CN117120627A - Affinity reagents with enhanced binding and detection characteristics - Google Patents

Abstract

An affinity reagent having: (a) a retention component, such as a structured nucleic acid particle; and (b) one or both of the following: (i) One or more labeling components attached to the retention component, and (ii) one or more binding components attached to the retention component.

Description

Affinity reagents with enhanced binding and detection characteristics

Cross reference

The present application claims the benefit of priority from U.S. provisional application Ser. No. 63/112,607, filed 11/2020, U.S. provisional application Ser. No. 63/132,170, filed 12/30/2020, and U.S. provisional application Ser. No. 63/227,080, filed 7/2021, which are incorporated herein by reference.

Background

Affinity reagents include a broad class of chemical reagents that form a detectable interaction with other molecules. Affinity reagents may include binding reagents that form temporary or reversible binding pairs with other molecules. Affinity reagents can be used to characterize the structure and properties of biomolecules such as polypeptides, nucleic acids, and polysaccharides. The affinity reagent may comprise a detectable label for the purpose of visualizing the affinity reagent. Typically these detectable labels are fluorescent labels. There is a need for labeled affinity reagents that will generate strong and reliable signals, including, for example, signals that are strong enough for single molecule detection and reliable enough for accurate quantification. There is also a need for affinity reagents that bind affinity to a target molecule with sufficient affinity to support detection of the target molecule at single molecule resolution, for example.

Disclosure of Invention

The present disclosure provides an affinity reagent having: (a) retaining the components; and (b) one or both of the following: (i) One or more labeling components, and (ii) one or more binding components. Optionally, one or more of the marker components are attached to the retention component. Alternatively or additionally, one or more of the binding components are attached to the retention component. The retention component may include a structured nucleic acid, such as a nucleic acid paper break.

The affinity reagent may comprise: (a) retaining the components; (b) One or more labeling components, and (ii) a plurality of binding components. Optionally, the equilibrium dissociation constant of the affinity reagent is less than the equilibrium dissociation constant of any of the plurality of binding members for the binding partner, or wherein the dissociation rate constant of the detectable probe is less than the dissociation rate constant of any of the plurality of binding members for the binding partner.

The affinity reagent may comprise: (a) retaining the components; (b) A plurality of labeling components, and (ii) one or more binding components.

The affinity reagents of the present disclosure may be configured as detectable probes. The detectable probe may comprise: (a) retaining the components; (b) one or more marker components; and (c) two or more binding components attached to the retention component, wherein the equilibrium dissociation constant of the detectable probe for a binding partner is less than the equilibrium dissociation constant of any of the two or more binding components for the binding partner, or wherein the dissociation rate constant of the detectable probe for a binding partner is less than the dissociation rate constant of any of the two or more binding components for the binding partner.

The present disclosure further provides a method of detecting an analyte comprising the steps of: (a) Contacting an analyte with a detectable probe, wherein the detectable probe comprises (i) a retention component; (ii) One or more labeling components, and (iii) one or more binding components; and (b) acquiring signals from the one or more marker components, thereby detecting the analyte.

Also provided is a method of detecting an analyte comprising the steps of: (a) Contacting the analyte with a first detectable probe comprising: (i) a first retention component, (ii) one or more labeling components, and (iii) a first set of two or more binding components attached to the retention components, wherein at least one of the binding components in the first set binds to a first epitope in the analyte; (b) Obtaining signals from the one or more labeled components of the first detectable probe; (c) Contacting the analyte with a second detectable probe comprising: (i) a second retention component, (ii) one or more labeling components, and (iii) a second set of two or more binding components attached to the retention components, wherein at least one of the binding components in the second set binds to a second epitope in the analyte, the second epitope having a different chemical composition than the first epitope; and (d) acquiring signals from the one or more labeled components of the second detectable probe, thereby detecting the analyte. Optionally, the second retention component has substantially the same structure as the first retention component.

The present disclosure also provides a composition comprising: (a) A probe having a first structured nucleic acid particle attached to a binding component; and (b) an analyte of a second structured nucleic acid particle having an epitope attached to the binding component, wherein the probe is attached to the analyte via binding of the binding component to the epitope. In a particular configuration, one or both of the structured nucleic acid particles comprises a nucleic acid fold.

Also provided is a composition comprising: (a) A plurality of different probes, each of the different probes having a first structured nucleic acid particle attached to a binding member, each of the different probes having a different binding member; and (b) a plurality of different analytes, each of the different analytes having a second structured nucleic acid particle attached to an epitope of a different binding component, wherein the different probes are attached to the different analytes via binding of the different binding components of the plurality of different probes to the epitopes of the plurality of different analytes. Optionally, the first structured nucleic acid particles are substantially identical for the different probes. As a further option, the second structured nucleic acid particles may be substantially identical for the different analytes. In a particular configuration, one or both of the structured nucleic acid particles comprises a nucleic acid fold. The nucleic acid folds of the different probes may optionally include the same scaffold nucleic acid structure, whether the staple structure is the same or different for the different probes. Alternatively or additionally, the nucleic acid folds of the different analytes may optionally include the same scaffold nucleic acid structure, whether the staple structure is the same or different for the different analytes.

Described herein is a detectable probe comprising a retention component comprising one or more label components (e.g., a detectable label) and two or more binding components coupled to the retention component, wherein the detectable probe has a dissociation constant for a binding partner that is less than the dissociation constant of any of the two or more binding components for the binding partner.

