such as no more than about 500 nm, 400 nm, 300 nm, 200nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm, 15 nm, 10 nm, 5 nm, 1 nm, or less than 1 nm. Alternatively or additionally, a separation distance between a first reactant and an analyte at an address may be at least about 1 nanometer (nm), 5 nm, 10 nm, 15 nm, 20 nm, 30 nm, 40 nm, 50 nm, lOOnm, 200 nm, 300 nm, 400 nm, 500 nm, or more than 500 nm.

[0051] Further provided herein is a probe composition, comprising: (a) an affinity reagent having a binding specificity for at least a first analyte, (b) a first reactant, wherein the first reactant has reactivity with a second reactant, and (c) a linker, wherein the linker couples the affinity reagent to the first reactant, wherein the linker permits the first reactant to react with the second reactant when the affinity reagent is bound to the analyte. In some cases, the length of a linker coupling an affinity reagent to a first reactant may be greater than a separation distance between an analyte and a second reactant that is co-localized with the analyte. In other cases, the length of a linker coupling an affinity reagent to a first reactant may be less than a separation distance between an analyte and a second reactant that is co-localized with the analyte. The linker length may be a useful parameter for controlling binding equilibrium and binding kinetics according to the methods set forth herein.

[0052] The present disclosure further provides binding assays that record the presence of binding interactions, facilitating detection of such binding interactions even if the binding interaction becomes dissociated. In some configurations, a binding interaction may be directly recorded by binding together a molecular complex between bound species for a sufficiently long period of time to detect the complex. In other configurations, a binding interaction may be indirectly recorded by providing a detectable label at an address that can be subsequently detected at the address after a molecular complex between bound species has been disrupted.

[0053] FIGs. 1 A and IB provides a diagrammatic representation of a first binding assay method. A section of a flow cell is shown in profile. Five proteins (white globules) are attached torespective addresses in an array that is formed on the bottom, inner surface of the flow cell. In the first step, a plurality of affinity reagents (Y-shapes) is delivered to the lumen of the flow cell. The fluid-phase affinity reagents are allowed to bind to proteins. In the example shown, the first, third and fourth proteins from the left are recognized by and bound to affinity reagents. Then at least some of the non-bound affinity reagents are removed from contact with the array, for example, being flowed out of the flow cell lumen. Moving to FIG. IB, the subset of affinity reagents that are retained in the flow cell are subjected to an applied stimulus that attracts them to the proteins in the array. Any of a variety of stimuli can be applied as set forth in further detail below herein, including for example, magnetic attraction of affinity reagents having magnetic or paramagnetic moieties, electrophoretic attraction of affinity reagents having charged moieties, colloidal attraction in response to volume exclusion agents, or pH-dependent attraction of protein moieties to polyacidic moieties. The result is an increased concentration of affinity reagents in the volume that is localized to the array of proteins. However, the relatively low concentration of affinity reagents in the volume of the lumen that is distal from the array surface can prevent or substantially reduce background signal that is produced from non-bound species in the assay. Typically, an applied stimulus that is used to attract an affinity reagent (or analyte) to an array is variably applied in a method set forth herein.

[0054] The step of removing non-bound affinity reagents shown in FIG. 1A is optional. For example, an assay can be configured to deliver a relatively low concentration of affinity reagents in the first step followed by attraction of the affinity reagents to the surface via the applied stimulus and then detecting binding. This configuration can provide the advantage of reducing the amount of affinity reagent used, thereby reducing costs and resources required to make and / or use the affinity reagents. The application of a stimulus that transfers affinity reagents toward the analyte can create a higher effective concentration adjacent to the analytes than the bulk concentration in the lumen of the flow cell, thereby driving equilibrium toward complex formation.

[0055] FIGs. 4A and 4B provide a diagrammatic representation of a second binding assay. Two addresses in an array are shown in FIG. 4A, each attached to a protein (white globule). In the first step, a plurality of affinity reagents (Y-shapes) is delivered to the array. Each of the affinity reagents is attached to a linker and the linker is reactive with an attachment moiety at each address of the array. In the example shown, an oligonucleotide serves as the attachment moietyat each of the addresses and the linkers include a nucleic acid sequence that is complementary to the oligonucleotide. Each address includes a single protein and a single linked affinity reagent. Non-linked affinity reagents are then removed from contact with the array. Turning to FIG. 4B, binding can then be allowed to occur, wherein the protein on the left bound to the affinity reagent thereby generating a signal (+) and the protein on the right is not bound by the affinity reagent and thus does not produce signal (-). The bound and unbound proteins can be distinguished using a detection technique that preferentially collects signal from a location at or near the protein relative to locations distal from the protein, or by collecting signals produced by energy transfer between the affinity reagent and protein. Other proximity-based detection methods can also be used. Exemplary methods for detecting complexes and distinguishing from non-bound components are set forth in further detail below.

[0056] The present disclosure further provides a method of detecting binding of an affinity reagent to an analyte by (a) contacting the analyte with the affinity reagent to form a complex, wherein the complex includes a first target nucleic acid that is co-localized with the affinity reagent and further includes a second target nucleic acid that is co-localized with the analyte; (b) contacting the complex with a set of oligonucleotides that hybridize to the first and second target nucleic acids, thereby forming a nicked double helix; and (c) detecting the nicked double helix, thereby detecting binding of the affinity reagent to the analyte.

[0057] Optionally, the set of oligonucleotides can include oligonucleotide species that are in a self-annealed state, such as a stem-loop conformation (also referred to as a hairpin conformation). Hybridization of oligonucleotides of the set to the first and second target nucleic acids can initiate a hybridization chain reaction in which regions of the oligonucleotides that had been self-annealed instead anneal to each other to form the nicked double-helix. An exemplary configuration for the oligonucleotides and chain reaction for forming the nicked double helix is shown in FIG. 15, and described in further detail below.

[0058] The compositions, methods and apparatus set forth herein are particularly useful for single molecule-resolved binding assays because the nicked double helix can be grown to effectively provide signal amplification. For example, oligonucleotides that are incorporated into a nicked double helix of an analyte-affinity reagent complex can include labels and as the helix grows more signal-producing labels are added to the complex. Even though the complex includes only a single affinity reagent and a single analyte, the nucleotide sequence of theoligonucleotides, type of labels used and conditions provided for growth of the nicked double helix can be optimized to allow for adequate signal production for any of a variety of detection apparatus.

[0059] The compositions, methods and apparatus set forth herein provide advantages for use in equilibrium binding conditions. A common dilemma with binding assays is being able to maintain equilibrium conditions while distinguishing bound complexes from unbound components. For a typical binding reaction, equilibrium constitutes a relatively large concentration of unbound components and a relatively small concentration of bound complex. Furthermore, one of the components of the binding reaction is typically labeled to facilitate detection. As a result, detection of the relatively small amount of signal arising from the labeled complex will often be overwhelmed or at least obscured by the signal arising from the labeled unbound component. In contrast, the methods set forth herein can be configured to detect the mass of the nicked double helix, which is distinguishable from the individual oligonucleotides from which it is generated due to its substantially larger mass. Optionally, the oligonucleotides can include labels that are quenched or otherwise not-competent to generate signal when the oligonucleotides are in the unbound state, but become competent for signal production when bound to each other in the nicked double helix. For example, an oligonucleotide can include a luminescent label that is quenched by proximity to a quenching moiety when the oligonucleotide is in a self-annealed conformation (thereby decreasing luminescence intensity of the luminescent label), but competent for signal production when the oligonucleotide changes conformation to hybridize to the nicked double helix.

[0060] Detection schemes set forth herein are particularly useful for cyclic assay in which a given analyte is repeatedly contacted with affinity reagents and detected to observe any resulting complexes. This is because a nicked double helix can be configured to dissociate readily from a complex once the affinity reagent and analyte components of the complex dissociate. More specifically, a nicked double helix can be co-localized with a complex via binding to both a first target nucleic acid that is co-localized with the affinity reagent and a second target nucleic acid that is co-localized with the analyte. After detecting the complex, the affinity reagent can be dissociated from the analyte to effectively separate the first and second nucleic acids, thereby destabilizing the nicked double helix and causing it to dissociate from the complex and its components. A nicked double helix, as it is non-covalently assembled of individualoligonucleotides, can be cooperatively disassembled into component monomers by strand displacement reaction, heat, denaturing agents or other techniques common to the art for dehybridizing short oligonucleotides. The nucleic acid sequences of the target nucleic acids and oligonucleotides can be tailored to optimize hybridization energetics for formation and dissociation of the nicked double helix at the appropriate steps of the method.

[0061] Methods set forth herein may be particularly useful for characterizing pluralities of analytes, in which the plurality of analytes is characterized by a measure of heterogeneity. Heterogeneity of a plurality of analytes may be characterized by the presence of chemical or structural diversity amongst the analytes. For example, a plurality of proteins may contain two or more species of proteins, in which the species of proteins are distinguished from each other by differing primary structures (e.g., a first species having a different amino acid sequence than a second species). Additionally or alternatively, a plurality of proteins may contain two or more species of proteins, in which the species of proteins are distinguished from each other by differing proteoforms (e.g., a first species having a different complement of post-translational modifications than a second species). Additionally or alternatively, a plurality of proteins may contain two or more species of proteins, in which the species of proteins are distinguished from each other by differing isoforms (e.g., a first species having a different amino acid sequence splicing than a second species). A method set forth herein may be useful for characterizing a heterogeneous plurality of analytes that contains at least about 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or more than 99% of the species diversity of a genome, transcriptome, and / or proteome.

[0062] A plurality of analytes may comprise at least about 1, 2, 5, 10, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000, 30000, 50000, 100000, 1000000, or more than 1000000 different species of analytes. Alternatively or additionally, a plurality of analytes may comprise no more than about 1000000, 100000, 50000, 30000, 20000, 10000, 5000, 2000, 1000, 500, 200, 100, 50, 10, 5, 2, or less than 5 different species of analytes.

[0063] Methods set forth herein may utilize one or more affinity reagents. It may be particularly useful to utilize an affinity reagent that binds to two or more differing species of analytes that are present in a plurality of analytes. In some cases, an affinity reagent may bind to an epitope that is present in two or more differing species of analytes that are present in a plurality of analytes. Forexample, an affinity agent may be configured to bind to an epitope (e.g., a trimer or tetramer amino acid sequence) that is common to two or more differing species of analytes.

[0064] An affinity reagent may bind to two or more differing binding targets with differing binding characteristics. For example, an affinity reagent may bind to a first binding target (e.g., a first analyte, a first epitope common to two or more differing analytes, etc.) with a first dissociation constant, and may further bind to a second binding target (e.g., a second analyte, a second epitope common to two or more differing analytes, etc.) with a second dissociation constant, in which the first dissociation constant differs from the second dissociation constant. Thus, the affinity reagent may dissociate, or separate, from one of the first binding target or the second binding target at a higher propensity than the other binding target. Useful distinguishing binding characteristics can also include the binding on-rate and / or the binding off-rate. Accordingly, for a plurality of analytes containing a first binding target and a second binding target for an affinity agent, there may exist two or more separate binding equilibria for the affinity agent. Some methods set forth herein may be useful for distinguishing between differing binding targets with a common affinity to an affinity reagent based upon the differences in binding equilibrium.

[0065] For affinity reagents described herein, a binding interaction with a binding target or plurality thereof having a highest characterized binding specificity (e.g., as determined by a lowest value of the affinity reagent’s dissociation constant) may be referred to as a superordinate binding interaction. Likewise, for affinity reagents described herein, a binding interaction with a binding target or plurality thereof having a lower characterized binding specificity (e.g., as determined by a higher value of the affinity reagent’s dissociation constant) relative to a superordinate binding interaction may be referred to as a subordinate binding interaction. An affinity reagent may have two or more superordinate binding interactions. An affinity reagent may have two or more subordinate binding interactions.

[0066] Binding equilibrium may depend in part on the relative concentrations of unbound affinity reagents and / or unbound analytes. For example, increasing the concentration of unbound affinity reagents may tend to increase the quantity of target analytes bound by the affinity reagents. Dissociation constant is a frequently used characterization of an affinity reagent for a binding target that can be utilized to estimate the steady-state fraction of binding targets that will be bound by an affinity reagent at binding equilibrium. A method set forth herein may compriseforming a binding equilibrium between affinity reagents and a plurality of binding targets (e.g., two or more species of analytes, analytes comprising a common epitope, etc.), in which at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more than 99% of binding targets are bound by an affinity reagent at equilibrium. Alternatively or additionally, a method set forth herein may comprise forming a binding equilibrium between affinity reagents and a plurality of binding targets, in which no more than about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than 10% of binding targets are bound by an affinity reagent at equilibrium. Some methods set forth herein may comprise a step of recording a binding interaction between an affinity reagent and a binding target such that the binding interaction can be detected even after the affinity reagent has dissociated from the binding target. Accordingly, a method may comprise detecting a binding interaction for a percentage of binding targets (e g., two or more species of analytes, analytes comprising a common epitope, etc.) that exceeds a percentage of binding targets bound by the affinity reagent at the steady-state binding equilibrium.

[0067] An affinity reagent may bind to at least about 1, 2, 5, 10, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000, 30000, 50000, 100000, 1000000, or more than 1000000 different species of analytes present in a plurality of analytes. Alternatively or additionally, an affinity agent may bind to no more than about 1000000, 100000, 50000, 30000, 20000, 10000, 5000, 2000, 1000, 500, 200, 100, 50, 10, 5, or less than 5 different species of analytes present in a plurality of analytes.

[0068] Methods set forth herein may be particularly well-suited to array-based techniques. In some cases, a method may utilize a single-analyte array, in which each individual analyte of a plurality of analytes is co-localized with a unique identifier, such that each individual analyte is identifiable by its unique identifier. In some cases, a unique identifier may comprise an address on a solid support, in which the address is a spatially resolvable distance from any other address of the solid support that contains an analyte. In some cases, an address on a solid support may contain a site that is configured to bind an analyte. Accordingly, the site may comprise one or more moieties that facilitate attachment of the analyte and / or unique identifier co-localized with the analyte. In some cases, a unique identifier may comprise a barcode (e.g., a nucleic acid barcode, a peptide barcode, etc.) or a tag (e.g., a nucleic acid tag, a peptide tag) that is colocalized with an analyte. A tag may be especially useful for methods that comprise a step ofrecording a binding interaction of an affinity agent to an analyte via transfer of a detectable label to a tag co-localized with the analyte.

[0069] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

[0070] In some of the implementations described herein, the term "address," when used in reference to an array, can mean a location in an array occupied by, or configured to be occupied by, a particular molecule or analyte such as a protein, nucleic acid, structured nucleic acid particle or reactive moiety. An address can contain a single analyte molecule, or it can contain a population of several analyte molecules of the same species (i.e. an ensemble of the molecules). Alternatively, an address can include a population of molecules that are different species such as an attached analyte and a linked affinity reagent. Addresses of an array are typically discrete. The discrete sites can be contiguous, or they can have interstitial spaces between each other. An array useful herein can have, for example, addresses that are separated by less than 100 microns, 10 microns, 1 micron, 0.5 micron, 0.1 micron, 0.01 micron or less. Alternatively or additionally, an array can have addresses that are separated by at least 0.01 micron, 0.1 micron, 0.5 micron, 1 micron, 10 microns, 100 microns or more. The addresses can each have an area of less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron or less. An array can include at least about IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, or more addresses, some or all of which are occupied by analytes or molecules. An address that is configured to bind an analyte, for example by the presence of attachment moieties, is referred to herein as a “site.”

[0071] In some of the implementations described herein, the term “affinity reagent” can refer to a molecule or other substance that is capable of specifically or reproducibly binding to an analyte (e.g., protein) or moiety (e.g., post-translational modification of a protein). An affinity reagent can be larger than, smaller than or the same size as the analyte. An affinity reagent may form a reversible or irreversible bond with an analyte. An affinity reagent may bind with an analyte in a covalent or non-covalent manner. Affinity reagents may include chemically reactive affinity reagents, catalytic affinity reagents e.g., kinases, proteases, etc.) or chemically non-reactive affinity reagents (e.g., antibodies or fragments thereof). An affinity reagent can be chemically non-reactive and non-catalytic, thereby not permanently altering the chemical structure of an analyte to which it binds. Affinity reagents that can be particularly useful for binding to proteinsinclude, but are not limited to, antibodies such as full-length antibodies or functional fragments thereof (e.g., Fab’ fragments, F(ab’)2 fragments, single-chain variable fragments (scFv), di-scFv, tri-scFv, or microantibodies), or aptamers, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, miniproteins, DARPins, monobodies, nanoCLAMPs, lectins, or functional fragments thereof. The term “affinity agent” is intended to be synonymous with the term “affinity reagent.”

[0072] In some of the implementations described herein, the term "antibody” can refer to a protein that binds to an antigen or epitope via at least one complementarity determining region (CDR). An antibody can include all elements of a full-length antibody. However, an antibody need not be full length and functional fragments can be particularly useful for many uses. The term “antibody” as used herein encompasses full length antibodies and functional fragments thereof.

[0073] In some of the implementations described herein, the term "array" can refer to a population of analytes (e.g., proteins) that are co-localized with unique identifiers such that the analytes can be distinguished from each other. A unique identifier can be a solid support (e.g., particle or bead), structured nucleic acid particle (SNAP), address on a solid support, tag, label (e.g., luminophore), or barcode (e.g., nucleic acid barcode) that is co-localized with an analyte and that is distinct from other identifiers in the array. Analytes can be co-localized with unique identifiers by attachment, for example, via covalent or non-covalent bonds. An array can include different analytes that are each attached to different unique identifiers. An array can include different unique identifiers that are attached to the same or similar types of analytes. An array can include separate solid supports, or separate sites on a given solid support, that each bear a respective analyte, wherein the respective analytes can be identified according to the locations of the solid supports or sites. Analytes that can be included in an array can be, for example, nucleic acids such as structured nucleic acid particles, proteins, enzymes, glycans, affinity reagents, ligands, or receptors.

[0074] In some of the implementations described herein, the term “artificial” when used in reference to a substance, can mean that the substance is made by human activity rather than occurring naturally. For example, a polymer that is made at least in part by human activity or that includes at least one artificial monomer unit is referred to as an “artificial polymer.” As used herein, the term “natural” when used in reference to a substance, means that the substance occursnaturally. A substance that is purified or refined from its naturally occurring state without further modification can be considered natural, but a substance that is chemically modified from its naturally occurring state can be considered artificial. For example, cellulose can be considered a natural polymer, but methyl cellulose can be considered an artificial polymer.

[0075] In some of the implementations described herein, the term "attached" can refer to the state of two things being joined, fastened, adhered, connected or bound to each other. Attachment can be covalent or non-covalent. For example, a particle can be attached to a protein by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, adhesion, adsorption, and hydrophobic interactions.

[0076] In some of the implementations described herein, the term “binding affinity” or “affinity” can refer to the strength or extent of binding between an affinity reagent and a binding partner. The binding affinity of an affinity reagent for a binding partner may be qualified as being “high affinity,” “medium affinity,” or “low affinity.” A binding affinity of an affinity reagent for a binding partner, affinity target, or target moiety may be quantified as being “high affinity” if the interaction has a dissociation constant of less than about 100 nM, “medium affinity” if the interaction has a dissociation constant between about 100 nM and 1 mM, and “low affinity” if the interaction has a dissociation constant of greater than about ImM. Binding affinity can be described in terms known in the art of biochemistry such as equilibrium dissociation constant (KD), equilibrium association constant (KA), association rate constant (kon), dissociation rate constant (koff) and the like. See, for example, SegeL Enzyme Kinetics John Wiley and Sons, New York (1975), which is incorporated herein by reference in its entirety.

[0077] In some of the implementations described herein, the term “binding interaction” can refer to a reaction that associates an affinity reagent to an analyte. A binding reaction may be a covalent or non-covalent interaction. A binding interaction may associate an affinity reagent to an analyte for a sufficient length of time to detect a complex formed by the affinity reagent and analyte.

[0078] In some of the implementations described herein, the term “biomolecule” can refer to a molecule that is produced by a living organism or capable of being produced by a living system.Biomolecules include, for example, proteins, amino acids, nucleic acids, nucleotides, nucleosides, polysaccharides, glycans, sugars, hormones, metabolites and the like. Biomolecules can encompass both natural and artificial biomolecules.

[0079] In some of the implementations described herein, the term “competent,” when used in reference to a label, can mean the label is in a state where it produces a detectable signal or is capable of producing a detectable signal. For example, a luminescent label is competent when it is able to produce a detectable photon when excited by light at an appropriate wavelength. A luminescent label that is quenched is considered to not be competent.

[0080] In some of the implementations described herein, the term “complex” can refer to two or more molecules held together by at least one non-covalent interaction. Two or more molecules of a complex can be dissociated from each other without necessarily breaking any covalent bonds. Rather, two or more molecules of a complex can be dissociated by breaking at least one non-covalent bond. It will be understood that in some cases covalent bonds can also be broken when dissociating two or more molecules of a complex.

[0081] The term "comprising" is intended herein to be open-ended, including not only the recited elements, but further encompassing any additional elements.

[0082] In some of the implementations described herein, the term “concatemer structure,” when used in reference to a nucleic acid, can mean the nucleic acid has at least two repeats of a nucleotide sequence. The repeated nucleotide sequence can include at least 5, 6, 8, 10, 12, 14, 16, 18, 20, 25 or more nucleotides. A concatemer structure can include at least two repeats of other structural elements. Exemplary structural elements include nicks or gaps in a doublestranded nucleic acid. Nicks or gaps can optionally separate repeated nucleotide sequence regions of a nicked double helix having a concatemeric structure. A concatemer can include at least 2, 3, 4, 5, 6, 8, 10, 15, 20, 25 or more repeats of a given sequence and / or other structural element.

[0083] In some of the implementations described herein, the term “covalent,” when used in reference to a bond between atoms or moieties of a molecule, can refer to bonding due to sharing of a pair of electrons between the two atoms or moieties. Covalent interaction can arise due to a chemical reaction between a first reactive moiety and a second reactive moiety, optionally in the presence of a third intermediary or catalytic moiety. Covalent binding interactions can form between two atoms or moieties due to various chemical mechanisms, including addition,substitution, elimination, oxidation, and reduction. In some cases, a covalent binding interaction may be formed by a Click-type reaction, as set forth herein (e.g., methyltetrazine (mTz) - tetracyclooctylene (TCO), azide - dibenzocyclooctene (DBCO), thiol-epoxy). In some cases, a ligand-receptor-type binding interaction can also form a covalent binding interaction. For example, Spy Catcher-Spy Tag, SnoopCatcher- SnoopTag, and SdyCatcher-SdyTag are receptorligand binding pairs that can form covalent binding interactions due to isopeptide bond formation. Additional useful covalent interactions can include coordination bond formation, such as between a metal-containing substrate and a ligand. Exemplary coordination bonds can include silicon-silane, metal oxi de-phosphate, and metal oxide-phosphonate. Useful reagents and mechanisms for forming covalent binding interactions, including bioorthogonal binding interactions, as set forth herein, are provided in U.S. Patent Nos. 11,203,612 and 11,505,796, each of which is herein incorporated by reference in its entirety.

[0084] In some of the implementations described herein, the term “docker” can refer to a molecule or moiety that is configured to interact with a tether or that is interacting with a tether. A docker can be a moiety of a substance, object, molecule, solid support, address, particle, or bead. A docker can include a polymer, nucleic acid strand, nucleic acid duplex, nucleotide sequence, protein, affinity reagent, epitope, paratope, receptor, ligand or the like. A docker can interact with a tether via covalent or non-covalent bonding.

[0085] In some of the implementations described herein, the term "each," when used in reference to a collection of items, can be intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0086] In some of the implementations described herein, the term “epitope” can refer to a molecule or part of a molecule, which is recognized by or binds specifically to an affinity reagent or paratope. Epitopes may include amino acid sequences that are sequentially adjacent in the primary structure of a protein or amino acids that are structurally adjacent in the secondary, tertiary or quaternary structure of a protein. An epitope can optionally be recognized by or bound to an antibody. However, an epitope need not necessarily be recognized by any antibody, for example, instead being recognized by an aptamer, miniprotein or other affinity reagent. An epitope can optionally bind an antibody to elicit an immune response. However, an epitope need not necessarily participate in, nor be capable of, eliciting an immune response.

[0087] In some of the implementations described herein, the term “fluid-phase” or “fluid phase,” when used in reference to a molecule or particle participating in a binding interaction, can mean the molecule or particle is in a state wherein it is mobile in a fluid, for example, being capable of diffusing through the fluid to another molecule or particle that is complementary to the molecule or particle in the binding interaction. A fluid-phase molecule or particle may be attached to a solid support provided the moiety that attaches the molecule or particle to the solid support permits sufficient movement of the molecule or particle through the fluid to contact another molecule or particle that is complementary to the molecule or particle in a binding interaction. A fluid-phase molecule or particle may be attached to a solid support by a linker, such as a flexible linker. An immobilized analyte that is provided in a denatured or partially-denatured state may contain one or more fluid-phase portions of its molecular structure.

[0088] In some of the implementations described herein, the term “gap,” when used in reference to a double stranded nucleic acid, can refer to a region in one strand of the double helix which occurs between two double stranded regions and which lacks at least one nucleotide, whereby the gap is incapable of forming a Watson-Crick base pair with the other strand of the double helix. A gap in one strand of a double helix can span at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or more nucleotide positions of the other strand of the double helix.

[0089] In some of the implementations described herein, the term “hairpin sequence” can mean a nucleotide sequence that includes a first region of the nucleotide sequence that is the reverse complement of a second region of the nucleotide sequence. The first region of the nucleotide sequence and second region of the nucleotide sequence are typically separated by an intermediate region that forms a single-stranded structure, that is referred to as a “loop,” when the nucleotide sequence self-anneals. The hairpin sequence can include a palindromic sequence of nucleotides or a gapped palindromic sequence of nucleotides. The gap in the palindrome can occur in the loop and / or portions of the palindromic sequence can occur in the loop. The reverse complementary regions of a hairpin sequence can anneal with the second region of the nucleotide sequence to form an intramolecular double helix that is referred to as a “stem” structure.

[0090] In some of the implementations described herein, the term “immobilized,” when used in reference to a molecule or particle that is in contact with a fluid phase, can refer to the molecule or particle or a portion thereof being prevented from diffusing in the fluid phase. For example, immobilization can occur due to confinement at, or attachment to, a solid phase. Immobilizationcan be temporary (e.g., for the duration of one or more steps of a method set forth herein) or permanent. Immobilization can be reversible or irreversible under conditions utilized for a method, system or composition set forth herein. A denatured molecule may be considered immobilized if the molecule as a whole cannot diffuse through the fluid phase, even if portions of the molecular structure have an ability to diffuse in regions of the fluid adjacent to the immobilization site of the molecule.

[0091] In some of the implementations described herein, the terms "label" and “detectable label” can refer synonymously to a molecule or moiety that provides a detectable characteristic. The detectable characteristic can be, for example, an optical signal such as absorbance of radiation, luminescence emission, luminescence lifetime, luminescence polarization, fluorescence emission, fluorescence lifetime, fluorescence polarization, or the like; Rayleigh and / or Mie scattering; binding affinity for a ligand or receptor; magnetic properties; electrical properties; charge; mass; radioactivity or the like. Exemplary labels include, without limitation, a luminophore (e.g., fluorophore), chromophore, nanoparticle (e.g., gold, silver, carbon nanotubes), heavy atoms, radioactive isotope, mass label, charge label, spin label, receptor, ligand, or the like. A label may produce a signal that is detectable in real-time (e g., fluorescence, luminescence, radioactivity). A label may produce a signal that is detected off-line (e.g., sequencing of, or hybridization to, a nucleic acid barcode) or in a time-resolved manner (e.g., time-resolved fluorescence). A label may produce a signal with a characteristic frequency, intensity, polarity, duration, wavelength, sequence, or fingerprint.