In some configurations, one or more label components of a detectable probe are coupled to the retention component of the detectable probe. In some embodiments, the retention component comprises a scaffold having closed single stranded nucleic acids and a plurality of oligonucleotides hybridized to the scaffold.

In some configurations, the retention component in the detectable probe or affinity reagent comprises a scaffold nucleic acid. The scaffold may comprise a strand from a phage genome or plasmid, for example from the M13 phage genome. Optionally, the retention component may further comprise a plurality of oligonucleotides. The oligonucleotides may be annealed to the scaffold, for example, to form staples in a folded paper structure. In some configurations, an oligonucleotide of the plurality of oligonucleotides may include at least one non-natural nucleotide. Optionally, the non-natural nucleotides in the oligonucleotides may have functional groups, such as functional groups for bioorthogonal or click reactions. In some configurations, one, two, or more binding components are attached to one or more of the plurality of oligonucleotides. In some configurations, one, two, or more labeling components are attached to one or more of the plurality of oligonucleotides. In some configurations, the scaffold can comprise at least one non-natural nucleotide. Optionally, the non-natural nucleotides in the scaffold may have functional groups, such as functional groups for bio-orthogonal or click reactions. In some configurations, one, two, or more binding components are attached to the scaffold. In some configurations, one, two, or more marker components are attached to the scaffold.

In some configurations, the dissociation constant of a detectable probe or affinity reagent having two or more binding components for a binding partner is less than the dissociation constant of any of the two or more binding components for the binding partner. For example, the dissociation constant of the detectable probe or affinity reagent for the binding partner may be less than or equal to 50%, 25%, 10% or less of the dissociation constant of any of the two or more binding components for the binding partner. In some configurations, the dissociation rate (off-rate) of the detectable probe or affinity reagent upon binding to the binding partner is lower than the dissociation rate of any of the two or more binding components alone upon binding to the binding partner. In some configurations, the binding rate (on-rate) of the detectable probe or affinity reagent to the binding partner is higher than the individual binding rate of any of the two or more binding components to the binding partner.

In some configurations, the detectable probe or affinity reagent has a binding affinity that is non-zero for the first type of epitope and a binding affinity that is non-zero for the second type of epitope. For example, the detectable probe or affinity reagent may have a first non-zero binding probability to a first type of epitope and a second non-zero binding probability to a second type of epitope. In some configurations, a first binding component of the two or more binding components of the detectable probe or affinity reagent has a first non-zero binding probability to a first type of epitope and also has a second non-zero binding probability to a second type of target moiety. In other configurations, a first binding component of the two or more binding components comprises a first non-zero probability of binding to a first type of target moiety and a second binding component of the two or more binding components comprises a second non-zero probability of binding to a second type of target moiety.

In some configurations, at least one of the binding components in the detectable probe or affinity reagent comprises an antibody or functional fragment thereof, wherein the binding partner of the detectable probe or affinity reagent has an epitope of the antibody or functional fragment thereof. In some configurations, at least one of the binding components in the detectable probe or affinity reagent comprises an aptamer, wherein the binding partner of the detectable probe or affinity reagent has an epitope of the aptamer.

In some configurations, the detectable probe or affinity reagent binds to the binding partner via at least one binding component. Optionally, the binding partner may bind to the detectable probe or affinity reagent via two or more binding components. The binding partner may be a polypeptide. At least one of the binding components may be configured to recognize a dimeric, trimeric or tetrameric amino acid sequence in the polypeptide. Optionally, the polypeptide may include post-translational modifications, such as being present within or outside of the epitope recognized by the binding component. In some embodiments, the binding partner comprises a non-polypeptide material, such as a polysaccharide, a polymer, a metal, a ceramic, or a combination thereof. In some embodiments, the non-polypeptide material comprises a polysaccharide, a polymer, a metal, or a ceramic nanoparticle. The detectable probe or affinity reagent may be non-covalently bound to the binding partner or covalently bound to the binding partner. The binding partner that binds to the detectable probe or affinity reagent may be in solution phase or attached to a solid support. Optionally, the binding partner may be attached to a structured nucleic acid particle other than the structured nucleic acid particle that is a component of the detectable probe or affinity reagent to which it binds. The structured nucleic acid particles can optionally mediate attachment of the binding partner to a solid support, e.g., at a site of an array.

The retention component of the detectable probe or affinity reagent having two or more binding components may be configured to limit a first binding component of the two or more binding components to not contact a second binding component of the two or more binding components. The retention component of the detectable probe or affinity reagent having two or more label components may be configured to limit a first label component of the two or more label components to not contact a second label component of the two or more label components. The retention component of the detectable probe or affinity reagent having a labeling component and a binding component may be configured to limit the labeling component from contacting the binding component. Optionally, the retained component may limit the first component of the two or more components to a specified distance, such as a distance no greater than 1nm, 5nm, 10nm, 20nm, or longer, from the second component of the two or more components. In some configurations, the limitation is due to an angular offset. For example, the angular offset may be at least about 90 ° or 180 °. In some configurations, the restriction is due to a blocking portion, such as a blocking portion attached to the retained component. Exemplary blocking moieties include, but are not limited to, polyethylene glycol (PEG), polyethylene oxide (PEO), linear or branched alkane chains, or dextran.

The retention component of the detectable probe or affinity reagent may comprise a three-dimensional structure having a first side offset from a second side. For example, the first side may be offset from the second side by an angle offset of at least about 90 ° or 180 °. Optionally, one, some or all of the binding components of the detectable probe or affinity reagent are confined to the first side and are not confined to the second side. In some configurations, the first side and the second side may each include one or more binding components. As a further alternative, one, some or all of the labeling components of the detectable probe or affinity reagent may be confined to a first side of the retention component and not to a second side of the retention component. In some configurations, the first side and the second side may each include one or more marking components. Thus, the detectable probe or affinity reagent may be configured to retain the binding component on a first side of the retention component while retaining the labeling component on the other side of the retention component. The detectable probes or affinity reagents may be configured to limit one, some, or all of the binding components to the side of the retention component on which one, some, or all of the labeling components are located. In addition, the detectable probes or affinity reagents may be configured to limit one, some, or all of the labeling components to the side of the retention component on which one, some, or all of the binding components are located. The retention component of the detectable probe or affinity reagent may comprise a Structured Nucleic Acid Particle (SNAP), such as a nucleic acid nanosphere or a nucleic acid fold.

The one or more label components of the detectable probe or affinity reagent can include any of a variety of labels, including, for example, an optical label (e.g., a fluorophore, a luminophore), a radiolabel, or a nucleic acid-based label (e.g., a sequence tag). In some configurations, two or more label components of a detectable probe or affinity reagent may produce overlapping or indistinguishable signals. For example, two or more of the label components may be fluorophores configured to emit at the same wavelength. In some configurations, two or more label components of a detectable probe or affinity reagent produce signals that are distinguishable from each other. For example, two or more of the label components may be fluorophores configured to emit at different wavelengths from one another.

In some configurations, the two or more label components of the detectable probe or affinity reagent include a donor and an acceptor in a Forster resonance energy transfer mechanism. Alternatively, two or more of the label components of the detectable probe or affinity reagent may be separated from each other by a distance that precludes quenching or Forster resonance energy transfer. The two or more label components of the detectable probe or affinity reagent may have a relative orientation that precludes quenching or Forster resonance energy transfer. For configurations that include SNAP, a first fluorescent label can be attached at a first nucleotide position in SNAP and a second fluorescent label attached at a second nucleotide position in SNAP, wherein the first nucleotide position is separated from the second nucleotide position by at least 3, 4, 5, 6, 7, 8, or 9 nucleotide positions in the primary sequence of the structured nucleic acid particle. Alternatively or additionally, the first nucleotide position may be separated from the second nucleotide position by at most 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide position in the primary sequence of the structured nucleic acid particle.

The binding or labeling component may be attached to the detectable probe or affinity reagent by a linker. The joint may be a rigid joint or a flexible joint. Exemplary flexible linkers include, but are not limited to, PEG, PEO, alkane chains, single stranded nucleic acids, or combinations thereof. Double-stranded nucleic acids or branched alkane strands may be used as rigid linkers.

In some configurations, the major diameter of the detectable probe or affinity reagent is greater than the major diameter of the binding partner to which it binds. Optionally, the volume of the detectable probe or affinity reagent is greater than the volume of the binding partner to which it binds.

In particular configurations, the detectable probe or affinity reagent may comprise an optically detectable retention component. For example, one, two, or more binding components may be attached to the optically detectable retaining component. In such a configuration, the detectable probe or affinity reagent need not include a labeling component, and the cocoa-baseDetection is performed by observing the signal generated by the optically detectable retention component. Particularly useful optically detectable retention components include, but are not limited to, fluorescent nanoparticles, fluoSpheres ^TM Or quantum dots.

In another aspect, described herein are detectable probes or affinity reagents comprising an optically detectable retention component and two or more binding components coupled to the optically detectable retention component, wherein the detectable probes or affinity reagents have a dissociation constant for a binding partner that is less than the dissociation constant of any of the two or more binding components for the binding partner.

In another aspect, described herein are detectable probes or affinity reagents comprising an optically detectable retention component and two or more binding components coupled to the optically detectable retention component, wherein the binding rate of the detectable probes or affinity reagents for binding to a binding partner is higher than the binding rate of any of the two or more binding components for binding to the binding partner.

In another aspect, described herein are detectable probes or affinity reagents comprising an optically detectable retention component and two or more binding components coupled to the optically detectable retention component, wherein the detectable probes or affinity reagents have a lower dissociation rate for binding to a binding partner than any of the two or more binding components.

In another aspect, described herein is a method comprising contacting an analyte with a detectable probe, wherein the analyte is a binding partner of the detectable probe, and acquiring a signal from the detectable probe, thereby detecting the analyte. The method may further comprise confirming the analyte from the acquired signal or determining the chemical composition of the analyte from the acquired signal. In some embodiments, the analyte comprises a polypeptide, and the determined chemical composition comprises the presence or absence of an amino acid sequence of at least a portion of the polypeptide, or the presence or absence of a post-translationally modified amino acid in the polypeptide. In some embodiments, the method further comprises quantifying the analyte from the obtained signal. In some embodiments, the method further comprises determining the location of the analyte on the solid support from the acquired signal, e.g., identifying the site in the array at which the analyte is located. In some embodiments, the signal is obtained from an optically detectable retention component of the detectable probe. In some embodiments, the signal is obtained from one or more labeled components of the detectable probe.