[0092] In some of the implementations described herein, the terms “linker” and “linking moiety” can refer synonymously to a moiety that connects two objects to each other. One or both objects can be a molecule (e.g., affinity reagent or analyte), solid support, address, particle or bead. The term can also refer to an atom, moiety or molecule that is configured to react with two objects to form a moiety that connects the two objects. The connection of a linker to one or both objects can be a covalent bond or non-covalent bond. A linker may be configured to provide a chemical or mechanical property to the moiety connecting two objects, such as hydrophobicity, hydrophilicity, electrical charge, polarity, rigidity, or flexibility. A linker may comprise two or more functional groups that facilitate coupling of the linker to the first and second objects. A linker may include a polyfunctional linker such as a homobifunctional linker, heterobifunctional linker, homopolyfunctional linker, or heteropolyfunctional linker. Exemplary compositions forlinkers can include, but are not limited to, a polyethylene glycol (PEG), polyethylene oxide (PEO), amino acid, polypeptide, nucleotide, nucleic acid, nucleic acid origami, dendrimer, peptide nucleic acid (PNA), polysaccharide, carbon, nitrogen, oxygen, ether, sulfur, or disulfide. A linker can be a bead or particle such as a structured nucleic acid particle.

[0093] In some of the implementations described herein, the term “moiety” can refer to a component or part of a molecule. The term does not necessarily denote the relative size of the component or part compared to the rest of the molecule, unless indicated otherwise. A moiety can include one or more atoms.

[0094] In some of the implementations described herein, the term “non-covalent,” when used in reference to a bond between atoms or moieties of a molecule, can refer to bonding due a mechanism other than electron pair-sharing between the two atoms or moieties. Non-covalent interaction can arise due to an electrostatic or magnetic interaction between moieties and / or atoms. Non-covalent binding interactions can include electrostatic interactions such as ionic bonding, hydrogen bonding, halogen bonding, Van der Waals interactions, Pi-Pi stacking, Pi-ion interactions, Pi-polar interactions, or magnetic interactions. In some cases, a non-covalent interaction may include hybridization of a first oligonucleotide to a complementary second oligonucleotide. In some cases, a non-covalent interaction may form between a receptor and ligand, such as streptavidin-biotin. Other useful non-covalent interactions can include affinity reagent-target interactions, such as antibody-epitope or aptamer-epitope interactions.

[0095] In some of the implementations described herein, the term “nucleic acid origami” can refer to a nucleic acid construct having an engineered tertiary or quaternary structure. A nucleic acid origami may include DNA, RNA, PNA, or modified or non-natural nucleic acids, or combinations thereof. A nucleic acid origami may include a plurality of oligonucleotides that hybridize via sequence complementarity to produce the engineered structuring of the origami. A nucleic acid origami may include sections of single-stranded or double-stranded nucleic acid, or combinations thereof. Exemplary nucleic acid origami structures may include nanotubes, nanowires, cages, tiles, nanospheres, blocks, and combinations thereof. A nucleic acid origami can optionally include a relatively long scaffold nucleic acid to which multiple smaller nucleic acids hybridize, thereby creating folds and bends in the scaffold that produce an engineered structure. The scaffold nucleic acid can be circular or linear. The scaffold nucleic acid can be single stranded but for hybridization to the smaller nucleic acids. A smaller nucleic acid(sometimes referred to as a “staple”) can hybridize to two regions of the scaffold, wherein the two regions of the scaffold are separated by an intervening region that does not hybridize to the smaller nucleic acid.

[0096] In some of the implementations described herein, the term “nick,” when used in reference to a double stranded nucleic acid, can refer to discontinuity in the sugar phosphate backbone of one strand of the double helix. In some cases, a nicked double stranded nucleic acid can further include a gap adjacent to the nick. Alternatively, nucleotide positions on either side of the discontinuity can occupied by nucleotides that are hybridized to nucleotides of the other strand in the double helix. As such, a nicked double stranded helix can include a gap adjacent to the site of the nick but need not necessarily include a gap adjacent to the site of the nick.

[0097] In some of the implementations described herein, the term “paratope” can refer to a molecule or part of an affinity reagent, which recognizes or binds to an epitope. A paratope may include an antigen binding site of an antibody. A paratope may include at least 1, 2, 3, or more complementarity-determining regions of an antibody. A paratope need not necessarily be present in nor derived from an antibody, for example, instead being present in a nucleic acid aptamer, lectin, streptavidin, miniprotein or other affinity reagent. A paratope need not necessarily participate in, nor be capable of, eliciting an immune response.

[0098] In some of the implementations described herein, the term "particle" can mean an object having a largest dimension between 10 nm and 1 mm. The object can be composed of a rigid or semi-rigid material. The particle can be insoluble in a fluid such as aqueous liquid. A particle can have a shape characterized, for example, as a sphere, ovoid, polyhedron, or other recognized shape whether having regular or irregular dimensions. Exemplary particles include, but are not limited to, structured nucleic acid particles (SNAPs) such as nucleic acid origami particles; optically detectable particles such as fluorescent nanoparticles, FluoSpheres™, and quantum dots; organic particles; inorganic particles; viral particles, such as phage particles having analytes displayed on their surfaces; gel particles; or particles made from solid support materials set forth herein or known in the art.

[0099] In some of the implementations described herein, the term “polyacid” can refer to a polymer having a plurality of repeating monomer subunits connected via a network of covalent bonds, wherein monomer subunits in the polymer each include a proton donating moiety. The network can include one or more chain(s) of the monomer subunits. The polymer can be linearor branched. A linear polymer includes only one chain in the network of covalent bonds. A branched polymer includes at least two chains in the network of covalent bonds. The polymer can include a single type of monomer subunit or multiple different types of monomer subunits. Accordingly, a polymer can include at least 1, 2, 3, 4, 5 or more different types of monomer subunits. Alternatively or additionally, a polymer can include at most 5, 4, 3, 2 or 1 different types of monomer subunits. A polymer having only one type of subunit in the network of covalent bonds is referred to as a “homopolymer.” In contrast, a “copolymer” includes two or more different types of subunits in the network of covalent bonds. The proton donating moiety can be, for example, a hydroxyl, carboxylic acid, sulfonic acid, or phosphoric acid. Exemplary polyacids include, but are not limited to, poly(styrene sulfonic acid) (PSS), polymethacrylic acid (PMAA) or poly(acrylic acid) (PAA).

[0100] In some of the implementations described herein, the term “polyacidic brush” can refer to a plurality of polyacids grafted to a surface. The polyacids can be grafted by one end of their polymer chains. The graft density on the surface can be at least 0.1 chains / nm2, 0.2 chains / nm2, 0.3 chains / nm2, 0.4 chains / nm2, 0.5 chains / nm2, 0.6 chains / nm2, 0.7 chains / nm2, 0.8 chains / nm2, 0.9 chains / nm2, 1 chain / nm2, 2 chains / nm2or higher. Polymer brushes are capable of transitioning from individually stable polymers to a volume-excluded or otherwise interacting system. Exemplary polyacidic brushes are set forth in Ritsema et al., ACS Appl. Polym. Mater. 4:3062-3087 (2022), which is incorporated herein by reference.

[0101] In some of the implementations described herein, the term “polymer” can refer to a molecule having a plurality of monomer subunits connected via a network of covalent bonds. The network may contain a single type of monomer subunit, or two or more types of monomer subunits. A polymer network may have a pattern of subunits or a random network of subunits (e g., a protein). The network can include one or more chain(s) of the monomer subunits. A polymer can be linear or branched. A linear polymer includes only one chain in the network of covalent bonds. A branched polymer includes at least two chains in the network of covalent bonds. For example, a branched polymer can include at least 2, 3, 4, 5, 6, 8, 10 or more chains in the network of covalent bonds. Alternatively or additionally, a branched polymer can include at most 10, 8, 6, 5, 4, 3 or 2 chains in the network of covalent bonds. A polymer can include a single type of monomer subunit or multiple different types of monomer subunits. Accordingly, a polymer can include at least 1, 2, 3, 4, 5 or more different types of monomer subunits.Alternatively or additionally, a polymer can include at most 5, 4, 3, 2 or 1 different types of monomer subunits. A polymer having only one type of subunit in the network of covalent bonds is referred to as a “homopolymer.” In contrast, a “copolymer” includes two or more different types of subunits in the network of covalent bonds.

[0102] In some of the implementations described herein, the term “pool,” when used in reference to a plurality of objects or molecules, can refer to the objects or molecules being in fluidic communication with each other. A pool can be in a fluid phase that is, in turn, in contact with a solid phase. For example, the solid phase can include immobilized objects or substances that are in communication with fluid-phase objects or substances.

[0103] In some of the implementations described herein, the term “protein” can refer to a molecule including three or more amino acids joined by peptide bonds. A protein may also be referred to as a polypeptide, oligopeptide or peptide. Although the terms “protein,” “polypeptide,” “oligopeptide” and “peptide” may optionally be used to refer to molecules having different characteristics, such as amino acid composition, amino acid sequence, amino acid length, molecular weight, origin of the molecule or the like, the terms are not intended to inherently include such distinctions in all contexts. A protein can be a naturally occurring molecule, or synthetic molecule. A protein may include one or more non-natural amino acids, modified amino acids, or non-amino acid linkers. A protein may contain D-amino acid enantiomers, L-amino acid enantiomers or both. Amino acids of a protein may be modified naturally or synthetically, such as by post-translational modifications.

[0104] In some of the implementations described herein, the term “recognize” can refer to the capability of two or more molecules to interact with each other through non-covalent bonding such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, 71-71 interactions, halogen bonding, or resonant interaction effects.

[0105] In some of the implementations described herein, the term “reaction,” when used in reference to a first molecule, particle, or moiety and a second molecule, particle, or moiety, can refer to a chemical interaction that involves both molecules, particle, or moieties. A reaction can include reversible or irreversible binding of a first molecule, particle, or moiety to the second molecule, particle or moiety. A reaction can include transfer of matter or energy from the first molecule, particle, or moiety to the second molecule, particle or moiety, such as transfer of one or more atoms, or transfer of a photon. A reaction can include a catalyzed change to the secondmolecule, particle, or moiety when it is associated to the first molecule, particle, or moiety. A reaction can include interactions of biomolecules, such as ligand-receptor binding, nucleic acid hybridization, protein-protein interactions, protein-nucleic acid interactions, etc.

[0106] In some of the implementations described herein, the term “recording,” when used in reference to a binding interaction, can refer to altering the presence of a detectable label at a unique identifier associated with an analyte, thereby providing a characteristic signal at the unique identifier until a detection event has occurred. Recording can include providing the detectable label to the unique identifier, thereby providing a characteristic presence of a signal from the detectable label at the unique identifier when the binding interaction has occurred at the unique identifier. Recording can include removing the detectable label from the unique identifier, thereby providing a characteristic absence of a signal from the detectable label at the unique identifier when the binding interaction has occurred at the unique identifier. Recording can include providing a reaction that binds an affinity reagent to an analyte in the presence of a detectable label until a detection event has occurred. Recording can include transferring a detectable label between an affinity reagent and an analyte or unique identifier, thereby providing a characteristic signal at the unique identifier until a detection event has occurred. A characteristic signal provided by the recording of a binding interaction may persist for any number of intermediate method steps that occur between the altering of a detectable label and a detection event of the characteristic signal.

[0107] In some of the implementations described herein, the term “quenched,” when used in reference to a label, can mean the label is inhibited or prevented from producing a detectable signal. For example, a luminophore can be quenched by physical proximity to a quenching agent that inhibits the luminophore from producing a detectable photon when excited by an appropriate wavelength of light. In other examples, a fluorophore can be quenched by excitation with light at a wavelength that differs from an excitatory wavelength for the fluorophore.

[0108] In some of the implementations described herein, the term "retaining component" can refer to a particle, molecule or material to which one or more moieties of an affinity reagent or analyte are attached. Exemplary retaining components include, but are not limited to, structured nucleic acid particles, nucleic acid origami, particles made of solid support materials, or polymers such as branched polymers or dendrimers. Affinity reagent moieties that can be attached to a retaining component, directly or indirectly, include for example, one or moreparatopes, one or more labels, one or more antibodies, one or more nucleic acid aptamers, one or more nucleic acid tags or the like.

[0109] In some of the implementations described herein, the term “single,” when used in reference to an object such as an analyte, can mean that the object is individually manipulated or distinguished from other objects. A single object can also be referred to as one, and only one, object. A single analyte can be a single molecule (e.g., single protein), a single complex of two or more molecules (e.g., a multimeric protein having two or more separable subunits, a single protein attached to a structured nucleic acid particle or a single protein attached to an affinity reagent), a single particle, or the like. Reference herein to a “single analyte” in the context of a composition, system or method herein does not necessarily exclude application of the composition, system or method to multiple single analytes that are manipulated or distinguished individually, unless indicated contextually or explicitly to the contrary.

[0110] In some of the implementations described herein, the term “single-analyte resolution” can refer to the detection of, or ability to detect, an analyte on an individual basis, for example, as distinguished from its nearest neighbor in an array. The term when used in reference to a singleanalyte array, refers to detection of a single-analyte under the conditions that: 1) the singleanalyte is detected by a signal with a magnitude that exceeds the magnitude of background signals for the detection system, and 2) the single-analyte is detected by a signal at a location that is spatially separated from the location of a signal corresponding to a different single-analyte (z.e., a spatial minimum of signal magnitude exists between a first single-analyte and a second single-analyte for the two single-analytes to be spatially resolved). In some cases, a signal corresponding to a first single-analyte may be considered spatially resolved from a signal corresponding to a second single-analyte if a signal minimum occurs between the locations of the two single-analytes with a magnitude that is substantially less than an average or peak signal maximum of one or both signal maxima corresponding to the first and second single analytes. For example, a signal minimum between two signal maxima corresponding respectively to a first single analyte and a second single analyte may have a magnitude that is no more than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less than 1% of an average or peak signal maximum of the two signal maxima. In some cases, signals corresponding to two or more analytes may be considered spatially resolved if a spatial resolution criterion is achieved, such as the Rayleigh Criterion. A signal magnitude (peak or average) corresponding to a single-analyte may have a signal -to-noise ratio relative to an average background signal of at least about 1.1 : 1, 1.5:1, 2:1, 2.5: 1, 3:1, 4: 1, 5:1, 10:1, 20: 1, 50: 1, 100:1 or more than 100:1.

[0111] In some of the implementations described herein, the term "solid support" can refer to a rigid substrate that is insoluble in aqueous liquid. The substrate can be non-porous or porous. The substrate can optionally be capable of taking up a liquid (e.g., due to porosity) but will typically be sufficiently rigid that the substrate does not swell substantially when taking up the liquid and does not contract substantially when the liquid is removed by drying. A nonporous solid support is generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor™, silica or silica-based materials including silicon, quartz, fused silica and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

[0112] In some of the implementations described herein, the term “structured nucleic acid particle” or “SNAP” can refer to a single- or multi-chain polynucleotide molecule having a compacted three-dimensional structure. The compacted three-dimensional structure can optionally be characterized in terms of hydrodynamic radius or Stoke’s radius of the SNAP relative to a random coil or other non- structured state for a nucleic acid having the same sequence length as the SNAP. The compacted three-dimensional structure can optionally be characterized with regard to tertiary structure. For example, a SNAP can be configured to have an increased number of internal binding interactions between regions of a polynucleotide strand, less distance between the regions, increased number of bends in the strand, and / or more acute bends in the strand, as compared to a nucleic acid molecule of similar length in a random coil or other non- structured state. Alternatively or additionally, the compacted three-dimensional structure can optionally be characterized with regard to quaternary structure. For example, a SNAP can be configured to have an increased number of interactions between polynucleotide strands or less distance between the strands, as compared to a nucleic acid molecule of similar length in a random coil or other non-structured state. In some configurations, the secondary structure (i.e. the helical twist or direction of the polynucleotide strand) of a SNAP can be configured to be more dense than a nucleic acid molecule of similar length in a random coil or other non-structured state. A SNAP can optionally be modified to permit attachment ofadditional molecules to the SNAP. A SNAP may contain DNA, RNA, PNA, or modified or nonnatural nucleic acids, or combinations thereof. A SNAP may include a plurality of oligonucleotides that hybridize to form the SNAP structure. The plurality of oligonucleotides in a SNAP may include oligonucleotides that are attached to other molecules e.g., probes, analytes such as proteins, reactive moieties, or detectable labels) or are configured to be attached to other molecules (e.g., by functional groups). A SNAP may include engineered or rationally designed structures. Exemplary SNAPs include nucleic acid origami and nucleic acid nanoballs.

[0113] As used herein, the term “tether” refers to a molecule or moiety that is configured to interact with a docker or that is interacting with a docker. A tether can be a moiety of a substance, object, molecule (e.g., affinity reagent or analyte), solid support, address, particle, or bead. A tether can include a polymer, nucleic acid strand, nucleic acid duplex, nucleotide sequence, protein, affinity reagent, epitope, paratope, receptor, ligand or the like. A tether can interact with a docker via covalent or non-covalent bonding.

[0114] In some of the implementations described herein, the term “unique identifier” can refer to a moiety, object or substance that is co-localized with an analyte and that is distinct from other identifiers, throughout one or more steps of a process. The moiety, object or substance can be, for example, a solid support such as a particle or bead; a location on a solid support; a site in an array; a tag; a label such as a luminophore; a molecular barcode such as a nucleic acid having a unique nucleotide sequence or a protein having a unique amino acid sequence; or an encoded device such as a radiofrequency identification (RFID) chip, electronically encoded device, magnetically encoded device or optically encoded device. A unique identifier can be covalently or non-covalently attached to an analyte. A unique identifier can be exogenous to a co-localized analyte, for example, being synthetically attached to the co-localized analyte. Alternatively, a unique identifier can be endogenous to the analyte, for example, being attached or co-localized with the analyte in the native milieu of the analyte.

[0115] In some of the implementations described herein, the term “vessel” can refer to an enclosure that contains a substance. The enclosure can be permanent or temporary with respect to the timeframe of a method set forth herein or with respect to one or more steps of a method set forth herein. Exemplary vessels include, but are not limited to, a well (e.g., in a multiwell plate or array of wells), test tube, channel, tubing, pipe, flow cell, bottle, vesicle, droplet that is immiscible in a surrounding fluid, or the like. A vessel can be entirely sealed to prevent fluidcommunication from inside to outside, and vice versa. Alternatively, a vessel can include one or more ingress or egress to allow fluid communication between the inside and outside of the vessel.

[0116] The embodiments set forth below and recited in the claims can be understood in view of the above definitions.Methods of Detecting Binding Interactions

[0117] In an aspect, provided herein is a method, comprising: (a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, in which the equilibration period is sufficient to form a binding equilibrium between affinity reagents of the plurality of affinity reagents and analytes of the plurality of analytes, and (b) after the equilibration period, detecting complexes comprising an affinity reagent bound to an analyte, in which the quantity of detected complexes is greater than a steady-state quantity of complexes that exists at the binding equilibrium.

[0118] In another aspect, provided herein is a method, comprising: (a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, in which the equilibration period is sufficient to form a binding equilibrium between affinity reagents of the plurality of affinity reagents and analytes of the plurality of analytes, and (b) after the equilibration period, detecting which analytes of the plurality of analytes formed a binding interaction with an affinity reagent of the plurality of affinity reagents during the equilibration period, in which the quantity of binding interactions is greater than a steady-state quantity of binding interactions that exists at the binding equilibrium.

[0119] In another aspect, provided herein is a method, comprising: (a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, in which each analyte of the plurality of analytes is individually paired to a unique identifier, (b) during the equilibration period, recording binding interactions at unique identifiers containing an affinity reagent bound to an analyte, and (c) detecting presence of absence of signals at a subset of the unique identifiers, thereby identifying analytes bound by affinity reagents during the equilibration period at the subset of unique identifiers.

[0120] In another aspect, provided herein is a method, comprising: (a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, thereby coupling affinity reagents of the plurality of affinity reagents to analytes of the plurality of analytes, wherein eachanalyte of the plurality of analytes is paired to a unique identifier, (b) forming a detectable reaction for analytes bound by an affinity reagent during the equilibration period, and (c) detecting presence of the detectable reaction at a subset of unique identifiers, thereby detecting the subset of analytes of the plurality of analytes bound by an affinity reagent of the plurality of affinity reagents.

[0121] Methods set forth herein may contain the steps of: (i) forming a binding interaction between an affinity reagent and an analyte, and (ii) forming a reaction between a first moiety and a second moiety, in which the first moiety is co-localized with the affinity reagent and the second moiety is co-localized with the analyte. A moiety may be co-localized with an affinity reagent or analyte if the moiety is attached to the affinity reagent or analyte, or if the moiety and affinity reagent or analyte are each attached to a solid support, molecule, or particle at a separation distance such that the moiety and the affinity reagent or analyte are not optically resolvable from each other. Preferably, a moiety co-localized with a first affinity reagent or analyte may not be optically resolvable from the affinity reagent or analyte, but will be optically resolvable from a moiety co-localized with a second affinity reagent or analyte.

[0122] A moiety may be co-localized with an analyte if a separation distance between the moiety and the analyte is no more than about 100 nanometers (nm), 80 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, 1 nm, or less than 1 nm. Alternatively or additionally, a moiety may be co-localized with an analyte if a separation distance between the moiety and the analyte is at least about 1 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 80 nm, 100 nm, or more than 100 nm.

[0123] A reaction may be any suitable interaction between a first moiety and a second moiety. A reaction may be characterized by at least one of: (i) inhibiting dissociation of an affinity reagent from an analyte due to the interaction of the first moiety with the second moiety, and (ii) facilitating transfer of a detectable label associated with a binding interaction between the affinity reagent and the analyte (e.g., transferring a detectable label to an analyte or a unique identifier co-localized with the analyte, transferring a detectable label from an analyte or a unique identifier co-localized with the analyte). Preferably, a reaction may co-localize a detectable label at an address containing an analyte or a unique identifier such that the detectable label remains detectable at the address in the presence of a condition that disrupts a binding equilibrium between an affinity reagent and the analyte (e.g., rinsing unbound affinity reagentsfrom contact with the analyte), or in the presence of a condition that dissociates the binding interaction between the affinity reagent and the analyte (e.g., contacting with a denaturing species, altering a pH, altering an ionic strength, altering a temperature, etc.).

[0124] Affinity reagents may be contacted to analytes for an equilibration period, in which the equilibration period is sufficient for a binding equilibrium to occur between the affinity reagents and the analytes. A binding equilibrium can occur when the rate of affinity reagents associating to analytes is equal to the rate of affinity reagents dissociating from analytes. Accordingly, for fixed quantities of affinity reagents and analytes, the total quantity of complexes formed by affinity reagents associated to analytes will be substantially constant at binding equilibrium, although the specific associated or unassociated molecules may differ between two time points during the binding equilibrium. For affinity reagents contacted to immobilized analytes, binding equilibrium may be disrupted by removal of unbound affinity reagents. The decrease in concentration of affinity reagents around the analytes may facilitate dissociation of bound affinity reagents from analytes. Likewise, binding equilibrium may be altered by changes in the chemical environment surrounding the analytes and affinity reagents. Changes in pH, ionic strength, buffer composition, surfactant concentration, and temperature can increase or decrease the quantity of affinity reagent / analyte complexes occurring at steady state.

[0125] The amount of time necessary to achieve binding equilibrium can depend upon the binding characteristics of affinity reagents and analytes, as well as surrounding chemical environment. Binding on-rates and off-rates can vary depending upon the affinity reagent and its binding target. Methods of measuring binding characteristics of affinity reagents are known in the art, and the skilled person can readily determine a sufficient equilibration period to provide a binding equilibrium between affinity reagents and analytes. An equilibration period may occur for at least about 30 seconds (s), 1 minute (min), 2 mins, 3 mins, 4 mins, 5 mins, 10 mins, 15 mins, 30 mins, 60 mins, or more than 60 mins. Alternatively or additionally, an equilibration period may occur for no more than about 60 mins, 30 mins, 15 mins, 10 mins, 5 mins, 4 mins, 3 mins, 2 mins, 1 min, 30 s, or less than 30 s.

[0126] Some methods set forth herein may comprise a step of recording a binding interaction between an affinity reagent and an analyte. A binding interaction may be directly recorded if a reaction inhibits dissociation of the affinity reagent from the analyte, thereby facilitating detection of the complex comprising the affinity reagent and analyte (e.g., by detection of adetectable label co-localized with the affinity reagent or co-localized with a moiety that forms a reaction). A binding interaction may be indirectly recorded if a reaction transfers a detectable label between the affinity reagent and analyte, thereby providing a change in detectable signal that is detectable after the affinity reagent has dissociated from the analyte.

[0127] FIG. 18 illustrates a potential advantage of recording binding interactions by methods set forth herein. Steady-state binding between a plurality of analytes (1801, 1802, 1803, 1804, and 1805) and a plurality of affinity agents (1811, 1812, 1813) is shown at three time points, ti, t2, and t3, respectively. At time point ti, affinity agent 1811 is bound to analyte 1802, affinity agent 1812 is bound to analyte 1805, and affinity agent 1813 is unbound. At time point t2, affinity agent 1811 is bound to analyte 1801, affinity agent 1813 is bound to analyte 1804, and affinity agent 1812 is unbound. At time point t3, affinity agent 1812 is bound to analyte 1804, affinity agent 1813 is bound to analyte 1802, and affinity agent 1811 is unbound. Under a steady-state equilibrium condition, 40% of analytes are observed to be bound by an affinity reagent at any given time point. In scenario A (upper), detection of the system can provide the equilibrium bound fraction, but provides no information on which analytes formed binding interactions with affinity reagents at time points before or after the detection event. In scenario B (lower), each binding interaction between an analyte and an affinity reagent is recorded by attaching a detectable label 1820 to the analyte. In scenario B, at time point t3, a greater fraction of analytes can be detected to have participated in a binding interaction with an affinity agent than a fraction of analytes bound to affinity reagents at any given time point in the steady-state equilibrium condition. If analytes are provided in a spatially-resolvable format (e.g., an array of analytes), it may be possible to detect individually whether each analyte formed a binding interaction with an affinity reagent during an equilibration period by recording the binding interactions at unique identifiers co-localized with each individual analyte.

[0128] Accordingly, for binding interactions between a plurality of analytes and a plurality of affinity reagents, binding interactions between analytes and affinity reagents may be detected for a fraction of analytes that exceeds the steady-state fraction of analytes that are bound by the affinity reagents at equilibrium. The fraction of analytes of a plurality of analytes with a detected binding interaction may exceed the steady-state fraction of analytes of the plurality of analytes that are bound by the affinity reagents at equilibrium by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more than 50%. For example, ifthe fraction of analytes of a plurality of analytes with a detected binding interaction exceeds the steady-state fraction of analytes that are bound by the affinity reagents at equilibrium by 5%, and the steady-state fraction of analytes of a plurality of analytes that are bound by the affinity reagents at equilibrium is 10%, the fraction of analytes with a detected binding interaction may be about 15% of the plurality of analytes. Alternatively or additionally, the fraction of analytes of a plurality of analytes with a detected binding interaction may exceed the steady-state fraction of analytes of the plurality of analytes that are bound by the affinity reagents at equilibrium by no more than about 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or less than 0.1%. In some cases, binding interactions between analytes and affinity reagents may be detected for a fraction of analytes that is substantially the same as or less than the steady-state fraction of analytes that are bound by the affinity reagents at equilibrium.

[0129] A method may comprise a step of removing unbound affinity reagents from contact with a plurality of analytes before detecting which analytes have formed a binding interaction with an affinity reagent during an equilibration period. Such a step may be useful if the binding interaction has been directly recorded. Alternatively, a method may comprise a step of dissociating complexes, each complex comprising an analyte associated to an affinity reagent, after contacting affinity reagents to analytes and before detecting which analytes have formed a complex with an affinity reagent during an equilibration period. Such a step may be useful if the binding interaction has been indirectly recorded. Alternatively, a method set forth herein may not comprise a step of removing unbound affinity reagents before detecting which analytes have formed a binding interaction with an affinity reagent during an equilibration period. Accordingly, detection of the binding interaction between affinity reagents and analytes may occur in the presence of unbound affinity reagents.