In another aspect, described herein is a method comprising (a) contacting a plurality of different analytes with a first plurality of detectable probes, wherein detectable probes from the first plurality of detectable probes bind to different analytes from a first subset of the plurality of different analytes, (b) acquiring signals from the first plurality of detectable probes, (c) contacting the plurality of different analytes with a second plurality of detectable probes, wherein detectable probes from the second plurality of detectable probes bind to different analytes from a second subset of the plurality of different analytes, wherein different analytes from the first subset are different from analytes of the second subset, (d) acquiring signals from the second plurality of detectable probes, and (e) validating analytes based on the signals acquired in step (b) and step (d). Optionally, the first plurality of detectable probes comprises substantially the same two or more binding members as the second plurality of detectable probes. Alternatively, the first plurality of detectable probes may comprise two or more binding members that are different from the two or more binding members of the second plurality of detectable probes. As a further option, one, some or all of the detectable probes of the first plurality may comprise a retention component. Optionally, the retention components may have a common structure for some or all of the detectable probes in the first plurality. For example, some or all of the remaining components of the detectable probes may comprise the same paper folding structure, such as a scaffold folding structure. Similarly, the retained components of one, some, or all of the detectable probes in the first plurality may have the same structure as the retained components of one, some, or all of the detectable probes in the second plurality. For example, the retained components of some or all of the detectable probes in the first and second pluralities may comprise the same paper folding structure, such as a scaffold folding structure.

In some configurations, the above-described method may further comprise the step of removing the first plurality of detectable probes from the plurality of different analytes prior to step (c). In some configurations of the above methods, the first plurality of detectable probes produces the same signal as the signal produced by the one or more labeling components of the second plurality of detectable probes. For example, the first plurality of detectable probes may comprise the same one or more label components as the second plurality of detectable probes. In some configurations of the above methods, the first plurality of detectable probes comprises one or more label components that are different from the one or more label components of the second plurality of detectable probes. In some configurations of the above methods, the first plurality of detectable probes comprises the same optically detectable retention component as the second plurality of detectable probes. In some configurations of the above methods, the first plurality of detectable probes comprises an optically detectable retention component that is different from an optically detectable retention component of the second plurality of detectable probes.

One or more different analytes bound to an affinity agent or detected by a detectable probe in the methods set forth herein may be attached to a solid support. For example, a single analyte of a plurality of different analytes may be attached to a corresponding site on a solid support, whereby the solid support comprises an array of analytes.

The present disclosure further provides methods of localizing a binding partner comprising (a) providing a material (e.g., a solid support) comprising a binding partner at discrete locations in the material; (b) Contacting the material with a detectable probe, wherein the detectable probe comprises (i) a retention component, (ii) one or more label components configured to produce a detectable signal, and (iii) two or more binding components coupled to the retention component, wherein the retention component and the two or more binding components form a detectable probe; (c) Detecting the detectable signal from the one or more marker components; and (d) identifying discrete locations of said detectable signal, thereby localizing said binding partner in said substance. Optionally, the detectable probe binds to the binding partner with a dissociation constant that is less than or equal to half the dissociation constant of the binding partner for binding to any of the binding components alone.

Also provided are methods of forming a detectable probe or affinity reagent comprising (a) providing a retention component comprising a plurality of coupling groups, and (b) attaching a coupling group of the plurality of coupling groups to a plurality of binding components. Optionally, the method further comprises (c) attaching a coupling group of the plurality of coupling groups to a plurality of labeling components.

The attachment between the components of the detectable probe or affinity reagent may be covalent or non-covalent. Nucleic acids provide particularly useful coupling groups. For example, the coupling groups in the above methods may comprise single stranded nucleic acid moieties that anneal to complementary nucleic acids attached to the binding and/or labeling components. The retention component may include a nucleic acid fold engineered to position the single stranded nucleic acid portion at a known location. For example, the single stranded nucleic acid portion may be included in a staple strand or oligonucleotide that is annealed to a scaffold strand. Other useful non-covalent attachments include, for example, those mediated by receptor-ligand pairs such as streptavidin-biotin, spyCatcher-SpyTag, sdyCatcher-SdyTag, and snoospcatcher-snootptag.

Any of a variety of functional groups may be used to covalently attach the detectable probe or component of the affinity reagent. Bio-orthogonal reactions and click reactions are particularly useful.

The method for preparing the detectable probe or affinity reagent may include the step of forming a passivation layer on the retained component. Passivation of the retention component may be performed, for example, prior to attaching a functional group (e.g., a coupling group), a labeling component, or a binding component to the retention component. Optionally, the passivation layer may include a metal, a metal oxide, an organic functional group, or a polymer. For example, the metal may include gold, silver, copper, titanium, or iron. Optionally, the metal oxide comprises titanium oxide, aluminum oxide, silicon dioxide or magnesium oxide. Optionally, the organic functional group comprises a phosphate, phosphonate, carboxylate, epoxide, or silane. In some embodiments, the polymer comprises a hydrocarbon polymer or a biopolymer. In some embodiments, the hydrocarbon polymer comprises polyethylene glycol (PEG), polyethylene oxide (PEO), or an alkane chain. In some embodiments, the biopolymer comprises a polysaccharide, polynucleotide, or polypeptide.

The detectable probes of the present disclosure can include a duplex region and an aptamer; wherein the double stranded region comprises two or more marker components. For example, the labeling component may be a fluorescently labeled nucleotide in one or both strands. Optionally, the fluorescently labeled nucleotides can be separated from each other by, for example, at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. Optionally, the double stranded region may comprise at least 3, 4, 5, 6, 7, 8, 9, 10 or more fluorophores or other labeling components. The two or more label components of the detectable probe may be the same as or different from each other.

An aptamer or other binding component optionally included in a detectable probe or affinity reagent can recognize or bind to some or all of the sequence of the form αxβ, where X is the desired epitope and α and β are any amino acid residues. Optionally, the aptamer may recognize or bind to at least 10%, 20%, 30%, 50%, 80% or 90% of the sequence of the form αxβ.

In some configurations, an aptamer or other binding component included in a detectable probe or affinity reagent can recognize or bind to a desired three amino acid epitope without specifically binding any other three amino acid sequences and bind with substantially similar affinity to the desired three amino acid epitope, regardless of flanking sequences surrounding the desired epitope.

In another aspect, described herein are switchable aptamers that bind to between 5% and 10% of all proteins in the human proteome; and wherein the switchable aptamer comprises two or more fluorescent moieties.

The present disclosure further provides a method of preparing a fluorescently labeled aptamer, the method comprising synthesizing an aptamer having a primer sequence at the 3 'end, hybridizing a template DNA strand to the primer sequence, wherein the template DNA strand comprises a segment complementary to the primer and a template region, and extending the 3' end of the aptamer molecule along the template using a polymerase. Optionally, the polymerase reaction is performed with a mixture of nucleotides comprising labeled nucleotides. For example, one or more types of nucleotides may include a tag, while other types of nucleotides lack a tag. For example, the polymerase reaction may be performed with four nucleotides (adenine, cytosine, guanine and thymine or uracil), three of which are unlabeled and the fourth of which are fluorescently labeled, and wherein the template is designed such that bases complementary to the fluorescently labeled nucleotides occur in a predetermined pattern. Optionally, the base complementary to the fluorescently labeled nucleotide can occur at every 2 nd, 3 rd, 4 th, 5 th, 6 th, 7 th, 8 th, 9 th, 10 th, 15 th, 20 th, 25 th, 30 th, 35 th, 40 th, 45 th or 50 th position along the template.

In another aspect, described herein are methods of making the above-described fluorescently labeled aptamers, comprising ligating the aptamers to a fluorescently labeled oligonucleotide. In another configuration, a method of preparing a fluorescently labeled aptamer can include synthesizing an aptamer having an extension sequence at the 3' end, hybridizing a splint nucleic acid strand to the extension sequence, and hybridizing a labeled oligonucleotide to the splint nucleic acid strand such that the end of the labeled oligonucleotide is adjacent to the end of the extension sequence, and ligating the labeled oligonucleotide to the aptamer via the extension sequence using a ligase.

Incorporated by reference

All publications, patents, and patent applications mentioned 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.

Drawings

Fig. 1A shows an entity with an angular offset between its two faces.

Fig. 1B shows an entity with an angular offset between its two faces.

Fig. 2A shows an approximately two-dimensional projection of a square retained component.

Fig. 2B shows an approximately two-dimensional projection of a circular retention component.

Fig. 3A shows the relative separation between two binding components.

Fig. 3B shows the relative separation between the two binding components.

Fig. 3C shows the relative separation between the two binding components.

Fig. 4A shows an arrangement for separating two binding components.

Fig. 4B shows an arrangement for separating two binding components.

Fig. 4C shows an arrangement for separating two binding components.

Fig. 4D shows an arrangement for separating two binding components.

Fig. 5A shows an arrangement for spacing marking components on a retained component.

Fig. 5B shows an arrangement for spacing marking components on a reserved component.

Fig. 6A shows the configuration of binding and labeling components on the retention component.

Fig. 6B shows the configuration of binding and labeling components on the retention component.

Fig. 6C shows the configuration of binding and labeling components on the retention component.

Fig. 6D shows the configuration of binding and labeling components on the retention component.

Fig. 6E shows the configuration of binding and labeling components on the retention component.

Fig. 6F shows the configuration of binding and labeling components on the retention component.

FIG. 7A shows an arrangement of detectable probes comprising a linker for binding a component.

FIG. 7B shows an arrangement of detectable probes comprising a linker for binding a component.

FIG. 8 shows a configuration of a detectable probe with additional affinity components.

Fig. 9A shows a configuration of a system for generating weak binding interactions of a detectable probe.

Fig. 9B shows a configuration of a system for generating weak binding interactions of a detectable probe.

FIG. 10A shows a method of generating weak binding interactions of a detectable probe.

FIG. 10B shows a method of generating weak binding interactions of a detectable probe.

FIG. 10C shows a method of generating weak binding interactions of a detectable probe.

FIG. 11A shows a method of generating weak binding interactions with two detectable probes.

FIG. 11B shows a method of generating weak binding interactions with two detectable probes.

FIG. 11C shows a method of generating weak binding interactions with two detectable probes.

FIG. 11D shows a method of generating weak binding interactions with two detectable probes.

FIG. 11E shows a method of generating weak binding interactions with two detectable probes.