[0130] A method may comprise a step of recording a binding interaction between an affinity reagent and an analyte, in which recording the binding interaction between the affinity reagent and the analyte comprises forming a reaction between a first moiety co-localized with the affinity reagent and a second moiety co-localized with the analyte. In some cases, the reaction can inhibit dissociation of the affinity reagent from the complex. For example, methods of recording a binding interaction by inhibiting dissociation are shown in FIGs. 10A - 10B, FIGs. 13C - 13D, FIG. 14C, and FIGs. 15 - 17. In other cases, the reaction can produce a detectable signal. For example, methods of recording a binding interaction that produces a detectable signal are shownin FIGs. 3A - 3C, FIGs. 5A - 5C, FIGs. 8A - 8C, FIGs. 9 A - 9C, FIGs. 10A - 1 OB, FIG. 12, FIGs. 13A, 13B and 13D, FIGs. 14A - 14C, and FIGs. 15 - 17. In yet other cases, the reaction can transfer a detectable label between an affinity agent and an analyte or a unique identifier colocalized with the analyte (e.g., transfer of the detectable label from the affinity reagent to the analyte or unique identifier, transfer of the detectable label from the analyte or unique identifier to the affinity reagent). For example, methods of recording a binding interaction by transferring a detectable label are shown in FIGs. 8A - 8C, FIGs. 9A - 9C, and FIG. 14C.

[0131] In another aspect, provided herein is a method, comprising: (a) binding an affinity reagent to an analyte at a fixed spatial address, (b) coupling a kinetically-controlled detection agent to the fixed spatial address, and (c) after coupling the kinetically-controlled detection agent to the fixed spatial address, detecting a signal at the fixed spatial address. It may be especially useful to incorporate a kinetically-controlled detection agent into methods that utilize an affinity reagent with characterized superordinate and subordinate binding interactions, as set forth herein. A kinetically-controlled detection reagent may facilitate detection of a superordinate or subordinate binding interaction between an affinity reagent and a binding target, thereby facilitating identification of the binding target. Examples of methods utilizing kinetically- controlled detection agents may be found, for example, in FIGs. 9A - 9C, FIG.l 1 - 12, FIG. 13C - 13D, and FIGs 14B - 14C, and FIGs. 15 - 17.

[0132] In some methods, a binding interaction between an affinity reagent and an immobilized analyte may be directly recorded by forming a reaction that immobilizes the affinity reagent with the analyte. A kinetically-controlled binding reagent may be utilized to control the rate of formation of the reaction. Preferably, the on-rate or forward rate of the reaction is slower than the on-rate of the affinity reagent to the analyte, thereby reducing the likelihood of the reaction occurring before a binding interaction has occurred. In some cases, a reaction may bind a first moiety co-localized with the affinity reagent to a second moiety co-localized with the analyte. In such cases, a kinetically-controlled detection agent (e.g., an oligonucleotide comprising a secondary structure, a reactive functional group, a component of a ligand-receptor binding pair, etc.) may be incorporated into the first moiety, the second moiety, or both the first and second moiety. In other such cases, a kinetically-controlled detection agent (e.g., an oligonucleotide comprising a secondary structure, a moiety comprising a plurality of reactive functional groups, a moiety comprising a plurality of components of receptor-ligand binding pairs) may beincorporated into a third moiety that binds to the first moiety and the second moiety. Binding of a third moiety (e g., a bridging oligonucleotide) may be kinetically controlled (e.g., via incorporation of secondary nucleic acid structures) with respect to its binding to the first moiety, the second moiety, or to both the first and second moiety.

[0133] In some methods, a binding interaction between an affinity reagent and an immobilized analyte may be directly recorded by forming a reaction that transfers a detectable label between the affinity reagent and the immobilized analyte or a unique identifier co-localized with the analyte). Kinetically-controlled indirect recording methods could include enzymatic labeling of nucleic acid or peptide tags, photo-catalyzed binding of detectable labels to analytes or unique identifiers co-localized therewith, or transfer of a transferrable moiety between an affinity reagent and an analyte or a unique identifier co-localized therewith. Oligonucleotide moieties comprising secondary structure may be useful transferrable moieties for controlling the rate of transfer of the transferrable moiety between an affinity reagent and an analyte or a unique identifier co-localized therewith.

[0134] In some cases, an affinity reagent may be contacted to an analyte in the presence of a kinetically-controlled detection agent. Such a configuration may be utilized if the on-rate or reaction rate of the kinetically-controlled detection agent is substantially slower than the on-rate of the affinity reagent to the analyte. Alternatively, a kinetically-controlled detection agent may be contacted to an analyte after contacting an affinity reagent to the analyte. Such a configuration may be utilized if the on-rate or reaction rate of the kinetically-controlled detection agent is substantially faster or equal to the on-rate of the affinity reagent to the analyte.

[0135] The on-rate or forward reaction rate of a kinetically-controlled detection agent may be chosen to distinguish superordinate binding interactions from subordinate binding interactions of an affinity reagent. For example, if a binding on-rate of a kinetically-controlled detection agent is slower than a binding off-rate of a subordinate binding interaction of the affinity agent, it is unlikely that the reaction facilitated by the kinetically-controlled detection agent will occur in the presence of the subordinate binding interaction. In another example, if a binding on-rate of a kinetically-controlled detection agent is faster than a binding off-rate of a superordinate binding interaction of the affinity agent, it is likely that the reaction facilitated by the kinetically- controlled detection agent will occur in the presence of the superordinate binding interaction. In some cases, a method may utilize two differing kinetically-controlled detection agents, in whichthe first kinetically-controlled detection agent has a slower on-rate than the off-rate of a subordinate binding interaction, in which the second kinetically-controlled detection agent has a faster on-rate than the off-rate of a subordinate binding interaction, and in which the first and second kinetically-controlled detection agents each have a faster on-rate than the off-rate of a superordinate binding interaction. In such cases, reactions facilitated by both kinetically- controlled binding reagents will only be detected for a superordinate binding interaction of an affinity reagent and an analyte, while only the reaction of the second kinetically-controlled detection agent will be detected for a subordinate binding interaction of the affinity reagent and an analyte. Many methods set forth herein may readily be modified to include two or more differing kinetically-controlled detection agents for identification of superordinate or subordinate binding interactions.

[0136] Binding kinetics of kinetically-controlled detection agents can readily be measured by various methods, including empirical measurement of binding or reaction rates, as well as in silico estimation of binding or reaction rates for certain types of kinetically-controlled detection agents (e.g., structured oligonucleotides, enzymes, etc.). Accordingly, measured binding kinetics of kinetically-controlled detection agents can be compared to binding kinetics of affinity reagents to identify suitable combinations for methods set forth herein.

[0137] In some cases, a kinetically-controlled detection agent can comprise a detectable label (e g., a detectable label attached to or co-localized with the kinetically-controlled detection agent). For example, a bridging oligonucleotide may be provided with one or more detectable labels that facilitate detection of a signal from the one or more detectable labels when the bridging oligonucleotide is coupled to docker and tether strands, as set forth herein. Alternatively, a kinetically-controlled detection agent may not comprise a detectable label. For example, enzymatic detection agents may attach substrates to an analyte, an affinity agent, or a moiety co-localized therewith. In some cases, an affinity reagent, an analyte, or a unique identifier may comprises a detectable label.

[0138] The present disclosure further provides systems for measuring binding interactions by methods set forth herein. In another aspect, provided herein is a system, comprising: (a) a solid support comprising a plurality of sites, in which each site of the plurality of sites is optically resolvable from any other site of the plurality of sites, and (b) a plurality of analytes immobilized on the solid support, in which each site of the plurality of sites is attached to one and only oneanalyte of the quantity of analytes, in which, in a first configuration, the system further comprises a plurality of affinity reagents, in which a fraction of the affinity reagents is bound to analytes of the plurality of analytes, in which a quantity of the fraction of affinity reagents is determined by a binding equilibrium of the affinity reagents for the analytes, and in which, in a second configuration, the system is substantially devoid of affinity reagents, in which each individual site of a fraction of sites of the plurality of sites comprises a detectable label, in which a quantity of the fraction of sites is greater than a quantity of the second fraction of affinity reagents.

[0139] In another aspect, provided herein is a system, comprising: (a) a solid support comprising a plurality of sites, in which each site of the plurality of sites is optically resolvable from any other site of the plurality of sites, (b) a plurality of analytes immobilized on the solid support, in which each site of the plurality of sites is attached to one and only one analyte of the quantity of analytes, and (c) a plurality of affinity reagents bound analytes of the plurality of analytes at sites of the plurality of sites, in which the affinity reagents have a known binding equilibrium for binding to analytes of the plurality of analytes, and in which a quantity of the sites of the plurality of sites is greater than a quantity of analytes bound to affinity reagents based upon the known binding equilibrium.Attracting Affinity Reagents to Analytes Via Applied Stimuli

[0140] The present disclosure provides a method of detecting analytes. The method can include steps of (a) providing a mixture of analytes with affinity reagents, wherein the analytes are immobilized on a surface and the affinity reagents are in fluid phase; (b) applying a stimulus to attract the affinity reagents to the surface; and (c) detecting binding of affinity reagents to analytes on the surface. For ease of explanation, several configurations of the method will be exemplified herein in the context of using immobilized analytes and fluid-phase affinity reagents. However, it will be understood that the method can be carried out using immobilized affinity reagents and fluid-phase analytes. Accordingly, the method can include steps of (a) providing a mixture of analytes with affinity reagents, wherein the affinity reagents are immobilized on a surface and the analytes are in fluid phase; (b) applying a stimulus to attract the analytes to the surface; and (c) detecting binding of analytes to affinity reagents on the surface.

[0141] In some configurations, a method of detecting analytes on an array can include steps of (a) contacting an array with affinity reagents in fluid phase, wherein analytes are attached at addresses in the array; (b) allowing affinity reagents in the fluid phase to bind to analytes attached at addresses in the array; (c) removing a plurality of the affinity reagents from contact with the array, thereby retaining a fraction of the affinity reagents in contact with the array; (d) applying a stimulus to attract the fraction of affinity reagents to the array; and (e) detecting binding of affinity reagents in the fraction to analytes attached at addresses in the array. Again, the method will be exemplified herein in the context of using immobilized analytes and fluidphase affinity reagents. However, the method can be carried out using immobilized affinity reagents and fluid-phase analytes. Accordingly, the method can include steps of (a) contacting an array with analytes in fluid phase, wherein affinity reagents are attached at addresses in the array; (b) allowing analytes in the fluid phase to bind to affinity reagents attached at addresses in the array; (c) removing a plurality of the analytes from contact with the array, thereby retaining a fraction of the analytes in contact with the array; (d) applying a stimulus to attract the fraction of analytes to the array; and (e) detecting binding of analytes in the fraction to affinity reagents attached at addresses in the array.

[0142] Any of a variety of analytes can be used in a method or composition set forth herein. For ease of explanation, various methods and compositions will be exemplified herein in the context of using proteins. It will be understood that the exemplified methods and compositions can be extended to other analytes. Exemplary analytes include, but are not limited to, a tissue, cell, organelle, virus, nucleic acid (e.g., DNA or RNA), carbohydrate (e.g., monosaccharide, oligosaccharide or polysaccharide), glycan, vitamin, enzyme cofactor, hormone, or small molecule such as a candidate therapeutic agent, metabolite, nucleotide, nucleoside, amino acid, sugar, lipid, or the like. In some configurations, a composition or method set forth herein can lack one or more of the analytes set forth herein.

[0143] A composition or method set forth herein can be configured for a single analyte or for a plurality of different analytes. A plurality of analytes can include, for example, a proteome, or substantial fraction thereof, including a variety of different proteins; a genome, or substantial fraction thereof, including a variety of different DNA sequences; a transcriptome, or substantial fraction thereof, including a variety of different RNA sequences; a metabolome, or substantial fraction thereof, including a variety of different metabolites; or a microbiome, or substantialfraction thereof, including a variety of different microbes. These and other analytes known in the art can be used in compositions and methods set forth herein.

[0144] A protein or other analyte provided to a method set forth herein can be derived from a natural or synthetic source. Exemplary sources include, but are not limited to a biological tissue, fluid, cell or subcellular compartment such as an organelle (e.g., nucleus, mitochondria, chloroplast, endoplasmic reticulum, vesicle, cytoskeleton, vacuole, lysosome, cell membrane, cytosol or Golgi apparatus). For example, a sample can be derived from a tissue biopsy, biological fluid (e.g., blood, plasma, extracellular fluid, urine, mucus, saliva, semen, vaginal fluid, sweat, synovial fluid, lymph, cerebrospinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, intracellular fluid, extracellular fluid, etc.), fecal sample, hair sample, cultured cell, culture media, fixed tissue sample (e.g., fresh frozen or formalin-fixed paraffin-embedded) or protein synthesis reaction. A primary source for a cancer biomarker protein may be a tumor biopsy sample. Other sources include environmental samples or forensic samples.

[0145] Exemplary organisms from which a protein or other analyte can be derived include, but are not limited to, a mammal such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate, non-human primate or human; a plant such as Arabidopsis thaliana, tobacco, com, sorghum, oat, wheat, rice, canola, or soybean; an algae such as Chlamydomonas reinhardtii, a nematode such as Caenorhabditis elegans,' an insect such as Drosophila melanogaster , mosquito, fruit fly, honey bee or spider; a fish such as zebrafish; a reptile; an amphibian such as a frog or Xenopus laevis,' a dictyostelium discoideunr, a fungi such as Pneumocystis carinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombe or a Plasmodium falciparum. A protein can also be derived from a prokaryote such as a bacterium, Escherichia coli, staphylococci or Mycoplasma pneumoniae, ' an archae,' a virus such as Hepatitis C virus, influenza virus, coronavirus, or human immunodeficiency virus; or a viroid. A protein or other analyte can be derived from a homogeneous culture or population of the above organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0146] In some cases, a protein or other analyte can be derived from an organism that is collected from a host organism. A protein or other analyte may be derived from a parasitic, pathogenic, symbiotic, or latent organism collected from a host organism. A protein or other analyte can be derived from an organism, tissue, cell or biological fluid that is known orsuspected of being associated with a disease state or disorder (e.g., an oncogenic virus). Alternatively, a protein or other analyte can be derived from an organism, tissue, cell or biological fluid that is known or suspected of not being associated with a particular disease state or disorder. For example, one or more proteins isolated from such a source can be used as a control for comparison to results acquired from a source that is known or suspected of being associated with the particular disease state or disorder. A sample may include a microbiome. A sample may include a plurality of proteins or other analytes of interest contributed by microbiome constituents. In some cases, one or more proteins (or other analytes) used in a method, composition or apparatus set forth herein may be obtained from a single organism (e.g., an individual human), single cell, single organelle, or single protein-containing particle (e.g., a viral particle).

[0147] In some cases, one or more proteins or other analytes of interest can be obtained from a single cell, protein-containing particle (e.g., a viral particle), or organelle. A single cell, proteincontaining particle, or organelle may be collected by any known method in the art, such as fluorescence assisted cell sorting, magnetic-assisted cell sorting, and buoyancy-assisted cell sorting. In some cases, a single cell, protein-containing particle, or organelle may be collected by an emulsion technique such as liposome or micellar capture.

[0148] One or more analytes can optionally be separated or isolated from other components of the source for the analyte(s). For example, one or more proteins can be separated or isolated from lipids, nucleic acids, hormones, enzyme cofactors, vitamins, metabolites, microtubules, organelles (e.g., nucleus, mitochondria, chloroplast, endoplasmic reticulum, vesicle, cytoskeleton, vacuole, lysosome, cell membrane, cytosol or Golgi apparatus), other proteins or the like. Protein separation can be carried out using methods known in the art such as centrifugation (e.g., to separate membrane fractions from soluble fractions), density gradient centrifugation (e.g., to separate different types of organelles), precipitation, affinity capture, adsorption, liquid-liquid extraction, solid-phase extraction, chromatography (e.g., affinity chromatography, ion exchange chromatography, reverse phase chromatography, size exclusion chromatography, electrophoresis (e.g, polyacrylamide gel electrophoresis) or the like. Useful protein separation methods are set forth in Scopes, Protein Purification Principles and Practice, Springer; 3rd edition (1993).

[0149] A protein that is used in a composition or method set forth herein can be in a native or denatured conformation. For example, a protein can be in a native conformation, whereby it is capable of performing native function(s) such as catalysis of its natural substrate(s) or binding to its natural substrate(s). Alternatively, a protein can be in a denatured conformation whereby it is incapable of performing certain native function(s) such as catalysis of its natural substrate(s) or binding to its natural substrate(s). A protein can be in a native conformation for some manipulations set forth herein and in a denatured conformation for other manipulations set forth herein. A protein may be denatured at any stage during manipulation, including for example, upon removal from a native milieu or at a later stage of processing such as a stage where the protein is separated from other cellular components, fractionated from other proteins, functionalized to include a reactive moiety, attached to a particle or solid support, contacted with an affinity reagent, detected, or other manipulation. Any of a variety of denaturants can be used such as heat (e.g., temperatures greater than about 40°C, 60°C, 80°C or higher), applied force such as magnetic force or fluidic force, excessive pH (e.g., pH lower than 4.0, 3.0 or 2.0; or pH greater than 10.0, 11.0 or 12.0); chaotropic agents (e.g., urea, guanidinium chloride, or sodium dodecyl sulfate), organic solvent (e.g., chloroform or ethanol), physical agitation (e.g., sonication) and / or radiation. A denatured protein may be refolded, for example, reverting to a native state for one or more steps of a process set forth herein.

[0150] An analyte, such as a protein, can be attached to a particle, solid support or other substance. An exemplary particle is a virus particle such as a phage. A particularly useful particle is a structured nucleic acid particle. Structured nucleic acid particles can optionally include nucleic acid origami. A nucleic acid origami can include one or more nucleic acids folded into a variety of overall shapes such as a disk, tile, cylinder, cone, sphere, cuboid, tubule, pyramid, polyhedron, or combination thereof. Examples of structures formed with DNA origami are set forth in Zhao et al. Nano Lett. 11, 2997-3002 (2011); Rothemund Nature 440:297-302 (2006); Sigle et al, Nature Materials 20: 1281-1289 (2021); or US Pat. Nos. 8,501,923 or 9,340,416, each of which is incorporated herein by reference. In some configurations, a structured nucleic acid particle can include a nucleic acid nanoball and the nucleic acid nanoball can include a concatemeric repeat of amplified nucleotide sequences. The concatemeric amplicons can include complements of a circular template amplified by rolling circle amplification. Exemplary nucleic acid nanoballs and methods for their manufacture aredescribed, for example, in US Pat. No. 8,445,194, which is incorporated herein by reference. Further examples of structured nucleic acid particles are set forth in US Pat. Nos. 11,203,612 or 11,505,796; US Pat. App. Pub. No. 2022 / 0162684 Al, or US Pat. App. Ser. No. 18 / 058,000, each of which is incorporated herein by reference.

[0151] A particle, such as a structured nucleic acid particle, may have any of a variety of sizes and shapes to accommodate use in a desired application. For example, a particle can have a regular or symmetric shape or, alternatively, a particle can have an irregular or asymmetric shape. The shape can be rigid or pliable. The size or shape of a particle can be characterized with respect to length, area (i.e. footprint), or volume. The size or shape of a particle can be smaller than an address in an array to which it will associate or attach. Optionally, the size or shape of particles in a population are configured to preclude more than one of the particles from occupying an address in an array.

[0152] Optionally, a particle (e.g., a structured nucleic acid particle) or population of particles can have a minimum, maximum or average length of at least about 10 nm, 25 nm, 50 nm, 100 nm, 250 nm, 500 nm, 1 micron, 5 micron or more. Alternatively or additionally, a particle or population thereof can have a minimum, maximum or average length of no more than about 5 micron, 1 micron, 500 nm, 250 nm, 100 nm, 50 nm, 25 nm, 10 nm or less.

[0153] Optionally, a particle, such as a structured nucleic acid particle, or population thereof can have a minimum, maximum or average volume of at least about 1 micron3, 10 micron3, 100 micron3, 1 mm3or more. Alternatively or additionally, a particle or population thereof can have a minimum, maximum or average volume of no more than about 1 mm3, 100 micron3, 10 micron3, 1 micron3or less.

[0154] Optionally, the minimum, maximum or average area (i.e. footprint) for a particle, such as a structured nucleic acid particle, can be at least about 10 nm2, 100 nm2, 1 micron2, 10 micron2, 100 micron2, 1 mm2or more. Alternatively or additionally, the minimum, maximum or average area for a particle footprint can be at most about 1 mm2, 100 micron2, 10 micron2, 1 micron2, 100 nm2, 10 nm2, or less. The footprint of a particle may have a regular shape or an approximately regular shape, such as triangular, square, rectangular, circular, ovoid, or polygonal shape.

[0155] A structured nucleic acid particle (e.g., having origami or nanoball structures) may include regions of single-stranded nucleic acid, regions of double-stranded nucleic acid, or combinations thereof. For example, a structured nucleic acid particle can have a nucleic acidorigami structure which includes a scaffold strand and a plurality of staple strands. The scaffold strand can be configured as a single, continuous strand of nucleic acid, and the staples can be formed by nucleic acid strands that hybridize, in whole or in part, with the scaffold strand.

[0156] In some configurations, a nucleic acid origami includes a scaffold composed of a nucleic acid strand to which a plurality of oligonucleotides is hybridized. A nucleic acid origami may have a single scaffold molecule or multiple scaffold molecules. A scaffold strand can be linear (i.e. having a 3’ end and 5’ end) or circular (z.e. closed such that the scaffold lacks a 3’ end and 5’ end). A scaffold strand can be derived from a natural source, such as a viral genome or a bacterial plasmid. For example, a nucleic acid scaffold can include a single strand of an Ml 3 viral genome. In other configurations, a scaffold strand may be synthetic, for example, having a non-naturally occurring nucleotide sequence in full or in part. A scaffold nucleic acid can be single stranded but for a plurality of oligonucleotides hybridized thereto or short regions of internal complementarity. The size of a scaffold strand may vary to accommodate different uses. For example, a scaffold strand may include at least about 100, 500, 1000, 2500, 5000 or more nucleotides. Alternatively or additionally, a scaffold strand may include at most about 5000, 2500, 1000, 500, 100 or fewer nucleotides.

[0157] A nucleic acid origami can include one or more oligonucleotides that are hybridized to a scaffold strand. An oligonucleotide can include two sequence regions that are hybridized to a scaffold strand, for example, to function as a ‘staple’ that restrains the structure of the scaffold. For example, a single oligonucleotide can hybridize to two regions of a scaffold strand that are separated from each other in the primary sequence of the scaffold strand. As such, the oligonucleotide can function to retain those two regions of the scaffold strand in proximity to each other or to otherwise constrain the scaffold strand to a desired conformation. Two sequence regions of an oligonucleotide staple that bind to a scaffold strand can be adjacent to each other in the nucleotide sequence of the oligonucleotide or separated by a spacer region that does not hybridize to the scaffold strand.

[0158] An oligonucleotide can include a first sequence region that is hybridized to a complementary sequence of a nucleic acid origami and a second region that provides a “handle” for attaching another moiety. For example, the moiety can include an analyte (e.g., protein), paratope, affinity moiety (e.g., antibody), organic linker, inorganic ion, chemical reactant such as a click chemistry reagent, docker or tether. Optionally, the moiety can be attached to anoligonucleotide that is complementary to the second region of the handle and the moiety can be attached to the nucleic acid origami via hybridization of the handle to the complementary oligonucleotide.

[0159] Oligonucleotides can be configured to hybridize with a nucleic acid scaffold, another oligonucleotide, a staple oligonucleotide, or a combination thereof. One or more regions of an oligonucleotide that hybridizes to another sequence of a nucleic acid origami or other structured nucleic acid particle can be located at or near the 5’ end of the oligonucleotide, at or near the 3’ end of the oligonucleotide, or in a region of the oligonucleotide that is between the end regions. The oligonucleotides can be linear (z.e. having a 3’ end and a 5’ end) or closed (i.e. circular, lacking both 3’ and 5’ ends). An oligonucleotide that is included in a nucleic acid origami or other structured nucleic acid particle can have any of a variety of lengths including, for example, at least about 10, 25, 50, 100, 250, 500, or more nucleotides. Alternatively or additionally, an oligonucleotide may have a length of no more than about 500, 250, 100, 50, 25, 10, or fewer nucleotides. An oligonucleotide may form a hybrid of at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 50 or more consecutive or total base pairs with another nucleotide sequence of a nucleic acid origami. Alternatively or additionally, an oligonucleotide may form a hybrid of no more than about 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, or fewer consecutive or total base pairs with another nucleotide sequence.

[0160] A structured nucleic acid particle (e. ., nucleic acid origami, or nucleic acid nanoball) may be formed by an appropriate technique including, for example, those known in the art. Nucleic acid origami can be designed, for example, as described in Rothemund, Nature 440:297- 302 (2006), or US Pat. Nos. 8,501,923 or 9,340,416, each of which is incorporated herein by reference. Nucleic acid origami may be designed using a software package, such as CADNANO (cadnano.org), ATHENA (github.com / lcbb / athena), or DAEDALUS (daedalus-dna-origami.org).

[0161] An analyte can be attached to a label, for example, using compositions and methods set forth herein in the context of affinity reagents.

[0162] Optionally, an analyte of the present disclosure can be attached to a unique identifier in an array of unique identifiers. An array can include a number or variety of unique identifiers, for example, to accommodate a desired sample complexity. In some configurations, the array is configured for single-molecule resolution. For example, individual addresses of an array can each be attached to one, and only one, analyte. Alternatively, individual addresses of the arraycan each be attached to an ensemble of analytes. Several configurations for arrays and their methods of use will be exemplified below in the context of arrays having addresses as unique identifiers. It will be understood that the configurations can be extended to arrays having unique identifiers other than addresses.

[0163] The addresses of an array can optionally be optically observable and, in some configurations, adjacent addresses can be distinguishable when detected optically. Addresses of an array are typically discrete. The discrete addresses can be contiguous, or they can have interstitial spaces between each other. An array can have, for example, addresses that are separated by less than 100 microns, 10 microns, 1 micron, 500 nm, 100 nm, 10 nm or less. Alternatively or additionally, an array can have addresses that are separated by at least 10 nm, 100 nm, 500 nm, 1 micron, 5 microns, 10 microns, 50 microns, 100 microns or more. The addresses can each have an area of less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron or less. An array can include at least about IxlO4, IxlO5, IxlO6, IxlO8, IxlO10, IxlO12, or more addresses (e.g., addresses to which at least one analyte is attached).

[0164] Arrays can be made using methods known in the art such as those that deposit analytes at predefined addresses on a surface or those that contact an array surface with a plurality of analytes in fluid phase such that the analytes are randomly distributed to addresses in the array. Exemplary arrays and methods for making and using arrays are set forth, for example, in US Pat. Nos. 11,203,612 or 11,505,796; or US Pat. App. Pub. No. US 2023 / 0167488 Al, each of which is incorporated herein by reference.

[0165] An array can have a size and complexity that is sufficient to accommodate a plurality of proteins such as those exemplified below. Arrays of other analytes or arrays of affinity reagents can also have size and complexity exemplified below for proteins. It will be understood that the pluralities of proteins set forth below need not be limited to array configurations.

[0166] A plurality of proteins, whether present in an array or other composition set forth herein, can be characterized in terms of total protein mass. The total mass of protein in a liter of plasma has been estimated to be 70 g and the total mass of protein in a human cell has been estimated to be between 100 pg and 500 pg depending upon cells type. See Wisniewski et al. Molecular & Cellular Proteomics 13: 10.1074 / mcp.Ml 13.037309, 3497-3506 (2014), which is incorporated herein by reference. A plurality of proteins can include at least 1 pg, 10 pg, 100 pg, 1 ng, 10 ng,100 ng, 1 ug, 10 ug, 100 ug, 1 mg, 10 mg, 100 mg or more protein by mass. Alternatively or additionally, a plurality of proteins may contain at most 100 mg, 10 mg, 1 mg, 100 ug, 10 ug, 1 ug, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg or less protein by mass.

[0167] A plurality of proteins can include or be obtained from a proteomic sample. A proteomic sample can include substantially all proteins from a given source or a substantial fraction thereof. For example, a plurality of proteins may contain at least 60%, 75%, 90%, 95%, 99%, 99.9% or more of the total protein mass present in the source from which the sample was derived. Alternatively or additionally, a plurality of proteins may contain at most 99.9%, 99%, 95%, 90%, 75%, 60% or less of the total protein mass present in the source from which the sample was derived.