FIG. 12A shows fluorescence intensity measurements for a set of sensor pixels with low intensity fluorescent markers.

FIG. 12B shows fluorescence intensity measurements of a set of sensor pixels with high intensity fluorescent markers.

FIG. 13 shows a cross-sectional view of a highly observable detectable probe bound to a binding partner.

Fig. 14A shows a configuration for generating a Fluorescence Resonance Energy Transfer (FRET) pair on a retained component.

Fig. 14B shows an arrangement for generating a Fluorescence Resonance Energy Transfer (FRET) pair on a detectable probe.

FIG. 15A shows a first step in a method for detecting binding interactions using a nucleic acid barcode and weak secondary interactions.

FIG. 15B shows a second step of the method for detecting binding interactions using nucleic acid barcodes and weak secondary interactions.

FIG. 15C shows a third step of the method for detecting binding interactions using nucleic acid barcodes and weak secondary interactions.

FIG. 15D shows a fourth step of the method for detecting binding interactions using nucleic acid barcodes and weak secondary interactions.

FIG. 16A shows a detectable probe composition comprising binding competitor.

FIG. 16B shows a detectable probe composition comprising binding competitor.

FIG. 17A shows a method of using a detectable probe or affinity reagent as a capture reagent.

FIG. 17B shows a method of using a detectable probe or affinity reagent as a capture reagent.

FIG. 17C shows a method of using a detectable probe or affinity reagent as a capture reagent for polypeptide assays.

FIG. 18A shows a first step in a method of detecting binding of more than one detectable probe to a binding partner.

FIG. 18B shows a second step of the method of detecting binding of more than one detectable probe to a binding partner.

FIG. 18C shows a third step of the method of detecting binding of more than one detectable probe to a binding partner.

FIG. 19A shows a method for determining peptide sequences using a detectable probe.

FIG. 19B shows a method of determining peptide sequences using a detectable probe.

FIG. 19C shows a method of determining peptide sequences using a detectable probe.

FIG. 19D shows a method of determining peptide sequences using a detectable probe.

FIG. 20A shows a method of altering binding interactions using binding competitors.

FIG. 20B shows a method of altering binding interactions using binding competitors.

FIG. 21A shows a method of making a detectable probe.

FIG. 21B shows a method of making a detectable probe.

FIG. 22 shows the use of detectable probes to characterize multiple binding partners.

FIG. 23 shows a simplified schematic of a detectable probe or affinity reagent comprising a DNA fold retention component.

Fig. 24A shows binding profile data for a quantum dot based detectable probe.

Fig. 24B shows binding profile data for a quantum dot based detectable probe.

FIG. 25 shows a simplified schematic of a detectable probe or affinity reagent comprising a DNA fold retention component.

Fig. 26 shows a scheme of attaching an antibody-based binding component to a paper folding retention component via a click reaction.

FIG. 27 shows an image of SDS-Page gel containing antibody-oligonucleotide conjugates.

Figure 28A shows binding data of a detectable probe to a polypeptide target.

Figure 28B shows binding data of detectable probes to polypeptide targets.

Fig. 28C shows binding data of the detectable probes to the polypeptide targets.

Figure 28D shows binding data of detectable probes to polypeptide targets.

Fig. 29A shows fluorescence microscopy images of binding of detectable probes to a negative control polypeptide array.

Fig. 29B shows fluorescence microscopy images of binding of detectable probes to a negative control polypeptide array.

Fig. 29C shows a fluorescence microscope image of the binding of a detectable probe to a polypeptide array.

FIG. 30 shows binding data of detectable probes to polypeptide targets.

FIG. 31A shows the configuration of non-nucleic acid retaining components.

FIG. 31B shows the configuration of non-nucleic acid retaining components.

FIG. 31C shows the configuration of non-nucleic acid retaining components.

FIG. 31D shows the configuration of non-nucleic acid retaining components.

FIG. 32A shows a first step in a method of forming a non-nucleic acid retaining component.

FIG. 32B shows a second step of the method of forming a non-nucleic acid retaining component.

FIG. 32C shows a first step in a method of forming a non-nucleic acid retaining component.

FIG. 32D shows a second step of the method of forming a non-nucleic acid retaining component.

FIG. 32E shows a third step of the method of forming a non-nucleic acid retaining component.

FIG. 33 shows a method of forming a non-nucleic acid retaining component.

FIG. 34A shows a configuration of a detectable probe that includes a non-nucleic acid retaining component.

FIG. 34B shows a configuration of a detectable probe that includes a non-nucleic acid retaining component.

FIG. 34C shows a configuration of a detectable probe that includes a non-nucleic acid retaining component.

FIG. 35A shows a multi-probe complex formed from a plurality of detectable probes.

FIG. 35B shows a multi-probe complex formed from a plurality of detectable probes.

FIG. 35C shows a multi-probe complex formed from a plurality of detectable probes.

FIG. 36A shows a multi-probe complex with a controlled conformation.

FIG. 36B shows a multi-probe complex with a controlled conformation.

FIG. 37A shows a first step in a method of forming detectable probes from non-nucleic acid retaining components.

FIG. 37B shows a second step in the method of forming detectable probes from non-nucleic acid retaining components.

FIG. 37C shows a third step in the method of forming detectable probes from non-nucleic acid retaining components.