[0168] A plurality of proteins can be characterized in terms of total number of protein molecules. The total number of protein molecules in a Saccharomyces cerevisiae cell has been estimated to be about 42 million protein molecules. See Ho et al., Cell Systems (2018), DOI: 10.1016 / j. cels.2017.12.004, which is incorporated herein by reference. A plurality of proteins used or included in a method, composition or apparatus set forth herein can include at least 1 protein molecule, 10 protein molecules, 100 protein molecules, 1 x 104protein molecules, 1 x 106protein molecules, 1 x 108protein molecules, 1 x 1010protein molecules, 1 mole (6.02214076 * 1023molecules) of protein molecules, 10 moles of protein molecules, 100 moles of protein molecules or more. Alternatively or additionally, a plurality of proteins may contain at most 100 moles of protein molecules, 10 moles of protein molecules, 1 mole of protein molecules, 1 x 1010protein molecules, 1 x 108protein molecules, 1 x 106protein molecules, 1 x 104protein molecules, 100 protein molecules, 10 protein molecules, 1 protein molecule or less.

[0169] A plurality of proteins can be characterized in terms of the variety of full-length amino acid sequences in the plurality. For example, the variety of full-length amino acid sequences in a plurality of proteins can be equated with the number of different protein-encoding genes in the source for the plurality of proteins. Whether or not the proteins are derived from a known genome or from any genome at all, the variety of full-length amino acid sequences can be counted independent of presence or absence of post translational modifications in the proteins. A human proteome is estimated to have about 20,000 different protein-encoding genes such that a plurality of proteins derived from a human can include up to about 20,000 different full-length amino acid sequences. See Aebersold et al., Nat. Chem. Biol. 14:206-214 (2018),which is incorporated herein by reference. Other genomes and proteomes in nature are known to be larger or smaller. A plurality of proteins used or included in a method, composition or apparatus set forth herein can have a complexity that includes substantially all different nativelength amino acid sequences from a given source or a subfraction thereof. A proteome or subfraction can have a complexity of at least 2, 5, 10, 100, 1 x 103, 1 x 104, 2 x 104, 3 x 104or more different native-length amino acid sequences. Alternatively or additionally, a proteome or subfraction can have a complexity that is at most 3 x 104, 2 x 104, 1 x 104, l x 103, 100, 10, 5 , 2 or fewer different native-length amino acid sequences.

[0170] The diversity of a plurality of proteins can include at least one representative for substantially all proteins encoded by a source from which the plurality of proteins was derived or a substantial fraction thereof. For example, a plurality of proteins may contain at least one representative for at least 60%, 75%, 90%, 95%, 99%, 99.9% or more of the proteins encoded by a source from which the plurality of proteins was derived. Alternatively or additionally, a plurality of proteins may contain a representative for at most 99.9%, 99%, 95%, 90%, 75%, 60% or less of the proteins encoded by a source from which the plurality of proteins was derived.

[0171] A plurality of proteins can be characterized in terms of the variety of full-length amino acid sequences in the plurality including transcribed splice variants. The human proteome has been estimated to include about 70,000 different full-length amino acid sequences when splice variants are included. See Aebersold et al., Nat. Chem. Biol. 14:206-214 (2018), which is incorporated herein by reference. A plurality of proteins used or included in a method, composition or apparatus set forth herein can have a complexity of at least 2, 5, 10, 100, l x 103, 1 x 104, 7 x 104, 1 x 105, 1 x 106or more different full-length amino acid sequences. Alternatively or additionally, a plurality of proteins can have a complexity that is at most 1 x 106, 1 x 105, 7 x 104, 1 x 104, 1 x 103, 100, 10, 5, 2 or fewer different full-length amino acid sequences.

[0172] A plurality of proteins can be characterized in terms of the variety of protein structures therein including, for example, different full-length amino acid sequences or different proteoforms among those sequences. Different molecular forms of proteins expressed from a given gene are considered to be different proteoforms. Proteoforms can differ, for example, due to differences in primary structure (e.g., shorter or longer amino acid sequences), different arrangement of domains (e.g., transcriptional splice variants), or different post translationalmodifications (e.g., presence or absence of phosphoryl, glycosyl, acetyl, or ubiquitin moieties). The human proteome is estimated to include hundreds of thousands of proteins when counting the different primary structures and proteoforms. See Aebersold et al., Nat. Chem. Biol. 14:206-214 (2018), which is incorporated herein by reference. A plurality of proteins used or included in a method, composition or apparatus set forth herein can have a complexity of at least 2, 5, 10, 100, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 5 x 106, l x 107or more different protein structures. Alternatively or additionally, a plurality of proteins can have a complexity that is at most 1 x 107, 5 x 106, 1 x 106, 1 x 105, 1 x 104, 1 x 103, 100, 10, 5, 2 or fewer different protein structures.

[0173] A plurality of proteins can be characterized in terms of the dynamic range for the different protein structures in the plurality. The dynamic range can be a measure of the range of abundance for all different protein structures in a plurality of proteins, the range of abundance for all different primary protein structures in a plurality of proteins, the range of abundance for all different full-length primary protein structures in a plurality of proteins, the range of abundance for all different full-length gene products in a plurality of proteins, the range of abundance for all different proteoforms expressed from a given gene, or the range of abundance for any other set of different proteins set forth herein. The dynamic range for all proteins in human plasma is estimated to span more than 10 orders of magnitude from albumin, the most abundant protein, to the rarest proteins that have been measured clinically. See Anderson and Anderson Mol Cell Proteomics 1 :845-67 (2002), which is incorporated herein by reference. The dynamic range for plurality of proteins set forth herein can be a factor of at least 10, 100, l x 103, 1 x IO4, l x 106, 1 x 108, 1 x 1010, or more. Alternatively or additionally, the dynamic range for plurality of proteins set forth herein can be a factor of at most 1 x 1010, 1 x 108, 1 x 106, 1 x 104, 1 x 103, 100, 10 or less.

[0174] A sample used herein, whether containing proteins or other analytes, need not be from a biological source and can instead be from an artificial source, such as a library from a combinatorial synthesis or a library from an in vitro synthesis that exploits biological components. An artificial sample can have a range of complexity similar to those set forth herein for proteomes. A method set forth herein can detect, identify or characterize some or all proteins in a proteome or other sample including, for example, at least about 1%, 5%, 10%, 25%, 50%, 75%, 90% or 99% of the proteins in the sample.

[0175] Any of a variety of affinity reagents can be used in a composition or method set forth herein. Particularly useful affinity reagents include, but are not limited to, antibodies whether full length or functional fragments thereof (e.g., Fab’ fragments, F(ab’)2 fragments, single-chain variable fragments (scFv), di-scFv, tri-scFv, or microantibodies), or aptamers, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, miniproteins, DARPins, monobodies, nanoCLAMPs, lectins, or functional fragments thereof. The exemplified affinity reagents can be used individually. Alternatively, an affinity reagent set forth herein can be used as a paratope or moiety of an affinity reagent having a plurality of paratopes or moi eties. For example, an affinity reagent set forth herein can provide one paratope (or a subset of paratopes) of an affinity reagent having a plurality of paratopes. In some configurations, a composition or method set forth herein can lack one or more of the affinity reagents set forth herein.

[0176] An antibody is a particularly useful affinity reagent for use in a composition or method set forth herein. The antibody can be any antigen-binding molecule or molecular complex having at least one complementarity determining region (CDR) that binds to a particular epitope with high affinity. An antibody can include four polypeptide chains: two heavy chains (HC1 and HC2) and two light chains (LC1 and LC2). HC1 and HC2 can be covalently connected by one, two or more disulfide bonds. HC1 can be covalently connected to LC1 by at least one disulfide bond. HC2 can be covalently connected to LC2 by at least one disulfide bond. Each heavy chain can include a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region can include three domains, CHI, CH2 and Cm. Each light chain can include a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can further include regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL can include three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0177] An antibody can include all elements of a full-length antibody, such as those enumerated above. However, an antibody need not be full length and functional fragments can be particularly useful for many applications. The term “antibody” as used herein encompasses full length antibodies and functional fragments thereof. A functional fragment can be naturally occurring, enzymatically obtainable, synthetic, or genetically engineered. An antibody can be obtained using any suitable technique such as proteolytic digestion or recombinant geneticengineering techniques involving the manipulation and expression of DNA encoding one or more antibody domains. Such DNA is readily available, for example, from commercial sources, DNA libraries (e.g., phage-antibody libraries), or can be synthesized. The DNA may be manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, introduce cysteine residues, remove cysteine residues, modify, add or delete other amino acids, etc.

[0178] A functional fragment of an antibody can include any fragment that is capable of binding to an epitope with a detectable affinity, such as a Fab, Fab’, F(ab’)2, Fd, Fv, dAb, single-chain variable (scFv), di-scFv, tri-scFv, microantibody, or minimal recognition unit consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR1, CDR2 or CDR3 peptide). Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains can also be useful.

[0179] A functional fragment of an antibody will typically include at least one variable domain. The variable domain may have any of a variety of sizes or amino acid compositions and will generally include at least one CDR which is adjacent to or in frame with one or more framework sequences. For antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, a functional fragment of an antibody may contain a monomeric VH or VL domain.

[0180] In particular configurations, a functional fragment of an antibody contains at least one variable domain covalently connected to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) VH - CHI; (ii) VH - Cm; (iii) VH - CHS; (iv) VH - CHI- Cm; (v) VH - CHI - Cm - Cm; (vi) VH - Cm - Cm; (vii) VH - CL; (viii) VL- CHI; (ix) VL - Cm; (x) VL - Cm; (xi) VL - Cm; (xii) VL - CHI- Cm- Cm; (xiii) VL - Cm- Cm; and (xiv) VL - CL. In any configuration of variable and constant domains, including any of theexemplary configurations listed above, the variable and constant domains may be either directly connected to one another or may be connected by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., at least 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody may include a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0181] An affinity reagent of the present disclosure can include one or more paratopes. For example, an affinity reagent can include at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or more paratopes. Alternatively or additionally, an affinity reagent can include at most 25, 20, 15, 10, 5, 4, 3, 2, or 1 paratopes. Multiple paratopes that are present in an affinity reagent can have the same function or different functions compared to each other. For example, the multiple paratopes can each have affinity for the same epitope or same set of epitopes. In some cases, the strength and / or specificity of the affinity can be substantially the same, for example, in cases where the paratopes have the same structure. In other cases, the strength and / or specificity of two or more paratopes for a given epitope or set of epitopes can overlap despite some differences in the functional or structural characteristics of the two or more paratopes. In some configurations, multiple paratopes of a given affinity reagent can have affinity for different epitopes. This can be the case, whether the different epitopes are found in the same analyte (e.g., two different amino acid trimer epitopes present in a given protein analyte) or in different analytes e.g., a first trimer epitope being found in a first protein that lacks a second trimer epitope, and a second trimer epitope being found in a second protein that lacks the first trimer epitope).

[0182] In some configurations of the methods, compositions or systems set forth herein, two or more affinity reagents can be present as moi eties of a multimeric affinity reagent. For example, an affinity reagent can include two or more affinity moieties, wherein the affinity moieties are selected from an affinity reagent set forth herein or known in the art. Two or more affinity moieties can be combined via attachment to any of a variety of retaining components including, for example, a structured nucleic acid particle (SNAP), nucleic acid origami, artificial polymer or particle. Other particles (e.g., particles composed of solid support material set forth herein or known in the art) or substances, such as those set forth herein in the context of mediatingattachment of a protein to a solid support, can be used as retaining components for affinity reagents. The presence of multiple affinity moieties in an affinity reagent can provide increased binding strength, for example, due to increased avidity as compared to any one of the affinity moieties when used as an individual affinity reagent. In some configurations an affinity reagent can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more affinity moieties. Alternatively or additionally, an affinity reagent can include at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or fewer affinity moieties. It may be convenient to characterize an affinity reagent with respect to the number of paratopes it includes. For example, an affinity reagent can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more paratopes. Alternatively or additionally, an affinity reagent can include at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or fewer paratopes. Typically, the affinity moieties or paratopes that are present in an affinity reagent will be structurally identical. For example, a plurality of antibodies in an affinity reagent can have identical amino acid sequences.

[0183] Whether or not a plurality of affinity moieties or a plurality of paratopes include structurally identical members, the members can recognize the same epitopes. In some cases, the members can recognize the same epitopes with substantially the same binding strength. It will be understood, however, that in some cases an affinity reagent can include two or more affinity moieties having different structures and different binding affinities compared to each other. Similarly, an affinity reagent can include two or more paratopes having different structures and different binding affinities compared to each other.

[0184] An affinity reagent of the present disclosure can include one or more labels. For example, an affinity reagent can include at least 1, 2, 3, 4, 5, 10, 15, 20 or more labels. Alternatively or additionally, an affinity reagent can include at most 20, 15, 10, 5, 4, 3, 2, or 1 labels. In some configurations, an affinity reagent can be attached to one or more labels. For example, an affinity reagent can include a particle (e.g., structured nucleic acid particle) that is attached to at least one paratope and further attached to at least one label. Methods and compositions set forth herein in the context of labels although exemplified for affinity reagents can be extended to other molecules such as analytes (e.g., proteins).

[0185] Multiple labels that are present in an affinity reagent can have the same structure as each other or they can differ structurally from each other. Optionally, multiple labels can have different detectable characteristics. For example, two or more optical labels can differ in terms of luminescence lifetime, luminescence polarity, extinction coefficient, quantum yield ofluminescence, spectral region for absorbance, spectral region for excitation or spectral region for emission. Alternatively, two or more optical labels can have overlapping detectable characteristics, for example, in terms of luminescence lifetime, luminescence polarity, extinction coefficient, quantum yield of luminescence, spectral region for absorbance, spectral region for excitation or spectral region for emission. This can result from the two or more optical labels having the same structure, but in some cases two or more labels can have the same or overlapping detection properties despite having different structures.

[0186] A label of a molecule, such as an affinity reagent, can be exogenous or endogenous to the molecule. Exogenous labels can be attached to an affinity reagent or other molecule using methods and compositions known in the art such as artificial linkers or genetic fusions. A wide variety of labels can be used in a composition or method set forth herein including, for example, optically detectable labels, such as luminophores (e. ., fluorophores), enzymes (e.g., enzymes which catalyze reactions with colored reagents or products), electrochemical labels (e.g., highly charged moieties). Magnetic contrast imaging moieties can be used such as gadoliniumdiethylenetriaminepentacetate (Gd-DTPA), gadolinium-dodecane tetraacetic acid (Gd-DOTA) or others used for magnetic resonance techniques. Moieties that are detected through subsequent processing, such as nucleic acid barcode labels, can also be useful. Nucleic acids can be detected or identified via nucleic acid amplification, sequencing or hybridization assays.

[0187] Any of a variety of luminophores may be used herein. Luminophores may include labels that emit in the ultraviolet, visible, or infrared region of the spectrum. In some cases, the luminophore may be selected from the group consisting of FITC, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750, Pacific Blue, Coumarin, BODIPY FL, Pacific Green, Oregon Green, Cy3, Cy5, Pacific Orange, TRITC, Texas Red, R- Phycoerythrin, Allophcocyanin (APC). In some cases, the label may be an Atto dye, for exampleAtto 390, Atto 425, Atto 430, Atto 465, Atto 488, Atto 490, Atto 495, Atto 514, Atto 520, Atto532, Atto 540, Atto 550, Atto 565, Atto 580, Atto 590, Atto 594, Atto 610, Atto 611, Atto 612,Atto 620, Atto 633, Atto 635, Atto 647, Atto 655, Atto 680, Atto 700, Atto 725, Atto 740, AttoMB2, Atto Oxal2, Atto RholOl, Atto Rhol2, Atto Rhol3, Atto Rhol4, Atto Rho3B, Atto Rho6G, or Atto Thiol2. In some cases, the luminophore may be a fluorescent protein such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP),blue fluorescent protein (BFP), orange fluorescent protein (OFP), and yellow fluorescent protein (YFP). A wide range of effective luminophores are commercially available, for example, from the Molecular Probes division of ThermoFisher Scientific and / or generally described in the Molecular Probes Handbook (11thEdition) which is hereby incorporated by reference. Label components may also include intercalation dyes, such as ethidium bromide, propidium bromide, crystal violet, 4',6-diamidino-2-phenylindole (DAPI), 7-aminoactinomycin D (7-AAD), Hoescht 33258, Hoescht 33342, Hoescht 34580, YOYO-1, DiYO-1, TOTO-1, DiTO-1, or combinations thereof.

[0188] Optionally, an affinity reagent can be attached to a solid support or particle. A particularly useful particle is a structured nucleic acid particle (e.g., nucleic acid origami), for example, having structural or functional characteristics set forth herein in the context of attachment to proteins and other analytes. An analyte, affinity reagent, docker, tether, label or other moiety can be attached to a nucleic acid origami via a scaffold component or oligonucleotide component. For example, the scaffold or oligonucleotide can include a nucleotide analog that forms a covalent or non-covalent bond with the attached moiety.

[0189] Any of a variety of chemistries can be used to attach an analyte, affinity reagent, docker, tether or other moiety to a solid support or particle (e.g., structured nucleic acid particle). The attachment can be covalent. Exemplary covalent chemistries include, but are not limited to, click chemistries or chemistries set forth in U.S. Pat. Nos. 11,203,612 or 11,505,796; or US Pat. App. Pub. No. 2022 / 0162684 Al, each of which is incorporated herein by reference. Another example is the Spy Tag / Spy Catch er system (See, Zakeri et al. Proceedings Nat 'I Acad. Sciences USA. 109 (12): E690-7 (2012)). In this system, a 13 amino acid tag polypeptide (Spy Tag) forms a first coupling handle, with a 12.3 kDa protein (Spy-Catcher) forming the other coupling handle. The SpyCatcher can function by irreversibly bonding to a SpyTag through an isopeptide bond. Any of a variety of non-covalent bonds can be used to attach an analyte, affinity reagent or other moiety to a solid support or particle (e.g., structured nucleic acid particle). Receptors and their ligands can be particularly useful. Examples include, but are not limited to, antibodies, antigens, (strept)avidin (or analogs thereof), biotin (or analogs thereof), affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, monobodies, nanoCLAMPs, nucleic acids, peptide nucleic acids, polypeptides, nucleic acid aptamers, protein aptamers, lectins (or analogs thereof), carbohydrates or functional fragments thereof. Complementary nucleic acids can beused to non-covalently attach a functional moiety to a solid support or particle (e.g., structured nucleic acid particle). Useful nucleic acids can have complementary sequences that are at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more nucleotides in length. Alternatively or additionally, nucleic acids can have complementary sequences that are at most 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5 or fewer nucleotides in length. Reagents and techniques that can be used to non-covalently attach an affinity reagent or other moiety to a particle (e.g., structured nucleic acid particle) are set forth in U.S. Pat. Nos. 11,203,612 or 11,505,796; or US Pat. App. Pub. No. 2022 / 0162684 Al, each of which is incorporated herein by reference.

[0190] A structured nucleic acid particle that is made or used in a method set forth herein can be suspended in a fluid, immobilized on a solid support, or immobilized in another material such as a gel or solid support material. This can be the case before, during or after being attached to a moiety of interest, such as a tether, docker, linker, label, analyte or affinity moiety. For example, a population of structured nucleic acid particles can be colloidal for some, or all steps of a method set forth herein. Alternatively, a population of structured nucleic acid particles can be immobilized in, or on, a solid support for some, or all steps of a method set forth herein. For example, analytes or affinity reagents can be attached to addresses (or other unique identifiers) of an array via structured nucleic acid molecules.

[0191] A particle need not be composed primarily of nucleic acid and, in some cases, may be devoid of nucleic acids. For example, an analyte, affinity reagent, label, linker, docker or tether can be attached to an artificial polymer that is configured to form a particle. In other examples, a particle can be composed of a solid support material, such as those set forth herein, glass, silicon, silica, carbon, cellulose, polyethylene glycol (PEG), upconversion nanocrystal, or a quantum dot.

[0192] Delivery and removal of fluids will be set forth below in the context of fluid-phase affinity reagents and immobilized analytes (e.g., proteins attached to addresses of an array). This is done for ease of explanation. It will be understood that similar compositions and methods can be used for fluid-phase analytes and immobilized affinity reagents.

[0193] A method of the present disclosure can include a step of contacting an array of analytes with affinity reagents. Typically, the affinity reagents are in a fluid phase that is delivered to the array and the analytes are immobilized to addresses in the array. Any of a variety of fluidics techniques or apparatus can be employed for delivery of the fluid phase. For example, a fluid phase can be provided in a tube, pipette tip, syringe or the like and delivered via fluiddisplacement. In some cases, the array can be present in the lumen of a flow cell or other vessel having an inlet and outlet, and a fluid phase can enter the lumen via displacement of fluid through the inlet. The fluid phase can also exit the flow cell, at least in part, via displacement through the outlet. Other methods can be employed to deliver fluid to an array such as dipping the array into a vessel containing the fluid, flowing fluid through a nozzle to the array, or spin coating fluid on the surface of the array.

[0194] A fluid phase that is in contact with an array can contain affinity reagents in a desired quantity as measured, for example, by concentration, mass or number of affinity reagent molecules. For example, the concentration of affinity reagents in contact with an array can be at least 10 nM, 100 nM, 1 pM, 10 pM, 100 pM, 1 mM, 10 mM, 100 mM or higher. Alternatively or additionally, the concentration of affinity reagents in contact with an array can be at most 100 mM, 10 mM, 1 mM, 100 pM, 10 pM, 1 pM, 100 nM, 10 nM or lower. The quantity of affinity reagents in a fluid phase is typically greater than the quantity of analytes in an array to which the fluid is in contact. For example, a fluid phase can contain a number of affinity reagent molecules that is at least 1.5x, 2x, 5x, lOx, 50x, lOOx, lOOOx, lOOOOx or greater relative to the number of analytes in an array to which the fluid phase is in contact. However, in some cases the fluid can contain an equivalent or lower quantity of affinity reagents compared to the quantity of analytes in the array. For example, a fluid phase can contain a number of affinity reagent molecules that is at most lx, 0.5x, O.lx, O.Olx or less relative to the number of analytes in an array to which the fluid phase is in contact.

[0195] In particular configurations of the methods set forth herein, a fluid phase is contacted with an array and the affinity reagents are present at a first concentration in the fluid phase. A plurality of the affinity reagents can then be removed, thereby retaining a fraction of the affinity reagents in contact with the array. Thus, the quantity of affinity reagents in contact with the array is reduced. The fraction of affinity reagents can be contained in a fluid and can be present at a second concentration that is higher, equal or lower compared to the first concentration. For example, the concentration of affinity reagents can be reduced for the retained fraction if the fluid volume is equivalent before and after removal of the plurality of affinity reagents. Alternatively, a higher concentration of affinity reagents can result for the retained fraction if the volume of fluid for the retained fraction is substantially lower after removal of the plurality of affinity reagents.

[0196] Removal of fluid from an array can be carried out using any of a variety of fluidics techniques or apparatus including, but not limited to, those set forth above in the context of delivering fluids to an array. For example, at least a portion of the fluid in contact with an array can be removed by fluid displacement. The fluid can be displaced with an equivalent volume of liquid or gas. Displacement with gas can be particularly useful for reducing the volume of fluid in contact with the array. Liquid displacement can be performed to result in replacement of at least a portion of a first fluid phase with a second fluid phase. Displacement with a second fluid phase can be useful for adding or replacing reagents, cofactors, salts, or other fluid components. For example, the second fluid can contain affinity reagents of the same or different type as the first fluid that is displaced. Other methods that can be employed to remove affinity reagents from an array or vessel include, for example, precipitation of the affinity reagents, solid-phase extraction of the affinity reagents, liquid-liquid extraction of the affinity reagents, capture of the affinity reagents via a solid-phase receptor or ligand that binds to the affinity reagents, or dialysis of the affinity reagents.

[0197] Optionally, a step of removing a plurality of the affinity reagents from contact with an array can be carried out after allowing affinity reagents to bind to analytes in the array. For example, an array can be in contact with a fluid that contains affinity reagents and, after allowing affinity reagents in the fluid to bind to analytes in the array, a plurality of the affinity reagents can be removed. Binding can be allowed to occur under passive diffusion conditions. Alternatively, binding can be facilitated by active transport, for example, by application of a stimulus, such as a variably applied stimulus. For example, an affinity reagent can include charged moieties and an electric field or electrophoretic force can be applied to actively transport the affinity reagents to addresses of an array to which analytes are attached. The electric field or electrophoretic force can be reversed or otherwise varied to reduce or reverse attraction of the affinity reagent to the array. In another example, an affinity reagent can include magnetic or paramagnetic moieties and a magnetic field or magnetic force can be applied to actively transport the affinity reagents to addresses of an array to which analytes are attached. Again, the magnetic field or magnetic force can be variably applied. In some configurations of the methods set forth herein, active transport is not employed during one or more binding step. For example, a method can be configured to include steps of (a) contacting an array of analytes with affinity reagents in fluid phase; (b) allowing affinity reagents in the fluid phase to bind to analytes in the arraywithout employing active transport (e.g., using passive diffusion of affinity reagents to the array); (c) removing a plurality of the affinity reagents from contact with the array, thereby retaining a fraction of the affinity reagents in contact with the array; (d) attracting the fraction of affinity reagents to the array via active transport or application of a force; and (e) detecting binding of affinity reagents in the fraction to analytes attached at addresses in the array.

[0198] Detection techniques and apparatus will be set forth below in the context of fluid-phase affinity reagents and immobilized analytes (e.g., proteins attached to addresses of an array). This is done for ease of explanation. It will be understood that similar compositions and methods can be used for fluid-phase analytes and immobilized affinity reagents.

[0199] A method of the present disclosure can include a step of detecting binding of affinity reagents to analytes in an array. Binding of affinity reagents with analytes can be detected using any of a variety of techniques that are appropriate to the assay components used. For example, an affinity reagent can be detected at a unique identifier (e.g., address) in an array by acquiring a signal from a label attached to the affinity reagent when bound to an analyte at the unique identifier. In some configurations, a complex between an affinity reagent and analyte need not be directly detected, for example, in formats where a nucleic acid tag or other moiety is created or modified as a result of binding. Optical detection techniques such as luminescent intensity detection, luminescence lifetime detection, luminescence polarization detection, or surface plasmon resonance detection can be useful. Other detection techniques include, but are not limited to, electronic detection such as techniques that utilize a field-effect transistor (FET), ionsensitive FET, or chemically-sensitive FET. Exemplary detection techniques and apparatus are set forth in US Pat. No. 10,473,654 or US Pat. App. Pub. No. 2022 / 0162684 Al, each of which is incorporated herein by reference. A detection technique used in a method set forth herein can be configured to resolve addresses (or other unique identifiers) of an array. For example, a detection technique can be configured for single-molecule resolution of analytes.

[0200] In some configurations of the methods set forth herein, affinity reagents that are not bound to analytes at addresses of an array are removed from contact with the array prior to detecting affinity reagents that are bound to the addresses. Removal of non-bound affinity reagents can provide the advantage of reducing unwanted background when detecting bound affinity reagents. This can be particularly helpful when affinity reagents have labels that produce detectable signals in both the bound and non-bound state. One option to avoid or reduceunwanted background signals is to remove non-bound affinity reagents. However, this approach may change the dynamics of binding between affinity reagent and analyte unless remediation is performed, such as detecting bound affinity reagents on a time scale that is faster than the rate at which binding equilibrium is substantially shifted. Another option for reducing unwanted signal from non-bound affinity reagents is to use a detection apparatus that is capable of spatially confined collection of signals. For example, total internal reflectance can be used to collect signals for affinity reagents bound at or near an array surface while rejecting signals produced further away from the surface of the array. Another example is the use of waveguides such as zero mode waveguides for spatially resolved excitation of labels and / or spatially resolved acquisition of emission signals from excited labels. Other examples of spatially resolved detection of labeled affinity reagents include, but are not limited to, detection of luminescent moieties using confocal fluorescence microscopy, detection of affinity reagents using surface plasmon resonance (SPR) or detection of charged moieties using field effect transistors.