Fig. 38A shows a first step in drug delivery using a detectable probe or affinity reagent complex.

Fig. 38B shows a second step of drug delivery using a detectable probe or affinity reagent complex.

Fig. 38C shows a third step of drug delivery using a detectable probe or affinity reagent complex.

Fig. 38D shows a fourth step of drug delivery using a detectable probe or affinity reagent complex.

FIG. 39A shows a multi-probe complex formed with a secondary retention component.

FIG. 39B shows a multiprobe complex formed with a secondary retention component.

FIG. 40 shows an affinity chromatography system using a detectable probe or affinity reagent as a capture agent.

FIG. 41 shows the preparation of FluoSphere-based ^TM Is provided.

FIG. 42A shows FluoSphere based ^TM Is provided for the detection of characteristic data of the probe.

FIG. 42B shows FluoSphere based ^TM Is provided for the detection of characteristic data of the probe.

FIG. 42C shows FluoSphere based ^TM Is provided for the detection of characteristic data of the probe.

FIG. 42D shows FluoSphere based ^TM Is provided for the detection of characteristic data of the probe.

FIG. 42E shows FluoSphere based ^TM Is provided for the detection of characteristic data of the probe.

FIG. 42F shows FluoSphere based ^TM Is provided for the detection of characteristic data of the probe.

FIG. 43A shows FluoSphere based ^TM Binding profile of the detectable probe.

FIG. 43B shows FluoSphere based ^TM Binding profile of the detectable probe.

FIG. 43C shows FluoSphere based ^TM Binding profile of the detectable probe.

FIG. 43D shows FluoSphere based ^TM Binding specificity of the detectable probes of (2)And (5) sign data.

FIG. 44A shows FluoSphere based ^TM Stability profile of the detectable probe.

FIG. 44B shows FluoSphere based ^TM Stability profile of the detectable probe.

FIG. 44C shows FluoSphere based ^TM Stability profile of the detectable probe.

FIG. 45A shows FluoSphere based ^TM Binding profile of the detectable probe.

FIG. 45B shows Alexa-based-Binding profile of the aptamer of (c).

FIG. 45C shows binding profile data for APC-based aptamers.

FIG. 45D shows SureLight based ^TM Binding profile of the aptamer of APC.

FIG. 46 shows FluoSphere based with different numbers of available affinity reagent attachment sites ^TM Binding data for the detectable probes.

FIG. 47 shows binding data for fluorescent nanoparticle B1 probes with his-labeled Her2 (on target) and myoglobin (off target) using either direct attachment or using a pre-annealing protocol.

FIG. 48 shows an exemplary list of immobilized targets for selection of affinity reagents and peptides comprising the targets.

FIG. 49A provides a schematic of a labeled affinity reagent as described herein.

FIG. 49B shows a method of attaching a labeling component to a nucleic acid probe by enzymatic extension.

FIG. 50 illustrates some exemplary ways of fluorescent labeling of affinity reagents. Figure 50A shows an aptamer with a single fluorophore attached. FIG. 50B shows an aptamer having a double-stranded nucleic acid labeling region with two fluorophores attached. FIG. 50C shows the attachment of a plurality of regularly spaced fluorophores to an aptamer using a template.

FIG. 51 shows a labeled probe comprising a single strand of nucleic acid comprising an aptamer that hybridizes to a single strand of nucleic acid comprising a fluorophore.

FIG. 52 shows a gel that appears at 488nm to reveal double stranded DNA and at 647nm to reveal incorporated fluorophores. These labeled affinity reagents are generated via enzymatic extension and incorporation of fluorophore-modified nucleotides.

FIG. 53A shows a gel that appears at 488nm to reveal double stranded DNA.

FIG. 53B shows a gel that appears at 647nm to show incorporated fluorophores. These labeled affinity reagents are produced via enzymatic extension and incorporation of chemically modified nucleotides. Subsequently, according to some embodiments, chemical conjugation is used to incorporate fluorophores

FIG. 54 shows labeled aptamer concentrations.

Fig. 55A shows the fluorophore concentration in the labeled aptamers of fig. 54.

FIG. 55B shows a dUTP-647 standard curve for determining fluorophore concentration in FIG. 8A.

FIG. 56 shows the fluorophore to DNA ratio calculated from FIGS. 54 and 55.

FIG. 57A shows labeled aptamers conjugated to immobilized peptides.

FIG. 57B shows the binding of a labeled aptamer to an immobilized target peptide.

FIG. 57C shows the binding of a labeled aptamer to an immobilized non-target peptide.

Fig. 57D shows microscopy of limited dilution of the labeled aptamer on an amine coated coverslip.

FIG. 58A shows the binding reaction between a detectable probe and SNAP attached polypeptide.

FIG. 58B shows the binding reaction between a plurality of detectable probes and an SNAP-attached array of polypeptides.

Detailed Description

Detecting interactions between the affinity reagent and the bound target may be used to provide spatial and/or temporal characterization and/or quantification of the physical system. For example, affinity reagent-based methods such as enzyme-linked immunosorbent assay (ELISA) can be used to determine the presence and potential amount of a biomarker within a biochemical sample. Affinity reagents may exhibit complex behavior, such as interaction with unintended or unlikely targets, or failure to interact with intended targets. In batch or large scale characterization, affinity reagents can be engineered to a sufficient extent that complex aspects of their behavior fall within experimental errors, resulting in useful interaction data. However, for single molecule assays using affinity reagents, complex affinity reagent behavior may produce abnormal results (e.g., false negatives, false positives) that complicate interpretation of single molecule affinity reagent interaction data.