[0201] Any of a variety of proximity -based detection techniques can be used to detect binding of affinity reagents to analytes. A particularly useful method is detection of energy transfer between a donor and acceptor. For example, Forster resonance energy transfer (FRET) can be detected between a first luminophore i.e. donor) and second luminophore (i.e. acceptor).Energy transfer techniques utilize a donor that is excited by an energy source, such as a laser or other radiation source, and an acceptor that receives energy from the donor to, in turn, produce a detectable signal, such as a luminescence signal. The efficiency of energy transfer is inversely proportional to distance between donor and acceptor, making energy transfer sensitive to proximity on a scale that is comparable to the distances between affinity reagents and the analytes to which they bind. Alternatively, methods described below and shown in FIGs. 15 - 17 may be useful for providing an amplified signal without rinsing unbound affinity reagents from an array.

[0202] FIG. 2 shows a cross section of a region of a flow cell having two protein analytes (white globules) immobilized on the lower surface and a fluid phase containing affinity reagents (Y shapes), wherein the affinity reagents are attached to a first luminophore (open circle) and the protein analytes are attached to a second luminophore (closed circle). The first luminophore and second luminophore are capable of FRET when in proximity to each other, with either being the donor and the other being the acceptor. Using appropriate optics including, for example, a filterto selectively collect emission from the acceptor and to reject emission from the donor, binding between the affinity reagent and protein can be detected in the presence of unbound affinity reagent. Thus, detection can occur while equilibrium is maintained due to the concentration of free affinity reagent in solution phase; however, emission produced by free affinity reagent does not produce substantial background interference. Returning to FIG. 2, FRET can be detected due to binding of the affinity reagent to the protein on the left and absence of signal from the address for the protein on the right is indicative of the affinity reagent not recognizing the protein nor binding to the protein. It will be understood that FRET can be used similarly if the protein is in fluid phase and the affinity reagent is immobilized on the lower surface of the flow cell. The use of FRET in the context of FIG. 2 is exemplary and it will be understood that other proximitybased detection techniques can be used instead.

[0203] Other proximity -based detection techniques that can be used in a method set forth herein include those that employ various pairs of components that produce unique signals when in proximity to each other compared to when they are apart. For example, a luminophore component and quencher component can be used to detect proximity via quenching of signal that would otherwise be produced by the luminophore. A two component luminophore systems can also be useful. For example, fluorogen-activated-proteins utilize a protein component and fluorogen component to produce unique fluorescent signal when in proximity to each other (see, for example, Gallo, Bioconjugate Chem. 31 : 16-27 (2020). In another example, an intercalating dye component and nucleic acid component can produce a unique optical signal when in proximity to each other. Also useful are detection components used in split protein assays such as two parts of an enzyme that are uniquely functional when in proximity to each other.Exemplary enzymes that can be split into components for use in proximity-based detection include, but are not limited to beta lactamase, dihydrofolate reductase, focal adhesion kinase, Green Fluorescent Protein (and variants thereof), horseradish peroxidase, and luciferase.

[0204] An analyte or a unique identifier (e.g., address) to which an analyte is attached can be colocalized with a first component of a proximity-based detection pair (e.g., a FRET donor), for example, via covalent or non-covalent attachment; and an affinity reagent can be co-localized with a second component of the proximity -based detection pair (e.g., a FRET acceptor), for example, via covalent or non-covalent attachment. The opposite placement of donor and acceptor is also possible (i.e. the analyte or its unique identifier can be co-localized with the acceptor, andthe affinity reagent can be co-localized with the donor). Using a pair of proximity-based detection components (e.g., donor and acceptor) allows interaction between affinity reagent and analyte to be distinguished even in the presence of unbound affinity reagent.

[0205] Proximity-based detection can be facilitated by dockers and tethers. An analyte used in a composition or method of the present disclosure can have a docker and the docker can be attached to a first component of a proximity-based detection pair (e.g., a FRET donor or acceptor used for energy transfer). An affinity reagent used in a composition or method of the present disclosure can have a tether and the tether can be attached to a second component of a proximitybased detection pair (e.g., a FRET donor or acceptor used for energy transfer). The docker and tether can be configured to position the components of the pair in proximity to each other when the affinity reagent is bound to the analyte. As such, the docker and tether can facilitate proximity -based detection (e.g. energy transfer) and, thus, facilitate detection of binding between the affinity reagent and analyte.

[0206] FIG. 3A shows a diagram of a protein (white globule) attached to a surface, wherein the protein is co-localized with a first luminophore (closed circle), and an affinity reagent (Y-shape) that is bound to the protein, wherein the affinity reagent is co-localized with a second luminophore (open circle). The first luminophore is co-localized with the protein via a nucleic acid docker that attaches the luminophore to the address where the protein resides. The second luminophore is co-localized with the affinity reagent via a nucleic acid tether that attaches the luminophore to the affinity reagent. In this example, short regions of the docker and tether are complementary to each other and function to position the first and second luminophores for FRET. It will be understood that FRET can be used similarly to FIG. 3A if the protein is in fluid phase and the affinity reagent is immobilized on the lower surface of the flow cell.

[0207] FIG. 3B shows a diagram of a protein (white globule) attached to a surface, wherein the protein is co-localized with a first luminophore (closed circle), and an affinity reagent (Y-shape) that is bound to the protein, wherein the affinity reagent is co-localized with a second luminophore (open circle). The first luminophore is co-localized with the protein via a nucleic acid docker that attaches the luminophore to the address where the protein resides. The second luminophore is co-localized with the affinity reagent via a nucleic acid tether that attaches the luminophore to the affinity reagent. In this example, the docker and tether are not complementary to each other, but are complementary to adjacent regions of a splintoligonucleotide. Hybridization of the docker and tether to the splint oligonucleotide positions the first and second luminophores for FRET. An advantage of using a splint oligonucleotide is that modifications can be made to the strength and timing of the interaction that brings the luminophores into proximity for FRET, for example, by adjusting the concentration of the splint oligonucleotide independently from the concentration of the affinity reagent. Thus, the concentration of affinity reagent can be maintained at a level that facilitates binding of the affinity reagent to the protein and changes can be made to the concentration of the splint oligonucleotide to reduce, or even minimize, the impact of docker-tether-splint oligonucleotide interactions on the apparent binding affinity of the affinity reagent for the protein. Similarly, the splint oligonucleotide can be added after formation of a binding complex between the affinity reagent and the protein and, optionally, after removal of unbound affinity reagents, thereby reducing the impact of the docker-tether-splint oligonucleotide interactions on the apparent binding affinity of the affinity reagent for the protein. It will be understood that FRET can be used similarly to the example of FIG. 3B if the protein is in fluid phase and the affinity reagent is immobilized on the lower surface of the flow cell.

[0208] Any of a variety of molecules or chemical moieties can be used as dockers or tethers. Polymers can be particularly useful and can be selected for use based on a variety of characteristics such as length, flexibility, chemical reactivity or inertness to various chemicals. A docker or tether can have a composition exemplified herein in the context of linkers. A particularly useful docker or tether is a nucleic acid. The nucleic acid can be single-stranded or double-stranded. Single-stranded nucleic acids are typically more flexible whereas doublestranded nucleic acids provide relative rigidity. Optionally, a docker strand can have a nucleotide sequence that complements a nucleotide sequences of a tether strand. Similarly, a tether strand can have a nucleotide sequence that complements a nucleotide sequences of a docker strand. The length of a complementary region formed between a docker and tether can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more base pairs. Alternatively or additionally, the complementary region can be at most 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 base pairs. The complementary region can include A:T basepairs and G:C basepairs, only G:C basepairs, at most 90% G:C basepairs, at most 80% G:C basepairs; at most 70% G:C basepairs, at most 60% G:C basepairs, at most 50% G:C basepairs, at most 40% G:C basepairs, at most 30% G:C basepairs, at most 20% G:C basepairs, at most 10% G:C basepairs, only A:T basepairs, at most 90% A:Tbasepairs, at most 80% A:T basepairs; at most 70% A:T basepairs, at most 60% A:T basepairs, at most 50% A:T basepairs, at most 40% A:T basepairs, at most 30% A:T basepairs, at most 20% A:T basepairs, or at most 10% A:T basepairs. In some configurations of the methods and compositions set forth herein a docker and tether are not substantially complementary with each other.

[0209] A docker can be co-localized with an analyte via covalent and / or non-covalent attachment of the docker to the analyte. Exemplary attachment chemistries include those set forth herein in the context of attaching analytes and affinity reagents to solid supports and particles. In some configurations, a docker can be attached to a particle (e.g. structured nucleic acid particle) or solid support to which an analyte is attached. Optionally, one or more dockers can be co-localized with an analyte via attachment to an address or other unique identifier to which the analyte is attached.

[0210] An analyte can be co-localized with a single docker or, alternatively, with a plurality of dockers. For example, an analyte can be co-localized with at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dockers. Alternatively or additionally, an analyte can be co-localized with at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or fewer dockers. The dockers can be substantially identical to each other.Alternatively, a plurality of dockers can include dockers that differ from each other. In some cases, the different dockers will have different lengths or different nucleotide sequences. In some configurations, an analyte and the docker with which it is co-localized will have binding characteristics that are orthogonal to each other. As such, a paratope of an affinity reagent that recognizes or binds to the analyte will not recognize or bind to the docker, and a tether will not recognize or bind to the analyte.

[0211] An array of analytes can include a plurality of unique identifiers (e.g. addresses) each of which is co-localized with a different analyte. For example, the unique identifiers in an array can each be attached to a different analyte, such as a different protein. Each unique identifier in an array can also be attached to at least one docker. Optionally, the array utilizes universal dockers, whereby the dockers are substantially the same for some or all unique identifiers in the array. As such, each unique identifier in an array can be co-localized with an analyte that differs from analytes at other unique identifiers in the array and a universal docker that is the same as universal dockers at other unique identifiers in the array. In an exemplary configuration, addresses in an array are each attached to a different protein selected from a protein sample, andthe universal docker that is attached to each of the addresses is the same. Accordingly, individual addresses can be distinguished based upon the unique structure of the attached analyte but the universal docker provides a structure that is universal across the addresses.

[0212] In some configurations, an array can include indexed dockers, wherein unique identifiers in one subset are distinguishable from unique identifiers in another subset based on detectable properties of the indexed dockers with which they are respectively co-localized. Accordingly, an array can include at least 1, 2, 3, 4, 5, 10 or more subsets of indexed dockers, wherein each subset of indexed dockers is co-localized with a respective subset of unique identifiers in the array. An array need not use universal nor indexed dockers. For example, individual addresses (or other unique identifiers) in an array can be attached to unique dockers, wherein the addresses are distinguishable based on the structure of the attached docker.

[0213] A tether can be co-localized with an affinity reagent via covalent and / or non-covalent attachment of the tether to the affinity reagent. Exemplary attachment chemistries include those set forth herein in the context of attaching analytes and affinity reagents to solid supports and particles. In some configurations, a tether can be attached to a particle (e.g. structured nucleic acid particle) or solid support to which an affinity reagent is attached. For example, one or more tethers can be co-localized with an affinity reagent via attachment to a structured nucleic acid particle to which the affinity reagent is attached.

[0214] An affinity reagent can be co-localized with a plurality of tethers. For example, an affinity reagent can be co-localized with at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more tethers. Alternatively or additionally, an affinity reagent can be co-localized with at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or fewer tethers. The tethers can be substantially identical to each other. Alternatively, a plurality of tethers can include tethers that differ from each other. In some cases, the different tethers will have different lengths or different nucleotide sequences. In some configurations, an affinity reagent and the tether with which it is co-localized will have orthogonal binding recognition. As such, an analyte that recognizes or binds to a paratope of the affinity reagent will not recognize or bind to the tether, and a docker will not recognize or bind to the paratope.

[0215] Optionally, an analyte, affinity reagent, docker, tether, label or other moiety can be attached to a solid support or particle. A particularly useful particle is a structured nucleic acid particle (e.g. nucleic acid origami), for example, having structural or functional characteristics set forth herein in the context of attachment to proteins and other analytes. An analyte, affinityreagent, docker, tether, label or other moiety can be attached to a nucleic acid origami via a scaffold component or oligonucleotide component. For example, the scaffold or oligonucleotide can include a nucleotide analog that forms a covalent or non-covalent bond.

[0216] Any of a variety of chemistries can be used to attach an analyte, affinity reagent, docker, tether or other moiety to a solid support or particle (e.g. structured nucleic acid particle). The attachment can be covalent. Exemplary covalent chemistries include, but are not limited to, click chemistries or chemistries set forth in U.S. Pat. Nos. 11,203,612 or 11,505,796; or US Pat. App. Pub. No. 2022 / 0162684 Al, each of which is incorporated herein by reference. Another example is the Spy Tag / Spy Catcher system (See, Zakeri et al. Proceedings Nat’l Acad. Sciences USA. 109 (12): E690-7 (2012)). In this system, a 13 amino acid tag polypeptide (Spy Tag) forms a first coupling handle, with a 12.3 kDa protein (Spy-Catcher) forming the other coupling handle. The SpyCatcher can function by irreversibly bonding to a SpyTag through an isopeptide bond. Any of a variety of non-covalent bonds can be used to attach an analyte, affinity reagent, docker, tether or other moiety to a solid support or particle (e.g., structured nucleic acid particle). Receptors and their ligands can be particularly useful. Examples include, but are not limited to, antibodies, antigens, (strept)avidin (or analogs thereof), biotin (or analogs thereof), affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, monobodies, nanoCLAMPs, nucleic acids, peptide nucleic acids, polypeptides, nucleic acid aptamers, protein aptamers, lectins (or analogs thereof), carbohydrates or functional fragments thereof.Complementary nucleic acids can be used to non-covalently attach a functional moiety to a solid support or particle (e.g. structured nucleic acid particle). Useful nucleic acids can have complementary sequences that are at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more nucleotides in length. Alternatively or additionally, nucleic acids can have complementary sequences that are at most 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5 or fewer nucleotides in length. Reagents and techniques that can be used to non-covalently attach an affinity reagent or other moiety to a particle (e.g., structured nucleic acid particle) are set forth in U.S. Pat. Nos. 11,203,612 or 11,505,796; or US Pat. App. Pub. No. 2022 / 0162684 Al, each of which is incorporated herein by reference.

[0217] A method of detecting analytes can include multiple cycles of binding the analytes with affinity reagents. In a first configuration, a multicycle method of detecting analytes can include steps of (a) providing a mixture of analytes with first affinity reagents, wherein the analytes areimmobilized on a surface and the first affinity reagents are in fluid phase; (b) applying a stimulus to attract the first affinity reagents to the surface; (c) detecting binding of first affinity reagents to analytes on the surface; and (d) repeating steps (a) through (c) using second affinity reagents instead of the first affinity reagents.

[0218] In a second configuration, a multicycle method of detecting analytes in an array can include steps of (a) contacting an array with first affinity reagents in fluid phase, wherein analytes are attached at addresses in the array; (b) allowing first affinity reagents in the fluid phase to bind to analytes attached at addresses in the array; (c) removing a plurality of the first affinity reagents from contact with the array, thereby retaining a fraction of the first affinity reagents in contact with the array; (d) applying a stimulus to attract the fraction of first affinity reagents to the array; (e) detecting binding of first affinity reagents in the fraction to analytes attached at addresses in the array, and (f) repeating steps (a) through (e) using second affinity reagents instead of the first affinity reagents.

[0219] For a method that utilizes first and second affinity reagents, such as the multicycle configurations above, the second affinity reagents can recognize the same epitopes or molecules as the first affinity reagents. For example, the second affinity reagents can include the same paratope(s) as the first affinity reagents. Alternatively, the second affinity reagents, although recognizing the same epitopes or molecules as the first affinity reagents, can have a different composition compared to the first affinity reagents, for example, having different paratope(s) compared to the paratope(s) present in the first affinity reagents. In some configurations of the methods, the second affinity reagents can recognize different epitopes or molecules from the first affinity reagents. Optionally, the second affinity reagents and first affinity reagents can recognize the same epitope(s) or molecule(s) but with different affinities. For example, the second affinity reagents can differ from the first affinity reagents with regard to KD, koff, konor binding probability for a given epitope or set of epitopes.

[0220] The first and second affinity reagents can have the same labels and / or tethers. This can be the case independent of the similarity or difference of the paratopes of the first and second affinity reagents. Alternatively, the first and second affinity reagents can differ with respect to their labels and / or tethers. Again, this can be the case independent of the similarity or difference of the paratopes of the first and second affinity reagents. Different labels or tethers, whether in number or kind, can allow tuning of the detectable properties of the affinity reagents, forexample, to provide for more uniform detector settings or to accommodate a range of affinities for various types of affinity reagents.

[0221] A method of the present disclosure can be configured to be repeated using first analytes and second analytes. For example, a multicycle method can include steps of (a) providing a mixture of first analytes with affinity reagents, wherein the affinity reagents are immobilized on a surface and the first analytes are in fluid phase; (b) applying a stimulus to attract the first analytes to the surface; (c) detecting binding of first analytes to affinity reagents on the surface; and (d) repeating steps (a) through (c) using second analytes instead of the first analytes.

[0222] Optionally, a multicycle method can include steps of (a) contacting an array with first analytes in fluid phase, wherein affinity reagents are attached at addresses in the array; (b) allowing first analytes in the fluid phase to bind to affinity reagents attached at addresses in the array; (c) removing a plurality of the first analytes from contact with the array, thereby retaining a fraction of the first analytes in contact with the array; (d) applying a stimulus to attract the fraction of first analytes to the array; (e) detecting binding of first analytes in the fraction to affinity reagents attached at addresses in the array, and (f) repeating steps (a) through (e) using second analytes instead of the first analytes.

[0223] For a method that utilizes first and second analytes, such as the multicycle configurations above, the second analytes can recognize the same paratopes as the first analytes. For example, the second analytes can include the same epitope(s) as the first analytes. Alternatively, the second analytes, although recognizing the same paratopes as the first analytes, can have a different composition compared to the first analytes, for example, having different epitopes(s) compared to the epitopes(s) present in the first affinity reagents. In some configurations of the methods, the second analytes can recognize different paratopes from the first analytes.Optionally, the second analytes and first analytes can recognize the same paratopes(s) but with different affinities. For example, the second analytes can differ from the first analytes with regard to KD, koff, konor binding probability for a given paratope or set of paratopes.

[0224] One or more steps of a method set forth herein can be repeated at least 1, 2, 3, 4, 5, 10, 50, 100, 200, 300, 400, 500 or more times. Alternatively or additionally, one or more steps of a method set forth herein can be repeated at most 500, 400, 300, 200, 100, 50, 10, 5, 4, 3, 2 or 1 times. Some or all of the repetitions can be carried out using different affinity reagents and / or different analytes from one cycle to the next. Some or all of the repetitions can be carried outusing affinity reagents and / or analytes having the same function from one cycle to the next. Some or all of the repetitions can be carried out using affinity reagents and / or analytes having the same composition from one cycle to the next. Some or all of the repetitions can be carried out using different conditions from one cycle to the next. Exemplary conditions that can differ include, but are not limited to, the composition of labels attached to affinity reagents or analytes, concentration of affinity reagents in fluid phase, concentration of analytes in fluid phase, quantity of affinity reagents in fluid phase, quantity of analytes is fluid phase, duration for one or more step in the cycle, temperature for one or more step in the cycle, ionic strength for one or more step in the cycle, pH for one or more step in the cycle, detector gain or sensitivity for one or more step in the cycle, presence or concentration of denaturants for one or more step in the cycle, or presence or one or more reagent set forth herein for one or more step in the cycle.

[0225] A multicycle method can include a step of removing first affinity reagents and then delivering second affinity reagents. In some configurations, a multicycle method can include a step of removing first analytes and then delivering second analytes. Typically, removal of affinity reagents or analytes is performed after a detection step. However, in some cases it may be desirable to remove affinity reagents or analytes prior to detection. Optionally, a removal step can be followed by one or more wash steps, wherein a wash fluid is used to remove residual analytes or affinity reagents left behind by the removal step.

[0226] Methods set forth herein may comprise the steps of (i) providing to an array a fluid comprising a plurality of fluid phase entities (e g., affinity reagents, analytes, etc.), in which the fluid is provided at an initial concentration, and (ii) applying a stimulus to the fluid phase entities, thereby increasing a concentration of the fluid phase entities adjacent to a surface of the array. Increasing the concentration of the fluid phase entities adjacent to the surface of the array may facilitate formation of affinity reagent / analyte complexes in greater quantity than would be predicted based upon the equilibrium that would occur at the initial concentration of fluid phase entities. Increasing the concentration of the fluid phase entities adjacent to the surface of the array may facilitate a faster rate of affinity reagent / analyte complex formation.

[0227] In particular configurations of the methods set forth herein, a magnetic force is applied as a stimulus to attract affinity reagents to a surface or array to which analytes are attached. For example, individual affinity reagents can each include a magnetic moiety or paramagnetic moiety that is responsive to a magnetic field. Similarly, a magnetic force can be applied to attractanalytes to a surface or array to which affinity reagents are attached. For example, individual analytes can each include a magnetic moiety or paramagnetic moiety that is responsive to a magnetic field. Optionally, the affinity reagents further include a linker and the magnetic force is applied to facilitate attachment of the linker to an address of an array. In some cases, the magnetic force is applied to an affinity reagent that is linked to a solid support, for example, at an address of an array. As such, a linked affinity reagent can be attracted to a solid support surface or address by a magnetic force.

[0228] A magnetic moiety or paramagnetic moiety can be attached directly to an affinity reagent or analyte. Alternatively, an affinity reagent or analyte can be attached to a particle, such as a structured nucleic acid particle, and the particle can in turn be attached to a magnetic moiety or paramagnetic moiety. A magnetic particle can be used such as any of a variety available form commercial sources. An affinity reagent or analyte can be attached to any of a variety of scaffolds whether or not the scaffold has a particle composition. For example, scaffolds having polymer structures such as nucleic acids or non-naturally occurring polymers can be useful. Particles and other scaffolds for analyte-scaffold composites and affinity reagent-scaffold composites, along with methods for making and using the composites are set forth, for example, in US Pat. Nos. 11,505,796 or 11,692,217; US Pat. App. Pub. No. 2023 / 0167488 Al; or US Patent Application No. 18 / 438,973, each of which is incorporated herein by reference.

[0229] Binding of affinity reagents to analytes can occur during application of a magnetic force or magnetic field. Optionally, the magnetic force or magnetic field can be pulsed or oscillated. The pulsing or oscillation can facilitate specificity of binding between binding partners by allowing them to interact multiple times. Also, the pulsing or oscillation can mitigate unwanted adhesion of fluid phase binding partners to surfaces that may otherwise occur is a constant force or field is applied. Detection of complexes formed between affinity reagents and analytes can be performed while applying a magnetic force or magnetic field. Alternatively, the magnetic force can be turned off during a detection step. It will be understood that the magnetic force or magnetic field can be applied before or after detecting the complexes.

[0230] A method of the present disclosure which includes a step of applying a magnetic field to attract fluid-phase affinity reagents to a surface or array to which analytes are attached, can further include a step of changing polarity of the magnetic field. In some cases, the force can be strong enough to remove affinity reagents from contact with the array. Similarly, a methodwhich includes a step of applying a magnetic field to attract fluid-phase analytes to a surface or array to which affinity reagents are attached, can further include a step of changing polarity of the magnetic field. Again, the force can optionally be strong enough to remove analytes from contact with the array. After reversing field polarity, one or more steps of the method can then be repeated using the surface or the array, and using a fluid phase having second affinity reagents or second analytes (instead of the previously used affinity reagents or analytes). Typically, the second affinity reagents or second analytes will be co-localized with magnetic moieties or paramagnetic moieties. However, this is not necessary for all configurations of the methods.

[0231] In particular configurations of the methods set forth herein, an electrophoretic force or electric field is applied as a stimulus to attract affinity reagents to a surface or array to which analytes are attached. For example, individual affinity reagents can each include a charged moiety (e.g. cationic or anionic moiety) that is responsive to an electrophoretic force or electric field. Similarly, an electrophoretic force or electric field can be applied to attract analytes to a surface or array to which affinity reagents are attached. For example, individual analytes can each include a charged moiety that is responsive to an electrophoretic force or electric field.

[0232] A charged moiety can be attached directly to an affinity reagent or analyte. Optionally, the affinity reagents further include a linker and the electrophoretic force or electric field is applied to facilitate attachment of the linker to a solid support, for example, at an address of an array. In some cases, the electrophoretic force or electric field is applied to an affinity reagent that is linked to a solid support, for example, at an address of an array. As such, a linked affinity reagent can be attracted to a solid support surface or address by an electrophoretic force or electric field. Alternatively, an affinity reagent or analyte can be attached to a particle, such as a structured nucleic acid particle, and the particle can in turn be attached to a charged moiety. An electrically charged particle can be used such as a structured nucleic acid particle or any of a variety or particles available form commercial sources. An affinity reagent or analyte can be attached to any of a variety of scaffolds whether or not the scaffold has a particle composition. For example, scaffolds having polymer structures such as nucleic acids or non-naturally occurring polymers can be useful. Particles and other scaffolds for analyte-scaffold composites and affinity reagent-scaffold composites, along with methods for making and using the composites are set forth, for example, in US Pat. Nos. 11,505,796 or 11,692,217; US Pat. App.Pub. No. 2023 / 0167488 Al ; or US Patent Application No. 18 / 438,973, each of which is incorporated herein by reference.

[0233] Binding of affinity reagents to analytes can occur during application of an electrophoretic force or electric field. Optionally, the electrophoretic force or electric field can be pulsed or oscillated. The pulsing or oscillation can facilitate specificity of binding between binding partners by allowing them to interact multiple times. Also, the pulsing or oscillation can mitigate unwanted adhesion of fluid phase binding partners to surfaces that may otherwise occur is a constant force or field is applied. Detection of complexes formed between affinity reagents and analytes can be performed while applying an electrophoretic force or electric field. Alternatively, the electrophoretic force or electric field can be turned off during a detection step. It will be understood that the electrophoretic force or electric field can be applied before or after detecting the complexes.

[0234] A method of the present disclosure which includes a step of applying an electrophoretic force or electric field to attract fluid-phase affinity reagents to a surface or array to which analytes are attached, can further include a step of changing polarity of the electrophoretic force or electric field. In some cases, the force can be strong enough to remove affinity reagents from contact with the array. Similarly, a method which includes a step of applying an electrophoretic force or electric field to attract fluid-phase analytes to a surface or array to which affinity reagents are attached, can further include a step of changing polarity of the electrophoretic force or electric field. Again, the force can optionally be strong enough to remove analytes from contact with the array. After reversing field polarity, one or more steps of the method can then be repeated using the surface or the array, and using a fluid phase having second affinity reagents or second analytes (instead of the previously used affinity reagents or analytes). Typically, the second affinity reagents or second analytes will be co-localized with charged moieties.However, this is not necessary for all configurations of the methods.

[0235] An exemplary substrate that is capable of generating an electrophoretic force to attract affinity reagents, analytes or other molecules of interest is shown in FIG. 7A. The figure shows two wells in cross-section. Trans electrodes (e.g. TiN) are embedded in the well walls and a cis electrode is in contact with fluid phase surrounding the wells. As shown for the well on the left, the silicon oxide layer at the bottom of the well can be coated with oligonucleotides. The oligonucleotides can bind to complementary sequences of a structured nucleic acid particle towhich an analyte is attached, thereby attaching the analyte to the well. The voltage can be applied (or ground compared to cis electrode) alternatively or constantly to attract affinity reagents to the wells. FIG. 7B shows a microwell design for attracting reagents or analytes via electrophoretic force. Here a trans electrode (e.g., TiN) is deposited at the bottom surface of the well. The bottom surface can be coated with oligonucleotides that interact with complementary strands on structured nucleic acid particles to which an analyte is attached as shown in the well on the right.

[0236] A surface of a well shown in FIG. 7A or 7B can be attached to any of a variety of moi eties for attachment of analytes or moieties for attachment of particles including, for example, nucleic acids (as shown) or click reactive moieties, receptors, ligand or other moieties used for attachment chemistries set forth herein or known in the art. A hydrogel such as an acrylamide based polymer can provide a useful coating.

[0237] In particular configurations of the methods set forth herein, analytes are co-localized with polyacids (e.g., polyacidic brushes), affinity reagents include protein moieties, and a stimulus is applied to attract the polyacids (or polyacidic brushes) to the protein moieties, wherein the applied stimulus includes a reduced pH. In another configuration, affinity reagents are colocalized with polyacids (e.g., polyacidic brushes), analytes include protein moieties, and a stimulus is applied to attract the polyacids (or polyacidic brushes) to the protein moieties, wherein the applied stimulus includes a reduced of pH. Although not wishing to be limited to mechanism, low pH can facilitate hydrogen bonding between protein moieties and polyacids (e.g., polyacidic brushes). See, for example, Ferrand-Drake del Castillo et al., Chem. Commun. 56:5889-5892 (2020), which is incorporated herein by reference. Optionally, the pH can be less than or equal to 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.0 or lower. In some configurations a reduction in pH can facilitate attraction between polyacids (e.g., polyacidic brushes) and protein moieties. For example, the magnitude of the reduction can be at least 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 or more pH units.

[0238] A protein moiety that interacts with a polyacid (e.g. polyacidic brush) at low pH can be an endogenous component of an affinity reagent or analyte. For example, the protein moiety can be an antibody that has affinity for an analyte of interest. Alternatively, the protein moiety can be an exogenous moiety that is attached to a particle, such as a structured nucleic acid particle, and the particle can in turn be attached to the affinity reagent or analyte. Optionally, a proteinmoiety can be attached to a surface, such as an address in an array, and the surface can in turn be attached to the affinity reagent or analyte. A polyacid (or polyacidic brush) that interacts with a protein moiety at low pH can be an endogenous component of an affinity reagent or analyte. Alternatively, a polyacid (or polyacidic brush) can be an exogenous moiety that is attached to a particle, such as a structured nucleic acid particle, and the particle can in turn be attached to the affinity reagent or analyte. Optionally, a polyacid (or polyacidic brush) can be attached to a surface, such as an address in an array, and the surface can in turn be attached to the affinity reagent or analyte. An affinity reagent or analyte can be attached to any of a variety of scaffolds whether or not the scaffold has a particle composition. Exemplary scaffolds and particles include, but are not limited to, those set forth herein in the context of magnetic moieties, paramagnetic moieties and charged moieties.

[0239] Binding of affinity reagents to analytes can occur under low pH. Optionally, detection of complexes formed between affinity reagents and analytes is performed under low pH. Alternatively or additionally, the pH can be low before or after detecting the complexes.

[0240] A method of the present disclosure which includes a step of applying low pH to attract fluid-phase affinity reagents to a surface or array to which analytes are attached, can further include a step of applying high pH or increasing the pH, thereby removing affinity reagents from contact with the array. Similarly, a method which includes a step of applying low pH to attract fluid-phase analytes to a surface or array to which affinity reagents are attached, can further include a step of applying high pH or increasing the pH, thereby removing analytes from contact with the array. One or more steps of the method can then be repeated using the surface or the array, and using a fluid phase having second affinity reagents or second analytes (instead of the previously used affinity reagents or analytes). High pH can be at least 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 or higher. An increase in pH can be at least 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 or more pH units. Typically, the second affinity reagents or second analytes will be co-localized with polyacids (e.g. polyacidic brushes) or protein moieties. However, this is not necessary for all configurations of the methods.

[0241] In particular configurations of the methods set forth herein, analytes are attached to first particles, affinity reagents are attached to second particles, and a stimulus is applied to attract the first particles to the second particles, wherein the applied stimulus includes volume exclusion, for example, via presence of a volume exclusion agent. Although not wishing to be limited tomechanism, the presence of volume exclusion agents can facilitate colloidal attraction between the particles. See, for example, Hudoba et al., ACS Nano 11:6566-6573 (2017), which is incorporated herein by reference.

[0242] Volume exclusion agents useful in the present methods may include any of a variety of polar water soluble or swellable agents including, for example, glycerol, poly(methacrylic acid), poly(acrylic)acid, polyethylene glycol (PEG), anionic polymers of polyacrylate or polymethylacrylate, or charged saccharidic polymers (e.g. anionic saccharidic polymers, such as dextran sulfate). In particular configurations, the polymer weight for a volume exclusion agent is at least 10,000, 100,000, 500,000, 1,000,000 or 2,000,000 Daltons. Alternatively or additionally, the polymer weight for a volume exclusion agent can be at most 2,000,000, 1,000,000, 500,000, 100,00 or 10,000 Daltons. Macromolecules such as proteins, nucleic acids and polysaccharides can also be useful as volume exclusion agents. Exemplary volume exclusion agents are set forth in US Pat. No. 4,886,741, which is incorporated herein by reference. In some configurations of the compositions and methods set forth herein, it may be desirable to omit one or more of the volume exclusion agents set forth herein. For example, a method that is configured to detect or manipulate protein analytes can be configured to omit proteins or peptides as volume exclusion agents.

[0243] Optionally, a particle to which an analyte or affinity reagent is attached can, in turn, be attached to a surface of a solid support. For example, the particle can be attached to the surface via a flexible linker. The linker can have a composition set forth herein for linkers used to attach affinity reagents to addresses of an array. Other compositions for the linker include those set forth herein for dockers or tethers. Exemplary particles include, but are not limited to structured nucleic acid particles such as those having nucleic acid origami structures.

[0244] Binding of affinity reagents to analytes can occur in the presence of volume exclusion agents. Optionally, detection of complexes formed between affinity reagents and analytes is performed in the presence of volume exclusion agents. Alternatively or additionally, volume exclusion agents can be present before or after detecting the complexes.

[0245] A method of the present disclosure which includes a step of attracting analytes to affinity reagents in the presence of volume exclusion reagents, can further include a step of removing volume exclusion reagents, thereby separating affinity reagents from analytes. One or more steps of the method can then be repeated using second affinity reagents or second analytes(instead of the previously used affinity reagents or analytes). In other configurations, the structure of a particle to which an affinity reagent or analyte is attached can be changed to facilitate colloidal attraction or to inhibit colloidal attraction. For example, one or more nucleic acid strands can be added or removed from a structured nucleic acid particle to change the composition of the particle. A nucleic acid strand can be added to hybridize to a structured nucleic acid particle, for example, the nucleic acid strand hybridizing to a scaffold, staple or other component of a nucleic acid origami. Optionally, a nucleic acid strand can be removed from a structured nucleic acid particle, for example, a scaffold, staple or other component being removed from a nucleic acid origami. The resulting change of the structured nucleic acid particle can cause increased colloidal attraction or inhibition of colloidal attraction between the changed particle and another particle.

[0246] Colloidal attraction can be achieved if the space (or smallest dimension) between two structured nucleic acid particles is smaller than the excluded volume (or smallest dimension) of the volume exclusion agent. For example, high molecular weight PEG can cause colloidal attraction of nucleic acid origami particles described in US Pat. Nos. 11,692,217 or 11,505,796, or US Pat. App. Ser. No. 18 / 744,286, each of which is incorporated herein by reference.Colloidal attraction can be achieved by screening charges (e.g. salt) and other properties of the solution and solute that impact solubility. This can be achieved in accordance with the DLVO theory (named after Boris Derjaguin, Lev Landau, Evert Verwey and Theodoor Overbeek)). See Gooch Encyclopedic Dictionary of Polymers, pp. 318 (2007) ISBN 978-1-4419-6246-1, which is incorporated herein by reference.Linked Binding Components

[0247] The present disclosure provides a method of detecting an analyte, including steps of (a) providing an analyte attached to a solid support or particle; (b) attaching an affinity reagent to the solid support or particle via a flexible linker; and (c) detecting binding between the affinity reagent and the analyte on the solid support or particle.

[0248] Also provided is a method of identifying an analyte in an array. The method can include steps of (a) providing an array of individually resolved analytes, wherein analytes are attached at addresses in the array, and wherein individual addresses in the array are each attached to a single analyte; (b) attaching affinity reagents to the addresses in the array via flexible linkers, whereby individual addresses in the array each have a single, attached affinity reagent and a single,attached analyte; (c) detecting a plurality of addresses in the array, thereby distinguishing a higher level of binding between an attached affinity reagent and an attached analyte at a first address in the array from a lower level of binding between an attached affinity reagent and an attached analyte at a second address in the array; and (d) identifying an analyte detected at an address in the array in step (c).

[0249] For ease of explanation the use of linked binding components will be exemplified herein for configurations in which an analyte is attached to a solid support (e.g. address of an array) and an affinity reagent is attached at or near the analyte via a flexible linker. It will be understood that the positions of the analyte and affinity reagent can be switched (i.e. the affinity reagent can be attached to the solid support or particle, and the analyte can be attached at or near the analyte via a flexible linker).

[0250] The above method can utilize any of a variety of analytes and arrays including, for example, those set forth previously herein in the context of applying various stimuli to attract fluid-phase affinity reagents toward array surfaces. Proteins are particularly well suited to the method and can be arrayed on a surface using techniques set forth herein or known in the art. For example, individual proteins can each be attached to a particle, such as a structured nucleic acid particle, and the protein-attached particles can be distributed to addresses on the surface of a solid support. The protein-attached particles can be delivered to a solid support, thereby providing a fluid-phase pool, and the particles in the pool can randomly land at addresses on the solid support to form an array. The particles can further include flexible linkers to which affinity reagents can be subsequently attached or the particles can contain an attachment moiety that is competent for attachment to a flexible linker. However, the particles need not necessarily include flexible linkers nor attachment moieties. Instead, flexible linkers or attachment moieties can be attached to a solid support at or near addresses of an array on the solid support. In some configurations, a first particle can be attached to an analyte and a second particle can be attached to a flexible linker or to an attachment moiety for a flexible linker. The first and second particle can be collocated at an address of an array.

[0251] A method of the present disclosure can include a step of attaching affinity reagents to addresses in an array via flexible linkers, whereby individual addresses in the array each have a single, attached affinity reagent and a single, attached analyte. Optionally, attachment is carried out by (i) contacting the array with a fluid phase including a plurality of affinity reagents,thereby attaching at least a subset of the affinity reagents to the addresses in the array via flexible linkers, whereby individual addresses in the array each comprise a single, attached affinity reagent and a single, attached analyte. The process can further include (ii) removing from contact with the array a second subset of affinity reagents that are not attached to the individual addresses in the array. Optionally, the affinity reagents that are in fluid phase during the contacting of step (i) can include flexible linkers that become attached to the addresses. For example, a flexible linker that is attached to an affinity reagent can react with an attachment moiety at a site in an array, thereby attaching the affinity reagent to the address via the flexible linker. Alternatively, individual addresses in the array can each be attached to a flexible linker and the immobilized flexible linker can react with a fluid-phase affinity moiety to attach the affinity reagent to the address via the flexible linker. In another configuration, a linker can be formed by the combination of linker regions present in the affinity reagent and in the address. For example, a first flexible linker region that is attached to an affinity reagent can react with a second flexible linker region that is attached at a site in an array, thereby forming a flexible linker that attached the affinity reagent to the address via a flexible linker that includes the first and second regions.

[0252] Optionally, attachment of affinity reagents to linkers at addresses or attachment of linkerbearing affinity reagents to addresses can be carried out while applying a magnetic force, magnetic field, electrophoretic force or electric field. The force or field can be applied consistently for some or all of the duration of the attachment step. Alternatively, the force or field can be pulsed or oscillated for some or all of the duration of the attachment step.

[0253] In some configurations, a flexible linker can be attached to a solid support, address of an array, particle (e.g., structured nucleic acid particle), affinity reagent, analyte or other substance using a chemical reaction that produces a covalent linkage. Click reactions such as those exemplified herein can be particularly useful. As such, a flexible linker can include a first reactive moiety for a click reaction and the attachment moiety with which it is to react can include a second reactive moiety for the click reaction. Alternatively, a flexible linker can be attached to a solid support, address of an array, particle (c.g, structured nucleic acid particle), affinity reagent, analyte or other substance using a non-covalent linkage. For example, a flexible linker can include a receptor (or a ligand), and the attachment moiety with which it is to bind can include a ligand for the receptor (or a receptor for the ligand). Exemplary pairs of receptors andligands that can be used include, but are not limited to, antibodies and their epitopes; (strept)avidin (or analogs thereof) and biotin (or analogs thereof), complementary nucleic acid strands, or lectins (or analogs thereof) and carbohydrates. Complementary nucleic acids can be used to non-covalently attach a functional moiety to a solid support or particle (e.g., structured nucleic acid particle). The nucleic acids can form a complementary region that is at least 5 base pairs (bp), 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp or longer. Alternatively or additionally, the nucleic acids can form a complementary region that is at most 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, 5 bp or shorter. Other moieties that can be used for covalent or non-covalent attachment are set forth in US Pat. Nos. 11,203,612, 11,505,796 or 11,692,217; US Pat. App. Pub. No. 2022 / 0050049 Al, 2023 / 0167488 Al, or 2024 / 0053333 Al, each of which is incorporated herein by reference.

[0254] A linker can be composed of any of a variety of substances that provide a flexible attachment. For example, a linker can be composed of a polyethylene glycol (PEG), polyethylene oxide (PEO), protein, non-protein polymer, nucleic acid (e.g., single-stranded nucleic acid or double-stranded nucleic acid), dendrimer, peptide nucleic acid (PNA), polysaccharide, or the like.

[0255] A linker can demonstrate differential flexibility in response to environmental conditions such as temperature, pH or light. For example, the flexibility of poly(N-isopropylacrylamide) (PNIPA) and various co-polymers thereof are temperature or pH responsive. See, for example, Schild Progress in Polymer Science. 17 (2): 163-249 (1992) and Okano et al., Journal of Controlled Release . 11 (1-3): 255-265 (1990), each of which is incorporated herein by reference. Exemplary light responsive polymers include, but are not limited to, those having azobenzene, stilbene, cyanostilbene, stiff-stilbene, diarylethene, spiropyrans, hydrozones, orthonitrobenzyl moieties, triarylamine or cinnamic acid derivatives. See, for example, Xu and Feringa^t / v. Mater. 35:2204413 (2023), which is incorporated herein by reference. A lightsensitive moiety, azobenzene, can be incorporated into polyacrylamide to yield copolymers that are light responsive. See, for example, Zhao ACS AppL Polym. Mater. 2:256-262 (2020), which is incorporated herein by reference. Differential flexibility can be employed in the course of a method set forth herein. For example, a polymer can be in a flexible state during binding and detection steps. This can facilitate interaction between an affinity reagent and analyte.Optionally, the polymer can be in a rigid or reduced flexibility state during steps used to (1)attach the polymer to a solid support, (2) attach an affinity reagent to the polymer, (3) remove the polymer from the solid support or particle, or (4) remove an affinity reagent from the solid support or particle.

[0256] A linker for methods set forth herein can be characterized in terms of its effective length. The effective length can be measured for a given conformation of a linker as the shortest distance between two moieties to which the linker is attached when the liker is in the conformation. For example, the effective length can be measured between an affinity reagent to which the linker is attached and a particle or solid support surface to which the linker is also attached. A linker can be characterized in terms of its maximum effective length, which is the effective length when the linker is in its most extended conformation. A linker that is used in a method or composition set forth herein can have a maximum effective length of at least 50 nm, 75 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 micron or longer. Alternatively or additionally, a linker that is used in a method of composition set forth herein can have a maximum effective length of at most 1 micron, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 75 nm, 50 nm, or shorter.

[0257] A linker can be characterized in terms of its average effective length, for example under a particular condition of use. A linker that is used in a method or composition set forth herein can have an average effective length of at least 50 nm, 100 nm, 250 nm, 500 nm, 750 nm, 1 micron or longer. Alternatively or additionally, a linker that is used in a method of composition set forth herein can have an average effective length of at most 1 micron, 750 nm, 500 nm, 250 nm, 100 nm, 50 nm or shorter.

[0258] The length of a linker used for a particular pair of affinity reagent and analyte can be selected based on the known or expected affinity between the pair. The affinity can be considered in terms of an equilibrium binding constant (e.g., KD or KA), half maximal effective concentration (EC50), kinetic binding constant e.g., kon or koff) or probability of binding. An equilibrium binding constant or EC50 for a given pair of affinity reagent and analyte can be expressed in terms of the average molecular distance between analytes at equilibrium. For example, an EC50 of 10 nM for an affinity reagent and analyte pair corresponds to an average distance between analytes of 550 nm. Accordingly, a linker that attaches the affinity reagent to an address where the analyte resides can have an average or maximum effective length of 550 nm to simulate an equilibrium in which the affinity reagent has a 50% probability of being boundto the analyte. The average or maximum linker length can be increased to simulate a lower concentration or the average or maximum linker length can be decreased to simulate a higher concentration. Thus, linker length can be increased as (1) KA decreases, (2) EC50 decreases or (3) KD increases. Linker length can be decreased as (1) KA increases, (2) EC50 increases or (3) KD decreases. As exemplified above, linker length can correspond to the average molecular distance between analytes at EC50, KA or KD. However, it may be beneficial in some configurations of the methods set forth herein to select a linker length that corresponds to an average distance that is at least 0.1 fold (x), 0.25x, 0.5x, 0.75x, 0.9x, lx, 2x, 3x or more higher than the length corresponding to the EC50, KA or KD for the linked affinity reagent.Alternatively or additionally, it may be beneficial in some configurations of the methods set forth herein to select a linker length that corresponds to an average distance that is at least 0.1 fold (x), 0.25x, 0.5x, 0.75x, 0.9x, lx, 2x, 3x or more lower than the length corresponding to the EC50, KA or KD for the linked affinity reagent.

[0259] An array can include a plurality of addresses, each of which is attached to an affinity reagent via a flexible linker. In such configurations, the effective length of the linkers can be shorter than the distance between nearest neighbor addresses. As such, an affinity reagent that is attached to a given address is prevented from binding to an analyte at the neighboring address. It may also be desirable for the sum of the lengths of the linkers attached to nearest neighbor addresses to be shorter than the distance between nearest neighbor addresses. For example, when the nearest neighbor addresses are attached to linkers having the same length, the length of the linkers can be shorter than half the distance between the addresses. This can be beneficial to prevent interactions between affinity reagents linked to neighboring addresses.

[0260] A method of the present disclosure can include a step of binding a flexibly linked affinity reagent to a surface immobilized analyte. Binding can optionally proceed via passive diffusion of the linked affinity reagent in the vicinity of the immobilized analyte. For example, the conditions can be similar to those employed when binding non-linked affinity reagents to surface immobilized analytes. Alternatively, binding of a flexibly linked affinity reagent to a surface immobilized analyte can proceed via active transport. For example, a linked affinity reagent can be transported toward the surface immobilized analyte under an applied force such as an electrophoretic force, electric field, magnetic force or magnetic field. A linked affinity reagent can include a paramagnetic moiety, magnetic moiety or charged moiety to facilitate activetransport under such a force or field. The force or field can be consistently applied, oscillated or pulsed during binding.

[0261] A method of the present disclosure can include a step of detecting binding between a surface immobilized analyte and a flexibly linked affinity reagent. For example, a method of the present disclosure can include a step of detecting binding between an analyte that is attached to an address of an array and an affinity reagent that is attached to the address via a flexible linker. A plurality of addresses can be detected to measure the level of binding between analytes and linked affinity reagents at individual addresses. For example, a method can include a step of detecting a plurality of addresses in an array, thereby distinguishing a higher level of binding between an attached affinity reagent and an attached analyte at a first address in the array from a lower level of binding between an attached affinity reagent and an attached analyte at a second address in the array. An electrophoretic force, electric field, magnetic force or magnetic field can be applied during a detection step. Alternatively, a force or field that is applied during a binding step can be turned off or reversed during a detection step.

[0262] In some configurations, detection of binding between an analyte and flexibly linked affinity reagent can utilize components of a proximity-based detection technique such as energy transfer between a donor and acceptor, or other technique set forth herein. For example, Forster resonance energy transfer (FRET) between a first luminophore attached proximal to the affinity reagent and a second luminophore attached proximal to the analyte. FIG. 5A shows an exemplary configuration in which analytes (white globules) are attached to addresses of an array and an affinity reagent (Y-shape) is attached to each address via a nucleic acid linker. A first fluorophore (open 12 point star) is attached proximal to the affinity reagent and a second fluorophore (closed 12 point star) is attached proximal to the protein. In the configuration shown, the first fluorophore is attached directly to the affinity reagent and the second fluorophore is attached to an attachment moiety to which the linker hybridizes. The affinity reagent is bound to the protein in the lefthand address and thus FRET can be detected when the address is excited. In contrast, the affinity reagent in the righthand address is dissociated from the protein and FRET is insubstantial or undetectable when the address is excited. A FRET donor can be attached in proximity to either of the analyte or linked affinity reagent and a FRET acceptor can be attached in proximity to the other member of the analyte and linked affinity reagent pair.

[0263] The positions for attachments of luminophores shown in FIG. 5 A is exemplary. Proximity-based detection components can be attached to other locations as well. FIG. 5B shows a configuration in which the first fluorophore (open 12 point star) is attached directly to the affinity reagent and the second fluorophore (closed 12 point star) is attached to the linker via a complementary oligonucleotide. Alternatively, the second fluorophore can be directly attached to the linker. For example, when using a nucleic acid linker, the second fluorophore can be attached to a nucleotide in the sequence of the linker. Attaching the second fluorophore to the linker allows the second fluorophore to be easily removed and replaced after use. This can provide a benefit for situations in which radiation used during FRET causes unwanted photobleaching of the luminophores. Other proximity -based detection components can be used in place of the FRET components. Exemplary configurations for associating or attaching the components to surfaces, analytes and affinity reagents are set forth herein.

[0264] Proximity-based detection can be facilitated by dockers and tethers. An analyte used in a composition or method of the present disclosure can have a docker and the docker can be attached to a first component of a proximity-based detection pair (e.g., a FRET donor or acceptor used for energy transfer). An affinity reagent used in a composition or method of the present disclosure can have a tether and the tether can be attached to a second component of a proximitybased detection pair e.g., a FRET donor or acceptor used for energy transfer). The docker and tether can be configured to position the components of the pair in proximity to each other when the linked affinity reagent is bound to the analyte. As such, the docker and tether can facilitate proximity -based detection (e.g. energy transfer) and, thus, facilitate detection of binding between the linked affinity reagent and analyte.

[0265] FIG. 5C shows an exemplary configuration in which analytes (white globules) are attached to addresses of an array and an affinity reagent (Y-shape) is attached to each address via a nucleic acid linker. A first fluorophore (open 12 point star) is attached to the affinity reagent via a nucleic acid tether and a second fluorophore (closed 12 point star) is attached to the linker via a nucleic acid docker. At the address on the left (“+’’), the affinity reagent is bound to the analyte and the tether is hybridized to the docker. A nucleotide sequence of the tether is complementary to a nucleotide sequence of the docker. Hybridization of the tether and docker positions the first and second fluorophore into proximity for FRET, thereby being capable of producing a signal indicative of binding between affinity reagent and analyte. At the address onthe right the affinity reagent is dissociated from the analyte and the tether is dissociated from the docker. Thus, a FRET signal is not produced, indicating the affinity reagent is not bound to the analyte.

[0266] FIGs. 14A - 14C depict additional configurations of methods and systems utilizing an affinity reagent attached to a linker. The left-hand configuration of FIG. 14A depicts an analyte 1401 that is immobilized to a fixed address of a solid support 1400. A first FRET dye 1402 is colocalized at the fixed address of the solid support 1400, optionally by a linking moiety. An affinity reagent 1420 is also co-localized at the fixed address by a linker 1421 that is attached to the solid support 1400. A second FRET dye 1422 is attached to the linker 1421, optionally by a branched linking moiety. In the right-hand configuration of FIG. 14A, binding of the affinity reagent 1420 to the analyte 1401 brings the first FRET dye 1402 in sufficient proximity to the second FRET dye 1422 to provide a detectable fluorescent signal 1430. FIG. 14B depicts a system in which the FRET dyes have been replaced by a luminescent protein 1423 (e.g., a luciferase) that provides a detectable signal 1430 when the luciferase protein 1423 binds to a cofactor 1403 (e.g., coelenterazine). The configurations of FIGs. 14A and 14B could also be substituted with a split fluorescent (e.g., split green fluorescent protein) or luminescent protein (e.g., split luciferase) that provides a detectable signal when two components of the split protein are brought into close enough proximity to bind to each other by the binding interaction of the affinity reagent and the analyte.

[0267] FIG. 14C depicts a system that is configured to indirectly record a binding interaction between an affinity reagent attached to a linker and an immobilized analyte. The upper left-hand configuration of FIG. 14C depicts an analyte 1401 that is immobilized to a fixed address of a solid support 1400. A first transferring moiety 1404 is co-localized at the fixed address of the solid support 1400. An affinity reagent 1420 is also co-localized at the fixed address by a linker 1421 that is attached to the solid support 1400. A second transferring moiety 1424 is attached to the linker 1421. A transferrable moiety 1425 comprising one or more detectable labels 1426 is attached to the second transferring moiety 1424. In the upper right-hand configuration of FIG. 14C, binding of the affinity reagent 1420 to the analyte 1401 facilitates binding of the transferrable moiety 1425 to the first transferring moiety 1404 due to the close proximity of the transferrable moiety 1425 to the first transferring moiety 1404. In the bottom center configuration, the affinity reagent 1420 and linker 1421 may be released, leaving thetransferrable moiety 1425 attached to the first transferring moiety 1404. A signal may from the one or more detectable labels 1426 may be detected at the fixed address of the solid support 1400, indicating the prior presence of a binding interaction between the affinity reagent 1420 and analyte 1401. Additional methods and system for forming reactions that record the presence of a binding interaction between an affinity reagent and analyte are set forth below in the section titled “Additional Methods of Proximity -Based Detection.” The skilled person can readily envision additional combinations of components that can be used to directly or indirectly identify presence of binding interactions between an affinity reagent and an analyte.

[0268] When using a linker-attached affinity reagent in combination with a docker and tether, the lengths for docker and tether can be substantially shorter than the length of the linker. As set forth above, the length of the linker can be selected based on the known or suspected strength of binding between the affinity reagent and analyte. Typically, the docker and tether are configured to position proximity-based detection components without substantially contributing to the binding of the affinity reagent to the analyte. As such, the length of the docker and tether can be selected to facilitate hybridization when the docker and tether are in close proximity without necessarily driving interactions from a larger distance. The relative lengths of the linker, docker and tether can be selected to balance binding interactions between affinity reagent and analyte with binding interactions between docker and tether. The relative lengths can be selected to correspond to the relative binding affinities. For example, the length for the docker and / or tether can be at most 50%, 25%, 20%, 10%, 5%, 1%, 0.1% or less of the effective length of the linker. The length of the complementary region formed by hybridization of a nucleic acid tether and nucleic acid docker can also be selected to balance binding interactions between affinity reagent and analyte with binding interactions between docker and tether. For example, the length of a complementary region formed between a docker and tether can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more base pairs. Alternatively or additionally, the complementary region can be at most 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 base pairs. The strength of binding between a tether and docker can also be selected to position proximity -based detection components without substantially contributing to the binding of the affinity reagent to the analyte. For example, a nucleic acid tether and docker can be configured to have a relatively low melting temperature (Tm). Melting temperature can be influenced by the GC and AT composition of the hybrid region formed between a docker and tether. The complementary region can include A:Tbasepairs and G:C basepairs, only A:T basepairs, only G:C basepairs, or various percentages of A:T basepairs and G:C basepairs such as those exemplified above herein. In some configurations, a nucleic acid docker and nucleic acid tether are not substantially complementary with each other

[0269] In some configurations, detection of binding between an analyte and linked affinity reagent can utilize a spatially selective detection technique. Any of a variety of spatially selective optical detection techniques can be used to detect binding of an analyte, at an address of an array, to an optically detectable affinity reagent that is linked at the address. The spatially selective optical detection technique can be configured to selectively filter, or even reject, signals produced by the affinity reagent when it is dissociated from the analyte and thus outside of a detection zone. For example, confocal fluorescence microscopy can be used to collect fluorescent signals from a spatially resolved region where an analyte binds to a fluorescently detectable affinity reagent while rejecting signals from the affinity reagent when it is outside of the region. Total internal reflection fluorescence can also be used to collect fluorescent signals proximal to a surface where an analyte binds to a fluorescently detectable affinity reagent while rejecting signals from the affinity reagent when it is located distal from the surface. Waveguides can be useful for exciting optically detectable affinity reagents to provide spatially selective optical detection. For example, a zero mode waveguide can be used.

[0270] FIG. 6 shows a section of an array of zero mode waveguides (ZMW) in which two ZMWs are shown in cross section. The array is configured to selectively deliver excitation radiation to a region of each ZMW that is near the bottom where a protein analyte (white globule) is attached. The bottom of the ZMW provides the highest detection sensitivity and the sensitivity decays with distance from the bottom. A detection zone can be delineated based on the parameters selected for detection, such as a threshold for signal intensity, threshold for signal-to-noise ratio, duration for signal acquisition etc. Both ZMWs have a protein analyte in a detection zone and both also have an affinity reagent (Y-shape) to which a fluorophore (12 point star) is attached. The affinity reagents are attached to the ZMW via a nucleic acid linker. The affinity reagent is bound to the protein in the lefthand ZMW and thus produces a detectable emission when the ZMW is excited. In contrast, the affinity reagent in the righthand ZMW is dissociated from the protein and no emission is produced when the ZMW is excited. ZMWs can be made and used as set forth, for example, in lizuka et al., Biophysics and Physicobiology 19,e!90032 (2002); Eid et. Al., Science 323: 133-138 (2009); US Pat. Nos. 7,313,308, or U.S. Pat. App. Pub. Nos. 2008 / 0128627 Al, 2012 / 0085894 Al, 2016 / 0334334 Al, 2016 / 0363728 Al, 2016 / 0273034 Al, 2016 / 0061740 Al, or 2017 / 0145498 Al, each of which is incorporated herein by reference.

[0271] Analytes can be distinguished in a method set forth herein according to the level of binding between the analytes and a given type of affinity reagent. In some cases, analytes can be distinguished according to the level of binding between the analytes and a plurality of different affinity reagents. For example, a method can include a step of distinguishing a higher level of binding between an attached affinity reagent and an attached analyte at a first address in an array from a lower level of binding between an attached affinity reagent and an attached analyte at a second address in the array. The level of binding can be determined or reported as the intensity of signal collected from a measurement of binding between affinity reagent and analyte (e.g., higher intensity is correlated with higher level of binding), the duration of signal production while measuring binding between affinity reagent and analyte (e.g., longer duration is correlated with higher level of binding), relative proportion of acceptor emission to donor emission in a FRET detection system (e.g., higher FRET is correlated directly with higher level of binding), or the like.

[0272] A method of detecting an analyte using a linked affinity reagent can include multiple cycles of binding the analyte with affinity reagents. A multicycle method can include steps of (a) providing an analyte attached to a solid support or particle; (b) attaching an affinity reagent to the solid support or particle via a flexible linker; (c) detecting binding between the affinity reagent and the analyte on the solid support or particle; (d) detaching the affinity reagent from the solid support or particle after step (c), and (e) repeating steps (b) and (c) using second affinity reagents instead of the affinity reagents. It will be understood that the positions of the analyte and affinity reagent can be reversed (i.e. the affinity reagent can be attached to the solid support or particle, and the analyte can be replaced when repeating steps (b) and (c))

[0273] A multicycle method can include steps of (a) providing an array of individually resolved analytes, wherein analytes are attached at addresses in the array, and wherein individual addresses in the array are each attached to a single analyte; (b) attaching affinity reagents to the addresses in the array via flexible linkers, whereby individual addresses in the array each have a single, attached affinity reagent and a single, attached analyte; (c) detecting a plurality ofaddresses in the array, thereby distinguishing a higher level of binding between an attached affinity reagent and an attached analyte at a first address in the array from a lower level of binding between an attached affinity reagent and an attached analyte at a second address in the array; (d) identifying an analyte detected at an address in the array in step (c); (e) detaching affinity reagents from the addresses in the array after step (c), and (f) repeating steps (b) and (c) using second affinity reagents instead of the affinity reagents. It will be understood that step (d) can be performed before or after steps (e) and (f). Again, the positions of the analyte and affinity reagent can be reversed (i.e. the affinity reagent can be attached to the solid support or particle, and the analyte can be replaced when repeating steps (b) and (c)).

[0274] For a method that utilizes first and second affinity reagents, such as the multicycle configurations above, the second affinity reagents that are linked to the solid support, address or particle can recognize the same epitopes or molecules as the first affinity reagents. For example, the second affinity reagents can include the same paratope(s) as the first affinity reagents.Alternatively, the second affinity reagents, although recognizing the same epitopes or molecules as the first affinity reagents, can have a different composition compared to the first affinity reagents, for example, having different paratope(s) compared to the paratope(s) present in the first affinity reagents. In some configurations of the methods, the second affinity reagents can recognize different epitopes or molecules from the first affinity reagents. Optionally, the second affinity reagents and first affinity reagents can recognize the same epitope(s) or molecules(s) but with different affinities. For example, the second affinity reagents can differ from the first affinity reagents with regard to KD, koff, kon or binding probability for a given epitope or set of epitopes.

[0275] For a method that utilizes first and second analytes, such as the multicycle configurations above, the second analytes can recognize the same paratopes as the first analytes. For example, the second analytes can include the same epitope(s) as the first analytes. Alternatively, the second analytes, although recognizing the same paratopes as the first analytes, can have a different composition compared to the first analytes, for example, having different epitopes(s) compared to the epitopes(s) present in the first affinity reagents. In some configurations of the methods, the second analytes can recognize different paratopes from the first analytes.Optionally, the second analytes and first analytes can recognize the same paratopes(s) but withdifferent affinities. For example, the second analytes can differ from the first analytes with regard to KD, koff, kon or binding probability for a given paratope or set of paratopes.

[0276] One or more steps of a method set forth herein can be repeated at least 1, 2, 3, 4, 5, 10, 50, 100, 200, 300, 400, 500 or more times. Alternatively or additionally, one or more steps of a method set forth herein can be repeated at most 500, 400, 300, 200, 100, 50, 10, 5, 4, 3, 2 or 1 times. Some or all of the repetitions can be carried out using different affinity reagents and / or different analytes from one cycle to the next. Some or all of the repetitions can be carried out using affinity reagents and / or analytes having the same function from one cycle to the next.Some or all of the repetitions can be carried out using affinity reagents and / or analytes having the same composition from one cycle to the next. Some or all of the repetitions can be carried out using different conditions from one cycle to the next. Exemplary conditions that can differ include, but are not limited to, the composition of labels attached to affinity reagents or analytes, effective length of the flexible linker attached to each affinity reagent or analyte, composition of the flexible linker attached to each affinity reagent or analyte, flexibility of the flexible linker attached to each affinity reagent or analyte, length of dockers and / or tethers, length of complementary sequences for a docker-tether pair, duration for one or more steps in the cycle, temperature for one or more steps in the cycle, ionic strength for one or more steps in the cycle, pH for one or more steps in the cycle, detector gain or sensitivity during detection, presence or concentration of denaturants for one or more step in the cycle, or presence or one or more reagent set forth herein for one or more step in the cycle.Additional Methods of Proximity-Based Detection

[0277] Methods set forth herein may comprise a step of recording a binding interaction between an affinity reagent and an analyte. Recording of a binding interaction may occur during an equilibration period between a plurality of affinity reagents and a plurality of analytes.Preferably, the equilibration period may occur for a sufficient time to establish a binding equilibrium between the affinity reagents and the analytes. A method of recording a binding interaction between an affinity reagent and an analyte may comprise forming a reaction, in which the reaction facilitates detection of a signal associated with a complex containing an analyte and an affinity reagent after an equilibration period has ended. Preferably, binding interactions may be recorded in a spatially resolved manner such that a binding interaction between a first analyte and a first affinity reagent can be resolved from a binding interaction between a second analyteand a second affinity reagent. Array-based methods set forth herein may be especially useful for resolving binding interactions between pluralities of analytes and pluralities of affinity reagents.

[0278] While numerous methods are exemplified by a signal becoming detectable when a binding interaction occurs, many described methods can easily be reversed to have a signal become undetectable when a binding interaction occurs (e.g., via removal or dissociation of a detectable label, etc.).

[0279] A method may comprise direct recording of a binding interaction between an affinity reagent and an analyte if a reaction of a first moiety co-localized with the affinity reagent with a second moiety co-localized with the analyte inhibits dissociation of the affinity reagent from the analyte, thereby facilitating detection of a signal associated with a complex containing the affinity reagent and the analyte. A method may comprise indirect recording of a binding interaction if a detectable label is retained with the affinity reagent or analyte after the affinity reagent has dissociated from the analyte, thereby facilitating detection of a signal associated with a complex containing the affinity reagent and analyte after the complex has dissociated.

[0280] FIG. 8A depicts a method of transferring a detectable label 826 to a site containing an immobilized analyte 810. Initially (left-hand side), the analyte 810 is immobilized at a fixed address of a solid support 800. An immobilized first transferring moiety 805 (e.g., an oligonucleotide, a member of a receptor-ligand binding pair, a covalently-reactive moiety, etc.) is immobilized at the fixed address of the solid support 800 containing the immobilized analyte 810. Preferably, the first transferring moiety 805 is co-localized at a distance from the immobilized analyte 810 that is not optically-resolvable. An affinity reagent 820 is bound to the immobilized analyte 810, thereby immobilizing the affinity reagent 820 at the fixed address of the solid support. The affinity reagent 820 is attached to a linking moiety 821 that contains a second transferring moiety 822 (e.g., an oligonucleotide). A transferrable moiety 825 is bound to the second transferring moiety 822. The transferrable moiety 825 comprises one or more detectable labels 826. In a second configuration (right-hand side of FIG. 8A), the transferrable moiety 825 has been dissociated from the second transferring moiety 822 and has become associated to the first transferring moiety 805 when the transferrable moiety 825 has been positioned in a close enough proximity to the first transferring moiety 805 to facilitate transfer. The transferrable moiety 825 and transferring moieties (805, 822) may be designed to have a greater stability when the transferrable moiety 825 is attached to the second transferring moiety822. For example, if the transferrable moiety 825 is a nucleic acid, a larger quantity of nucleotides of the transferrable moiety 825 may hybridize to the second transferring mioety 822 than hybridize to the first transferring moiety 805. Accordingly, the binding interaction of the affinity reagent 820 with the analyte 810 is recorded at the fixed address by the transfer of the transferrable moiety 825 from the second transferring moiety 822 of the affinity reagent 820 to the first transferring moiety 805 that is co-localized with the analyte 810. A detectable signal from the one or more detectable labels 826 can be detected at the fixed address after the affinity reagent 820 has dissociated from the analyte 810.

[0281] FIG. 8B depicts a similar method to the method of FIG. 8 A, with the addition of a second transferring moiety 823 to the affinity reagent 820. The affinity reagent 820 is provided an optional second linking moiety 829 containing the second transferring moiety 823. In another configuration, the linking moiety 821 could contain the second transferring moiety 823. In an initial configuration (left-hand side), a second transferrable moiety 828 is bound to the second transferring moiety 823. The second transferrable moiety 828 contains one or more second detectable labels 827. Preferably, signals from the second detectable labels 827 are distinguishable from the detectable labels 826 (e.g., by emission wavelength, by excitation wavelength, by luminescence lifetime, etc.). The fixed address of the solid support 800 further comprises an immobilized fourth transferring moiety 806. Preferably, the fourth transferring moiety 806 is co-localized at a distance from the immobilized analyte 810 that is not optically- resolvable. Preferably, the kinetics of transfer of the transferrable moiety 825 to the first transferring moiety 805 is faster than the kinetics of transfer of the second transferrable moiety 828 to the fourth transferring moiety 806. Accordingly, a time length of binding or binding strength of the affinity reagent 820 to the analyte 810 can be estimated by whether neither, one or both transferring moieties were transferred to the fixed address. In the second configuration (right-hand side), only the transferrable moiety 825 will be detectable at the fixed address of the solid support 800 due to insufficient binding time of the affinity reagent 820 to the analyte 810 to facilitate transfer of the second transferrable moiety 828 to the fourth transferring moiety 806.

[0282] For configurations of transferrable moieties comprising oligonucleotide transferrable moieties, such as those depicted in FIGs. 8A - 8B, it may be preferable to control the directionality of transfer of a transferrable moiety. For example, a transferrable moiety is likely to remain associated to a transferring moiety if it has greater number of nucleotides of sequencecomplementarity with the transferring moiety. Accordingly, a transferrable moiety may have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotides of additional sequence complementarity with a receiving transferring moiety with respect to its complementarity to a delivering transferring moiety.

[0283] Alternatively, directionality of transfer of a transferrable moiety can be controlled by attaching the transferrable moiety to a receiving transferring moiety by a non-dissociable binding interaction (e.g., a covalent bond, a non-dissociable receptor-ligand binding interaction). A non- dissociable binding interaction may comprise any binding interaction that is unlikely to spontaneously dissociate before a detection event has occurred. FIG. 8C depicts a similar configuration to FIG 8A, with a non-dissociable attachment moiety 807 attached to the transferring moiety 805. The transferrable moiety 825 further comprises a complementary non- dissociable attachment moiety 827. In a second configuration (right-hand side of FIG. 8C), the transferrable moiety 825 has been dissociated from the second transferring moiety 822 and has become associated to the first transferrable moiety 805 when the complementary non-dissociable attachment moiety 827 binds to the non-dissociable attachment moiety 807, thereby forming a non-dissociable interaction 809 between the transferrable moiety 825 and the transferring moiety 805.

[0284] In some cases, a transferrable moiety may be attached to a transferring moiety by an irreversible non-dissociable interaction. Exemplary irreversible non-dissociable binding interactions can include covalent binding interactions (e g., Click-type reactions) and receptorligand binding interactions (e.g., streptavidin-biotin, SpyCatcher-SpyTag, SnoopCatcher- SnoopTag, SdyCatcher-SdyTag, etc.). For methods having multiple possible detection events, it may be preferable to have a receiving transferring moiety be configured to be releasable so that the signal associated with transfer of the transferrable moiety can be erased after a detection event by release of the transferrable moiety -transferring moiety complex. For example, a non- dissociable attachment moiety may be attached to a first oligonucleotide, in which the first oligonucleotide is hybridized to a second oligonucleotide, and in which the second oligonucleotide is attached to a solid support. Accordingly, the first oligonucleotide can be released (e.g., by a toehold-mediated strand displacement reaction, by contacting with a chaotrope, by heating, etc.), thereby releasing any transferrable moiety attached to the first oligonucleotide.

[0285] Alternatively, a transferrable moiety may be attached to a transferring moiety by a reversible non-dissociable interaction. A reversible non-dissociable interaction can include certain photoreactive covalent bonds. Accordingly, a method may comprise contacting a complex comprising an analyte and an affinity reagent with light of a wavelength that activates a photoreactive reaction. Another useful reversible non-dissociable interaction can include use of immobilized metal affinity chromatography (IMAC)-type binding interactions. An IMAC -type binding interaction may be formed by attaching a histidine tag to a transferring moiety or a transferrable moiety, and attaching a metal-chelating complex (e.g., nitrilotriacetic acid, iminodiacetic acid, N-(carboxymethyl) aspartate, N,N,N’-tris(carboxymethyl)ethylenediamine, etc.) to the complement. The metal-chelating complex can strongly bind the histidine tag in the presence of metal ions (e.g., Cu2+, Ni2+, Zn2+, Co2+). The binding of the histidine tag to the metalchelating complex can be reversed by use of a low-pH solution (e.g., a pH of less than about 6.5, 6.0, 5.5, 5.0, 4.5, or 4.0), a solution comprising EDTA, a solution comprising imidazole, a solution comprising histidine or polyhistidine, or combinations thereof. An IMAC-type binding interaction may also be utilized for directly recording a binding interaction by attaching the histidine tag or metal-chelating complex to an affinity reagent, and attaching the complement to an analyte or a fixed address containing the analyte.

[0286] FIGs. 9A - 9C depict a method of recording a binding interaction via a catalyzed labeling reaction. FIG. 9A depicts in an initial configuration an analyte 910 that is immobilized at a fixed address of a solid support 900. A modifiable moiety 906 (e.g., an oligonucleotide, a peptide, a polymer chain, etc.) is immobilized at the fixed address of the solid support 900 containing the immobilized analyte 910. In some cases, the modifiable moiety may be releasably attached to the solid support 900. For example, the modifiable moiety 906 may comprise an oligonucleotide that is coupled to a complementary oligonucleotide 905, in which the complementary oligonucleotide is coupled to the solid support. Preferably, the modifiable moiety 906 is co-localized at a distance from the immobilized analyte 910 that is not optically-resolvable. An affinity reagent 920 is bound to the immobilized analyte 910, thereby immobilizing the affinity reagent 920 at the fixed address of the solid support 900. The affinity reagent 920 is attached to a linking moiety 921 that is also attached to a catalyzing moiety 922 (e.g., an enzyme, a catalyst particle). In a second configuration (right-hand side of FIG. 9A), the modifiable moiety 906 may be modified by the catalyzed formation of a detectable label 908 on the modifiable moiety.Exemplary methods of adding a detectable label can include polymerase extension of an oligonucleotide (e.g., by incorporation of modified or fluorescently-labeled nucleotides), ligation of a labeled nucleic acid to the modifiable moiety 906, or addition of ligands to the modifiable moiety (e.g., BirA-catalyzed biotinylation of a modifiable moiety comprising an avi-tag, addition or modification of amino-acid sidechains by kinases, etc.). Accordingly, the detectable label remains co-located at the fixed address containing the analyte 910 after the affinity reagent 920 has dissociated.

[0287] FIG. 9B depicts a method of secondary labeling to detect the detectable label 908 added to a modifiable moiety 906. In the left-hand configuration, a secondary detectable label 930 (e.g., a fluorophore, a luminophore, etc.) is contacted to the modifiable moiety 906. The secondary detectable label 930 is attached to a moiety 938 with a binding specificity for the detectable label 908. For example, a biotinylated avi-tag would be bound by a fluorescently-labeled streptavidin or avidin molecule. Similarly, an enzymatically-modified peptide (e.g., phosphorylated, ubiquinated, methylated, etc.) could be bound by an affinity agent that is specific to the modified peptide. Certain covalent modifications could be bound by complementary covalent compounds. The right-hand side configuration of FIG. 9B depicts the secondary detectable label 930 bound to the modifiable moiety 906 by binding of the moiety 938 to the detectable label 908. The lefthand side of FIG. 9C depicts detection of a signal 935 from the secondary detectable label 930 at the fixed address of the solid support 900 with an optical sensor 940. After detecting a signal from the modifiable moiety 906, the modifiable moiety may be optionally removed (e.g., by a toehold-mediated strand displacement reaction, by contacting with a chaotrope, etc.). After removing the modifiable moiety 906, an unmodified modifiable moiety 906 may be immobilized at the fixed address before another affinity reagent 920 (e.g., an affinity reagent with a different binding specificity) is bound to the analyte 910.

[0288] Methods and systems of the present disclosure set forth systems of dockers and tethers that may be useful for inhibiting dissociation of an affinity reagent from an analyte after the affinity reagent has become bound to the analyte. Oligonucleotide-based docker / tether systems may be particularly useful for methods and systems set forth herein. In some cases, it may be advantageous to provide an oligonucleotide in a fluid phase that is configured to bind to a docker nucleic acid strand and a tether nucleic acid strand.

[0289] FIGs. 10A - 10B illustrate a method of directly recording a binding interaction between an analyte 1010 and an affinity reagent 1020 utilizing a bridging nucleic acid strand. In an initial configuration (left-hand side), FIG. 10A depicts an analyte 1010 immobilized at a fixed address of a solid support 1000. A docker nucleic acid strand 1005 is immobilized at the fixed address of the solid support 1000 containing the immobilized analyte 1010. Preferably, the docker nucleic acid strand 1005 is co-localized at a distance from the immobilized analyte 1010 that is not optically-resolvable. An affinity reagent 1020 is bound to the immobilized analyte 1010, thereby immobilizing the affinity reagent 1020 at the fixed address of the solid support 1000. The affinity reagent 1020 is attached to a linking moiety 1021 that is also attached to a tether nucleic acid strand 1025. The complex comprising the affinity reagent 1020 bound to the analyte 1010 is contacted with a fluid phase bridging nucleic acid strand 1022. Optionally, the bridging nucleic acid strand 1022 is attached to one or more detectable labels 1026. Alternatively, the one or more detectable labels 1026 may be attached to the affinity reagent 1020 or the linking moiety 1021. In a subsequent configuration (right-hand side of FIG. 10A), the bridge nucleic acid strand 1022 is hybridized to the docker nucleic acid strand 1005 and the tether nucleic acid strand 1025, thereby inhibiting dissociation of the affinity reagent 1020 from the fixed address of the solid support 1000. Preferably, the bridging nucleic acid strand 1022 may be provided with one or more nucleotide sequences that do not hybridize to the docker nucleic acid strand 1005 and / or tether nucleic acid strand 1025. For example, as depicted in the right side of FIG. 10A, a portion of the bridge nucleic acid strand 1022 between the portions that hybridize to the tether nucleic acid strand 1025 and to the docker nucleic acid strand 1005 does not bind to either of the tether nucleic acid strand 1025 or the docker nucleic acid strand 1005. Such pendant nucleotide sequences may facilitate dissociation of the bridging nucleic acid strand 1022 (e.g., via toehold mediated strand displacement) after a signal from a detectable label 1026 has been detected at an address containing the bridging nucleic acid strand 1022. FIG. 10B depicts displacement of the bridging nucleic acid strand 1022 by contacting the complex containing the affinity reagent 1020 bound to the analyte 1010 with a fluid-phase displacing nucleic acid strand 1050 (left-hand side configuration). In a second figuration of FIG. 10B, the displacing nucleic acid strand 1050 has hybridized to the bridging nucleic acid strand 1022, thereby dissociating the bridging nucleic acid strand 1022 from the docker nucleic acid strand 1005 and / or the tether nucleic acid strand 1025. Once the bridging interaction has been dissociated, the affinity reagent 1020 maydissociate from the analyte 1010, optionally in the presence of a dissociation medium (e.g., a buffer comprising a chaotrope or surfactant).

[0290] A bridging nucleic acid strand may be configured to join a docker nucleic acid strand to a tether nucleic acid strand when the docker and tether are in proximity to each other. A bridging nucleic acid strand may comprise a first nucleotide sequence that is configured to hybridize to a nucleic acid sequence of a docker nucleic acid strand, a second nucleotide sequence that is configured to hybridize to a nucleic acid sequence of a tether nucleic acid strand, and optionally a linker (e.g., a linking nucleic acid, a linking non-nucleic acid) of a chosen length. A longer linker will facilitate bridging of a docker and a tether when there is a greater separation between the two strands (i.e., the docket nucleic strand and the tether nucleic strand). Accordingly, the length of a linker of a bridging nucleic acid strand can be chosen to provide a desired separation gap between a docker and tether when both nucleic acid strands are hybridized to the bridging nucleic acid strand. For nucleic acid linkers, a separation distance can be estimated as 0.6 - 0.7 nanometers per nucleotide.

[0291] A bridging nucleic acid strand may have a linker containing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 100, or more than 100 nucleotides. Alternatively or additionally, a bridging nucleic acid strand may have a linker containing no more than about 100, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less than 1 nucleotides. In some cases, a bridging nucleic acid strand may not have a linker.

[0292] FIGs. 13 A - 13D depict different configurations that may affect a chosen length of a linker in a bridging nucleic acid strand. In some cases, a linker of a bridging nucleic acid strand may be chosen to facilitate formation of an interaction between a first moiety and a second moiety. FIG. 13A depicts a bridging nucleic acid strand 1310 that can be utilized to control the separation distance between a pair of fluorescent FRET dyes. A docker strand 1301 attached to a first FRET dye 1303 may be held in proximity to a tether strand 1302 attached to a second FRET dye 1304 such that the FRET dye pair are sufficiently close (e.g., within about 5 nanometers of each other) to undergo a signal-producing FRET interaction in the presence of a stimuli (e.g., an exciting photon). FIG. 13B depicts a bridging nucleic acid strand 1310 that can be utilized to bring together two halves of a split protein system (e.g., a split fluorescent protein, a split luminescent protein). A docker strand 1301 attached to a first half of a split protein system 1305 may be held in proximity to a tether strand 1302 attached to a second half of a split proteinsystem 1306 such that the split protein components are sufficiently close to bind, thereby facilitating emission of a signal from the joined split protein components. FIG. 13C depicts a bridging nucleic acid strand 1310 with a zero-length linker moiety that joins a docker nucleic acid strand 1301 to a tether nucleic acid strand 1302. In the left-hand configuration, the nucleic acid complex may be bound by a ligase enzyme 1350, thereby producing a linkage between the docker strand and tether strand. Such a system may be useful for retaining co-localization of an affinity reagent and an analyte even after a bridging nucleic acid strand has dissociated.

[0293] Alternatively, it may be preferable to provide a larger separation gap between a docker strand and a tether strand with a bridging nucleic acid strand. In the left-hand configuration, FIG. 13D depicts a bridging nucleic acid strand 1310 hybridized to a docker nucleic acid strand 1301 and a tether nucleic acid strand 1302. The complex containing the bridged nucleic acid strands is coupled to a polymerase enzyme 1360. In the presence of modified nucleotides, the polymerase 1360 may extend the docker nucleic acid strand 1301 or tether nucleic acid strand 1302, thereby incorporating modified nucleotides into the extended nucleic acid strand. The right-hand configuration of FIG. 13D depicts an extended nucleic acid strand that has had incorporated detectable labels 1307 attached to the modified nucleotides. The size of the separation between the docker and tether and the concentration of modified nucleotides may be utilized to control the extent of incorporation of the modified nucleotides, thereby controlling the strength of a signal from the extended nucleic acid strand. In some cases, a polymerase may be utilized to incorporated fluorescently-labeled modified nucleotides. In other cases, a polymerase may be utilized to incorporate modified nucleotides that are subsequently labeled via the covalent attachment of fluorescent dye molecules.

[0294] A separation gap between a docker nucleic acid strand and a tether nucleic acid strand, when coupled by a bridging nucleic acid strand, may be at least about 0.1 nanometers (nm), 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, or more than 50 nm. Alternatively or additionally, a separation gap between a docker nucleic acid strand and a tether nucleic acid strand, when coupled by a bridging nucleic acid strand, may be no more than about 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0.1 nm, or less than 0.1 nm. For docker and tether systems containing FRET-based dye pairs, it may be preferable to provide a separation gap of 5 nanometers or less. A separation gap between a docker nucleic acid strand and a tether nucleic acid strand, when coupled by a bridging nucleic acid strand, mayinclude a single-stranded region of the bridging nucleic acid strand of at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, or more than 50 nucleotides. Alternatively or additionally, a separation gap between a docker nucleic acid strand and a tether nucleic acid strand, when coupled by a bridging nucleic acid strand, may include a single-stranded region of the bridging nucleic acid strand of no more than about 50, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, or 0 nucleotide.

[0295] Although some reactions between a first reactant and a second reactant have been exemplified with respect to a bridging nucleic acid strand, it will be recognized that other types of bridging molecules may be utilized. A bridging molecule may be configured to form a non- covalent binding interaction with a first reactant, a second reactant, or both the first reactant and the second reactant. For example, a bridging molecule may comprise a first affinity reagent attached to a second affinity reagent (optionally by a linking moiety), in which the first affinity reagent binds to a ligand (e.g., an affinity tag) of the first reactant and the second affinity reagent binds to a ligand of the second reactant, thereby coupling the first reactant to the second reactant. A bridging molecule may be configured to form a covalent binding interaction with a first reactant, a second reactant, or both the first reactant and the second reactant. For example, a bridging molecule may comprise covalent cross-linking moiety comprising a first reactive moiety and a second reactive moiety, in which the first reactive moiety bonds to a reactive moiety of the first reactant and the second reactive moiety bonds to a reactive moiety of the second reactant, thereby coupling the first reactant to the second reactant.

[0296] FIG. 12 depicts another method of directly recording a binding interaction between an affinity reagent and an analyte. In an initial configuration (left-hand side), FIG. 12 depicts an analyte 1210 immobilized at a fixed address of a solid support 1200. A linking moiety 1205 is immobilized at the fixed address of the solid support 1200 containing the immobilized analyte 1210. The linking moiety 1205 is attached to a cofactor 1206 or a plurality thereof for a luminescent protein 1222. Preferably, the linking moiety 1205 is co-localized at a distance from the immobilized analyte 1210 that is not optically-resolvable. An affinity reagent 1220 is bound to the immobilized analyte 1210, thereby immobilizing the affinity reagent 1220 at the fixed address of the solid support 1200. The affinity reagent 1220 is attached to a linking moiety 1221 that is also attached to the luminescent protein 1222. In the second configuration (right-hand side) of FIG. 12, the luminescent protein 1222 binds to the cofactor 1206, thereby inducingemission of photons 1235 from the luminescent protein 1222. The luminescent signal may be detected at the fixed address of the solid support 1200 by a detector 1240.

[0297] Certain methods set forth herein for recording a binding interaction between an affinity reagent and an analyte may be amenable to a method of kinetic control. Kinetic control may comprise any suitable method for controlling the rate of a secondary binding reaction that records the binding interaction between the affinity reagent and the analyte. Preferably, the binding on-rate for the reaction is slower than the binding on-rate for the affinity reagent with the analyte, thereby limiting the likelihood of a false detection event. It will be recognized that an affinity reagent may have a known and / or characterized tendency to bind to certain epitopes or analytes other than its primary target (i.e., off-target binding). Off-target binding may be characterized by a faster off-rate of the affinity reagent from an analyte having a secondary binding target relative to its off-rate from an analyte having a primary binding target for the affinity reagent. In such cases, it may be preferable for a reaction to have a binding on-rate that is slower than the binding off-rate for an affinity reagent with a secondary binding target. Accordingly, the reaction will be more likely to record binding interactions between an affinity reagent and its primary binding target.

[0298] Oligonucleotide systems may be especially useful for providing kinetic control to reactions. Oligonucleotides having internal complementarity of their nucleotide sequences can fold into secondary structures in which a rate of unfolding is governed by the secondary structure morphology and extent of internal complementarity. FIG. 11 illustrates oligonucleotide systems that may have different kinetic rates of hybridization due to differences in oligonucleotide structure. In case A, a first oligonucleotide attached to moiety Ri (e.g., an analyte, an affinity reagent, a linking moiety, a docker strand, a tether strand, etc.) has a loop structure formed by internal complementarity of nucleotide sequence 1101 with nucleotide sequence 1102. The first oligonucleotide is contacted with a second oligonucleotide attached to moiety R2 (e.g., an analyte, an affinity reagent, a linking moiety, a docker strand, a tether strand, etc.) also contains nucleotide sequences 1101 and 1102. The kinetic rate of unfolding the first oligonucleotide and hybridizing it to the second oligonucleotide may be very slow due to the absence of a toehold sequence for the second oligonucleotide to bind to, thereby initiating dehybridization of the internally complementary nucleotide sequences. In case B, the first oligonucleotide further comprises nucleotide sequences 1103 and 1104, whose lack of complementarity introduces asmaller loop structure into the secondary structure of the first oligonucleotide. The second oligonucleotide comprises a toehold nucleotide sequence 1105 that can hybridize to nucleotide sequence 1103 of the first oligonucleotide, thereby initiating dehybridization of the secondary structure of the first oligonucleotide. Case C depicts a first oligonucleotide with a larger loop structure due to the non-complementarity of nucleotide sequences 1106 and 1107. The second oligonucleotide comprises a larger toehold nucleotide sequence 1108 that can hybridize to nucleotide sequence 1106 of the first oligonucleotide, thereby initiating dehybridization of the secondary structure of the first oligonucleotide. The rate of binding of the first oligonucleotide to the second oligonucleotide in FIG. 11 from slowest to fastest can be ordered as A < B < C. Incorporation of secondary structures into one or more oligonucleotides similar to those depicted in FIG. 11 may be utilized to control the binding on-rates of oligonucleotides in any system or method depicted herein.

[0299] Other methods and systems may be designed to introduce kinetic control in a secondary binding reaction. For example, binding reactions or chemical reactions can be controlled by the concentration of enzymes, reactants, or substrates, as well as the use of modified enzymes, catalysts or substrate derivatives that can increase or decrease the binding on-rate of a secondary binding reaction. For example, use of a biotin ligase enzyme to label an affinity tag for the indirect recording of a binding interaction between an affinity reagent and an analyte may be kinetically throttled by the length of the linker moiety attaching the biotin ligase to the affinity reagent or affinity tag, as well as the concentration of free biotin in solution. Likewise, the biotin ligase enzyme may be modified to have a faster or slower rate of biotin attachment, thereby affecting the enzyme’s processivity. Further, the affinity tag can be modified by amino acid sequence or presence of modified or unnatural amino acids to control the rate of biotin attachment.

[0300] It will be understood that methods and systems exemplified herein via a single analyte and a single affinity reagent can readily be expanded into methods and system containing pluralities of analytes and / or affinity reagents. Array-based methods may be especially useful for observing binding interactions between pluralities of analytes and pluralities of affinity reagents at single-analyte resolution. For some array-based methods, particles (e.g., nucleic acid nanoparticles) may be useful for co-localizing certain components at a single fixed address of the array. For example, a single analyte and a docker strand, as set forth herein, may be attached to asingle nucleic acid nanoparticle, in which the nucleic acid nanoparticle is attached to a fixed address of a solid support. Accordingly, each individual address of a plurality of addresses of a single-analyte array may comprise a single particle, in which the single particle is attached to one and only one analyte, and at least one other moiety that is configured to facilitate formation of a reaction, as set forth herein.

[0301] It will be further understood that methods and systems exemplified by a single set of moi eties configured to form a reaction may further comprise additional moieties configured to form reactions. For example, an analyte may be co-localized with a plurality of docker strands, and / or an affinity reagent may be attached to a plurality of tether strands. In another example, a linking moiety may comprise a plurality of FRET dyes that can be brought into proximity of a plurality of complementary FRET dyes.Hybridization Chain Reactions

[0302] The present disclosure further provides a method of detecting binding of an affinity reagent to an analyte. The method can include steps of (a) contacting an analyte with an affinity reagent, thereby forming a complex, wherein the affinity reagent includes a paratope and a first target nucleic acid, wherein the analyte includes an epitope and a second target nucleic acid, and wherein the complex includes the analyte bound to the affinity reagent via interaction between the paratope and the epitope; (b) contacting the complex with a set of first oligonucleotides and a set of second oligonucleotides, thereby forming a nicked double helix, wherein the first oligonucleotides include a first hairpin nucleotide sequence, wherein the second oligonucleotides include a second hairpin nucleotide sequence, and wherein the nicked double helix includes (i) a first oligonucleotide of the set of first oligonucleotides hybridized to the first target nucleic acid and further hybridized to the second target nucleic acid, and (ii) a plurality of second oligonucleotides of the set of second oligonucleotides hybridized to a plurality of first oligonucleotides of the set of first oligonucleotides; and (c) detecting the nicked double helix, thereby detecting binding of the affinity reagent to the analyte.

[0303] The present disclosure also provides a molecular complex, including (a) an affinity reagent, wherein the affinity reagent includes a paratope and a first target nucleic acid; (b) an analyte, wherein the analyte includes an epitope and a second target nucleic acid, and wherein the affinity reagent is bound to the analyte via interaction between the paratope and the epitope; and (c) a nicked double helix, wherein the nicked double helix includes: (i) a plurality of firstoligonucleotides, each of the first oligonucleotides having a first hairpin nucleotide sequence, and (ii) a plurality of second oligonucleotides, each of the second oligonucleotides having a second hairpin nucleotide sequence, wherein a first oligonucleotide of the nicked double helix is hybridized to the first target nucleic acid and further hybridized to the second target nucleic acid, and wherein a plurality of second oligonucleotides of the nicked double helix is hybridized to a plurality of first oligonucleotides of the nicked double helix.

[0304] It will be understood that embodiments and configurations of the invention exemplified herein for proteins can be extended to other analytes. Sources for proteins and techniques for manipulating proteins are exemplified below. The exemplified sources can provide other analytes using techniques similar to those set forth herein for protein isolation or other techniques known by those skilled in the art. Moreover, an analyte that is used in a method or composition set forth herein can include any of a variety of epitopes. For example, an epitope can include one or more amino acids, nucleotides, or sugars. Conversely, an epitope that is utilized in a method or composition of the present disclosure can lack amino acids, nucleotides or sugars.

[0305] An analyte can be attached to a target nucleic acid. Several methods and reagents will be exemplified herein for attachment of an analyte to a target nucleic acid. It will be understood that similar methods and reagents can be used to attach an analyte to other entities such as retaining components, structured nucleic acid particles, nucleic acid origami or artificial polymers. Moreover, similar methods and reagents can be used to attach affinity reagents to a variety of entities such as target nucleic acids, retaining components, structured nucleic acid particles, nucleic acid origami or artificial polymers.

[0306] A protein that is to be attached to a target nucleic acid can include at least one amino acid that is reactive with an attachment moiety on the moiety of interest. Optionally, attachment can exploit a reactive moiety that is present in a natural amino acid. For example, attachment can occur between an attachment moiety and (A) an amine that is present at the amino terminus of a protein or in the side chain of a lysine, histidine or arginine; (B) a sulfur that is present in the side chain of a cysteine or methionine; (C) a carboxylate that is present at the carboxy terminus of a protein or in the side chain of an aspartic acid or glutamic acid; (D) an oxygen that is present in the side chain of a serine, threonine or tyrosine; or (E) an amide that is present in the side chain of a glutamine or asparagine.

[0307] Optionally, a protein can be modified to incorporate an exogenous moiety that is reactive with an attachment moiety on a target nucleic acid or other entity. For example, one or more amino acids of a protein can be modified to include an exogenous moiety that forms a covalent bond with a chemically reactive attachment moiety on a target nucleic acid. Optionally, one or more amino acids of a protein can be modified to include an exogenous moiety that participates in a binding reaction to form a non-covalent bond with a target nucleic acid or other entity. An amino acid can be functionalized with an exogenous moiety by exploiting reactivities of amines, sulfurs, carboxylates, oxygens, amides or other reactive moieties found in native amino acids. Exemplary reactive moieties (e.g., native or exogenous to proteins) and attachment moieties with which they react are set forth below or in WO 2019 / 195633 Al; US Pat. App. Pub. Nos.2021 / 0101930 Al, 2022 / 0162684 Al or 2022 / 0227890 Al; or US Pat. Nos. 11,203,612 or 11,505,796, each of which is incorporated herein by reference.

[0308] A protein and an attachment moiety with which the protein will react can include components of a SpyTag / SpyCatcher system (See, Zakeri et al. Proceedings Nat 'I Acad. Sciences USA. 109 (12): E690-7 (2012)). In this system, a 13 amino acid tag protein (Spy Tag) forms a first coupling handle, with a 12.3 kDa protein (Spy-Catcher) forming the partner to the first coupling handle. Optionally, the SpyCatcher can be attached to a protein. The SpyCatcher can irreversibly bond to a SpyTag on another entity through an isopeptide bond. As will be appreciated, either the SpyTag or SpyCatcher can be on a protein and the entity to which the protein is to be attached can be functionalized with the other.

[0309] In some configurations, an attachment moiety on a protein can be reactive in a click reaction. Attachment can be accomplished in part by chemical reaction of a click moiety with a reactive moiety on another entity. The chemical conjugation may proceed via an amide formation reaction, reductive amination reaction, N-terminal modification, thiol Michael addition reaction, disulfide formation reaction, copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction, strain-promoted alkyne-azide cycloaddtion reaction (SPAAC), Strain-promoted alkyne- nitrone cycloaddition (SPANC), inverse electron-demand Diels-Alder (IEDDA) reaction, oxime / hydrazone formation reaction, free-radical polymerization reaction, or a combination thereof.

[0310] Moieties that participate in cycloaddition reactions may be utilized to attach a protein to another entity such as a target nucleic acid. In cycloaddition reactions, two or more unsaturatedmoi eties form a cyclic product with a reduction in the degree of unsaturation, these reaction partners are typically absent from natural systems, and so the use of cycloadditions for conjugation utilizes the introduction of unnatural functionality within a coupling partner. Exemplary moieties and their attachment reactions include:

[0311] In some cases, moieties that participate in Copper-Catalyzed Azide-AlkyneCycloadditions (CuAAC) may be utilized to attach a protein to a target nucleic aci...

Claims

CLAIMSWhat is claimed is:

1. A method of detecting a first reaction, comprising:(a) providing immobilized on a solid support:(i) an analyte; and(ii) a first reactant, the first reactant being immobilized on the support within a first distance from the analyte;(b) contacting the immobilized analyte with a probe, the probe comprising an affinity reagent and a second reactant, the affinity reagent having binding specificity for the analyte, and the second reactant being capable of a second reaction with the first reactant when within a second distance from the first reactant;(c) forming a first reaction between the analyte and the affinity reagent, thereby bringing the second reactant within the second distance of the first reactant;(d) after forming the first reaction, forming the second reaction between the first reactant and the second reactant; and(e) detecting the first reaction.

2. The method of claim 1, wherein the first distance is an optically non-resolvable distance between the analyte and the first reactant.

3. The method of claim 2, wherein the first distance is less than 300 nanometers (nm).

4. The method of claim 3, wherein the first distance is less than 50 nm.

5. The method of any one of claims 1 - 4, wherein the second distance is less than the first distance.

6. The method of claim 5, wherein the second distance is no more than 10 nm.

7. The method of any one of claims 1 - 5, wherein the probe further comprises a linker, wherein the linker couples the affinity reagent to the second reactant.

8. The method of claim 7, wherein a length of the linker is greater than the first distance.

9. The method of claim 7, wherein a length of the linker is less than the first distance.

10. The method of any one of claims 1 - 9, wherein forming the second reaction comprises forming a non-covalent binding reaction.

11. The method of claim 10, wherein the first reactant comprises a first oligonucleotide, and wherein the second oligonucleotide comprises a second oligonucleotide.

12. The method of claim 11, wherein forming the non-covalent binding interaction comprises hybridizing the first oligonucleotide to the second oligonucleotide.

13. The method of claim 11, wherein forming the non-covalent binding interaction comprises hybridizing a third oligonucleotide to the first oligonucleotide and the second oligonucleotide, thereby coupling the first oligonucleotide to the second oligonucleotide.

14. The method of any one of claims 1 - 9, wherein forming the second reaction comprises forming a covalent binding reaction.

15. The method of claim 14, wherein the second reactant comprises an enzyme, wherein forming the covalent binding interaction comprises attaching a detectable label to the first reactant with the enzyme.

16. The method of claim 14, wherein the first reactant comprises an enzyme, wherein forming the covalent binding interaction comprises attaching a detectable label to the second reactant with the enzyme.

17. The method of any one of claims 1 - 9, wherein forming the second reaction comprises forming a photon transfer reaction.

18. The method of claim 17, wherein detecting the first reaction comprises detecting a photon from the photon transfer reaction.

19. The method of claim 17 or 18, wherein the first reactant comprises a first fluorescent label, wherein the second reactant comprises a second fluorescent label, wherein forming the photon transfer reaction comprises forming a Forster Resonance Energy Transfer (FRET) interaction between the first fluorescent label and the second fluorescent label.

20. The method of any one of claims 1 - 19, wherein the probe further comprises a detectable label.21 . The method of claim 20, wherein detecting the first reaction comprises detecting a signal from the detectable label.

22. The method of any one of claims 1 - 21, further comprising binding a bridging molecule to the first reactant and the second reactant.

23. The method of claim 22, wherein the bridging molecule brings the second reactant within the second distance of the first reactant.

24. The method of claim 22 or 23, wherein the bridging molecule further comprises a detectable label.

25. The method of claim 24, wherein detecting the first reaction comprises detecting a signal from the detectable label.

26. The method of any one of claims 22 - 25, wherein the bridging molecule non-covalently binds to the first reactant, the second reactant, or to both the first reactant and the second reactant.

27. The method of any one of claims 22 - 25, wherein the bridging molecule covalently binds to the first reactant, the second reactant, or to both the first reactant and the second reactant.

28. A method, comprising:(a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, wherein the equilibration period is sufficient to form a binding equilibrium between affinity reagents of the plurality of affinity reagents and analytes of the plurality of analytes; and(b) after the equilibration period, detecting complexes comprising an affinity reagent bound to an analyte, wherein the quantity of detected complexes is greater than a steady-state quantity of complexes that exists at the binding equilibrium.

29. The method of claim 28, further comprising: (c) after step (a) and before step (b), removing unbound affinity reagents from contact with the plurality of analytes.

30. The method of claim 28 or 29, further comprising: (d) after step (a) and before step (b), dissociating the complexes.31 . The method of claim 30, further comprising: (e) before dissociating the complexes, recording for each complex the presence of a binding interaction between the affinity reagent and the analyte.

32. The method of claim 31, wherein recording the binding interaction between the affinity reagent and the analyte comprises forming a reaction between a first moiety co-localized with the affinity reagent and a second moiety co-localized with the analyte.

33. The method of claim 32, wherein the reaction inhibits dissociation of the affinity reagent from the complex.

34. The method of claim 32, wherein the reaction produces a detectable signal.

35. The method of claim 32, wherein the reaction transfers a detectable label to a unique identifier co-localized with the analyte.

36. The method of claim 32, wherein the reaction attaches a detectable label to the affinity agent.

37. The method of any one of claims 28 - 36, wherein each individual affinity reagent of the plurality of affinity reagents has a binding specificity for an epitope that is present in the plurality of analytes.

38. The method of claim 37, wherein, at the binding equilibrium, the steady-state quantity of complexes is no more than 50% of a quantity of analytes containing the epitope.

39. The method of claim 38, wherein step (b) comprises detecting a quantity of complexes exceeding 50% of the quantity of analytes containing the epitope.

40. The method of any one of claims 28 - 39, wherein each individual affinity reagent of the plurality of affinity reagents has a binding specificity for two or more different species of analytes that are present in the plurality of analytes.

41. The method of claim 40, wherein each individual species of the two or more species of analytes differs from any other species of the two or more species of analytes with respect to primary structure, proteoform, or isoform.

42. The method of claim 40 or 41, wherein, at the binding equilibrium, the steady-state quantity of complexes is no more than 50% of a quantity of the two or more different species of analytes.

43. The method of claim 42, wherein step (b) comprises detecting a quantity of complexes exceeding 50% of the quantity of the two or more different species of analytes.

44. A method, comprising:(a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, wherein each analyte of the plurality of analytes is individually paired to a unique identifier;(b) during the equilibration period, recording detection proxies at unique identifiers containing an affinity reagent bound to an analyte; and(c) detecting signals from detection proxies at a subset of the unique identifiers, thereby identifying analytes bound by affinity reagents during the equilibration period.

45. A method, comprising:(a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, thereby coupling affinity reagents of the plurality of affinity reagents to analytes of the plurality of analytes, wherein each analyte of the plurality of analytes is paired to a unique identifier;(b) forming a detectable reaction for analytes bound by an affinity reagent during the equilibration period; and(c) detecting presence of the detectable reaction at a subset of unique identifiers, thereby detecting the subset of analytes of the plurality of analytes bound by an affinity reagent of the plurality of affinity reagents.

46. The method of claim 45, wherein detecting presence of the detectable reaction at a unique identifier of the subset of unique identifiers comprises identifying a superordinate binding interaction between an affinity reagent and the analyte paired to the unique identifier.

47. The method of claim 45, wherein detecting presence of the detectable reaction at a unique identifier of the subset of unique identifiers comprises identifying a subordinate binding interaction between an affinity reagent and the analyte paired to the unique identifier.

48. The method of any one of claims 45 - 47, wherein each individual unique identifier is located at an address of a solid support, wherein an address of a unique identifier is spatially resolvable from an address of any other unique identifier.

49. The method of claim 48, wherein the unique identifier comprises a site of the solid support.

50. The method of claim 48, wherein the unique identifier comprises a barcode immobilized at the address of the solid support.

51. The method of any one of claims 45 - 50, wherein the equilibration period is at least 1 minute.

52. The method of claim 51, wherein the equilibration period is at least 10 minutes.

53. The method of any one of claims 45 - 52, wherein each analyte is co-located with a first detection moiety.

54. The method of claim 53, wherein each affinity reagent of the plurality of affinity reagents is attached to a second detection moiety.

55. The method of claim 54, wherein forming the detectable reaction comprises coupling the first detection moiety to the second detection moiety.

56. The method of claim 54, wherein coupling the first detection moiety to the second detection moiety further comprises producing a detectable signal from a complex of the first detection moiety and the second detection moiety.

57. The method of claim 55 or 56, wherein coupling the first detection moiety to the second detection moiety comprises coupling a third detection moiety to the first detection moiety and the second detection moiety.

58. The method of claim 57, wherein the third detection moiety is attached to a detectable label.

59. The method of claim 57 or 58, wherein coupling the third detection moiety to the first detection moiety and the second detection moiety comprises:(i) coupling the third detection moiety to the second detection moiety; and(ii) after coupling the third detection moiety to the second detection moiety, coupling the third detection moiety to the first detection moiety.

60. The method of claim 59, further comprising, after step (ii), dissociating the third detection moiety from the second detection moiety.

61. The method of claim 54, wherein forming the detectable reaction comprises positioning the first detection moiety within a proximity of the second detection moiety.

62. The method of claim 61, wherein the proximity is less than 10 nanometers.

63. The method of claim 62, wherein the proximity is less than 5 nanometers.

64. The method of any one of claims 45 - 63, wherein the plurality of analytes comprises two or more species of analytes.

65. The method of claim 64, wherein the two or more species of analytes comprises at least 100 species of analytes.

66. The method of claim 65, wherein the two or more species of analytes comprises at least 1000 species of analytes.

67. The method of any one of claims 64 - 66, wherein the subset of analytes bound by the plurality of affinity reagents comprises two or more species of analytes.

68. The method of claim 67, wherein the subset of analytes bound by the plurality of affinity reagents comprises at least 100 species of analytes.

69. The method of any one of claims 45 - 68, further comprising, before detecting presence of the detectable reaction at the subset of unique identifiers, removing unbound affinity reagents of the plurality of affinity reagents from contact with the plurality of analytes.

70. The method of any one of claims 45 - 68, wherein detecting presence of the detectable reaction at the subset of unique identifiers occurs in the presence of unbound affinity reagents of the plurality of affinity reagents.

71. A method, comprising:(a) contacting a plurality of affinity reagents to a plurality of analytes for an equilibration period, wherein the equilibration period is sufficient to form a binding equilibrium between affinity reagents of the plurality of affinity reagents and analytes of the plurality of analytes; and(b) detecting during the equilibration period complexes comprising an affinity reagent bound to an analyte, wherein the quantity of detected complexes is greater than a steady-state quantity of complexes that exists at the binding equilibrium.

72. A method, comprising:(a) binding an affinity reagent to an analyte at a fixed spatial address;(b) coupling a kinetically-controlled detection agent to the fixed spatial address; and(c) after coupling the kinetically-controlled detection agent to the fixed spatial address, detecting a signal at the fixed spatial address.

73. The method of claim 72, wherein step (a) is performed in the presence of the kinetically- controlled detection agent.

74. The method of claim 73, wherein a binding on-rate of the kinetically-controlled detection agent binding to the fixed spatial address is slower than a binding on-rate of the affinity agent binding to the analyte.

75. The method of claim 74, wherein the binding on-rate of the kinetically-controlled detection agent binding to the fixed spatial address is slower than a binding off-rate of the affinity agent binding to the analyte.

76. The method of claim 74, wherein the binding on-rate of the kinetically-controlled detection agent binding to the fixed spatial address is faster than a binding off-rate of the affinity agent binding to the analyte.

77. The method of claim 72, wherein the kinetically-controlled detection agent is contacted to the fixed spatial address after step (a).

78. The method of any one of claims 72 - 77, wherein the kinetically-controlled detection agent comprises a detectable label.

79. The method of any one of claims 72 - 77, wherein the kinetically-controlled detection agent does not comprise a detectable label.

80. The method of claim 79, wherein the affinity reagent or the fixed spatial address comprises a detectable label.

81. The method of claim 80, wherein the affinity reagent and the fixed spatial address comprises a detectable label.

82. The method of any one of claims 72 - 81, wherein coupling the kinetically-controlled detection agent to the fixed spatial address comprises non-covalently coupling the kinetically- controlled detection agent to the fixed spatial address.

83. The method of claim 82, wherein the kinetically-controlled detection agent comprises a nucleic acid.

84. The method of claim 83, wherein the nucleic acid comprises a region of internal complementarity.

85. The method of claim 82, wherein coupling the kinetically-controlled detection agent to the fixed spatial address further comprises dissociating the region of internal complementarity of the nucleic acid.

86. The method of claim 85, wherein dissociating the region of internal complementarity of the nucleic acid comprises hybridizing the nucleic acid to a complementary nucleic acid, wherein the complementary nucleic acid is immobilized at the fixed spatial address.

87. The method of any one of claims 72 - 81, wherein coupling the kinetically-controlled detection agent to the fixed spatial address comprises covalently coupling the kinetically- controlled detection agent to the fixed spatial address.

88. The method of claim 87, wherein the covalently coupling the kinetically-controlled detection agent to the complex comprises covalently coupling a reactive moiety of the kinetically- controlled detection agent to a complementary reactive moiety, wherein the complementary reactive moiety is immobilized at the fixed spatial address.

89. A system, comprising:(a) a solid support comprising a plurality of sites, wherein each site of the plurality of sites is optically resolvable from any other site of the plurality of sites; and(b) a plurality of analytes immobilized on the solid support, wherein each site of the plurality of sites is attached to one and only one analyte of the quantity of analytes; wherein, in a first configuration, the system further comprises a plurality of affinity reagents, wherein a fraction of the affinity reagents is bound to analytes of the plurality of analytes, wherein a quantity of the fraction of affinity reagents is determined by a binding equilibrium of the affinity reagents for the analytes; and wherein, in a second configuration, the system is substantially devoid of affinity reagents, wherein each individual site of a fraction of sites of the plurality of sites comprises a detectable label, wherein a quantity of the fraction of sites is greater than a quantity of the second fraction of affinity reagents.

90. The system of claim 89, wherein the binding equilibrium of the plurality of affinity agents has a characterized dissociation constant.91 . The system of claim 89 or 90, wherein the affinity reagents bind to epitopes present in the plurality of analytes.

92. The system of claim 91, wherein each individual affinity reagent of the plurality of affinity reagents binds to an epitope that is common to two or more differing analytes of the plurality of analytes.

93. The system of claim 92, wherein, at the binding equilibrium, affinity reagents of the plurality of affinity reagents are bound to no more than 90% of analytes containing the epitope.

94. The system of claim 93, wherein the quantity of the fraction of sites is greater than 90% of a quantity of analytes comprising the epitope.

95. A system, comprising:(a) a solid support comprising a plurality of sites, wherein each site of the plurality of sites is optically resolvable from any other site of the plurality of sites;(b) a plurality of analytes immobilized on the solid support, wherein each site of the plurality of sites is attached to one and only one analyte of the quantity of analytes; and(c) a plurality of affinity reagents bound analytes of the plurality of analytes at sites of the plurality of sites, wherein the affinity reagents have a known binding equilibrium for binding to analytes of the plurality of analytes, and wherein a quantity of the sites of the plurality of sites is greater than a quantity of analytes bound to affinity reagents based upon the known binding equilibrium.

96. A method of detecting a first reaction, comprising:(a) providing immobilized on a solid support:(i) an analyte; and(ii) a first reactant, the first reactant being immobilized on the support within a first distance from the analyte;(b) contacting the immobilized analyte with a probe, the probe comprising an affinity reagent and a second reactant, the affinity reagent having binding specificity for the analyte, and the second reactant being capable of a second reaction with the first reactant when within a second distance from the first reactant;(c) forming a first reaction between the analyte and the affinity reagent, and forming a second reaction between the first reactant and the second reactant; and(d) detecting the first reaction.

97. A composition, comprising:(a) an affinity reagent having a binding specificity for at least a first analyte;(b) a first reactant, wherein the first reactant has reactivity with a second reactant; and(c) a linker, wherein the linker couples the affinity reagent to the first reactant, wherein the linker permits the first reactant to react with the second reactant when the affinity reagent is bound to the analyte.

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