Binding may be transient due to a number of factors that affect the binding interaction between the affinity reagent and the binding partner. In some cases, the affinity reagent may associate and dissociate freely with the binding partner. In other cases, the affinity reagent may interact with the binding partner with sufficient strength to produce a stable or quasi-stable complex. Furthermore, in some cases, the affinity reagent may associate with unintended or unlikely binding partners.

Some affinity reagent interactions may not fit into the conventional binary interaction framework (i.e., single binding partner, bound or unbound) due to the complex nature of the interaction between the affinity reagent and the binding partner. For example, interactions of the affinity reagent with available binding partners may not be observed for a variety of reasons, including 1) the affinity reagent is not sufficiently accessible to the binding partner; 2) During the time interval when the association observations are made, the affinity reagent cannot remain associated with the binding partner; or 3) interrupting or attenuating the change in the environment of the affinity interaction (e.g., conformational change of the binding partner or affinity reagent). Also, for various reasons, such as 1) environmental changes that enhance affinity interactions; or 2) transient associations that occur during the time interval when the association observations are made, it is possible to observe that the affinity reagent binds to an unintended or unlikely target.

Thus, it may be preferable to describe some affinity reagent interactions within a probabilistic or stochastic framework (e.g., multiple binding partners, non-zero probability of binding to each target). The probability or random model of affinity reagent binding may be particularly useful for single molecule assays where a single interaction measurement may not necessarily provide a definitive characterization. In such a case, multiple rounds or cycles of measurement may increase the measurement confidence.

While affinity reagents may not always perform optimally under certain conditions, affinity reagents may be engineered to increase or improve their behavior for certain purposes. Two aspects of affinity reagent behavior that can be modified include affinity and observability.

Avidity can be understood as the tendency of an affinity agent to remain bound to a binding partner due to the presence of a plurality of, typically synergistic, binding interactions between the affinity agent and the binding partner. The plurality of binding interactions may occur, for example, due to the binding partner having a plurality of different epitopes recognized by the affinity reagent, and/or due to the probe having a plurality of binding components that recognize epitopes in the binding partner. One common example of such a phenomenon is the avidity of an antibody, where avidity is typically achieved by weak binding of multiple binding sites on a single antibody to a particular target to produce an apparent binding strength that is greater than the binding strength between the binding partner and any individual binding sites on the antibody.

Observability can be understood as the ability to detect binding interactions between an affinity reagent and its binding partner. For example, observability may refer to the tendency of an affinity reagent to be detected during the interaction between the affinity reagent and its binding partner. In some configurations, observability may refer to the tendency of an affinity reagent to generate a signal or a signal-generating tag (e.g., a nucleic acid tag) that can be detected upon interaction between the affinity reagent and its binding partner. Observability may be affected by both the ability of the affinity reagent to maintain interaction for a sufficient time interval during detection, as well as the resistance of affinity to mechanisms that may attenuate the detectable signal of the interaction (e.g., photobleaching, cleavage of the labeling component).

Described herein are compositions comprising probes having enhanced affinity for binding partners. In some configurations, the probe is detectable and optionally has enhanced observability. Compositions described herein, such as affinity reagents and detectable probes, can be particularly useful in single molecule characterization assays, including, for example, in configurations in which multiple rounds or cycles of affinity reagent interactions are measured. The composition optionally has the property of enhanced affinity for one or more binding partners while also being configured for reversible binding, e.g., to allow removal of the binding partners, thereby allowing for multiple cycles or rounds of binding measurements. The composition may optionally have an adjustable detection label that allows the label to be observed and measured at a signal level that substantially exceeds the background signal of the system in which the affinity agent interaction is measured.

In some configurations, the detectable probes or affinity reagents described herein can be characterized as comprising a retention component associated with one or both of a plurality of binding components and a plurality of label components (e.g., detection labels). The plurality of binding components may be displayed on the retention component in a configuration that allows for enhanced affinity of the detectable probe or affinity reagent for binding to a particular binding partner. The affinity of the detectable probe or affinity reagent may exceed the affinity of any of the individual binding components for that particular binding partner. In some configurations, the affinity of the detectable probe or affinity reagent for the binding partner may exceed the sum of the affinities of the plurality of binding components for the binding partner. In addition, a plurality of label components may be displayed on the retention component of the detectable probe or affinity reagent in a manner that enhances the detectable signal produced by the detection label. For example, the signal may be enhanced in intensity, duration, or specificity. In particular configurations, the detectable probe or affinity reagent may further have the property of being stable in the presence of a chemical (e.g., surfactant, denaturant) that would otherwise disrupt the interaction between the affinity reagent and its binding partner, thereby allowing the detectable probe or affinity reagent to be moved away from the binding partner without damaging or disrupting the structure of the detectable probe or affinity reagent.

In some configurations, the detectable probes or affinity reagents employ nucleic acid break paper as a retention component coupled to a plurality of binding components (e.g., antibodies, antibody fragments, miniproteins, DARPin, DNA aptamers, RNA aptamers, etc.) and/or a plurality of labeling components (e.g., fluorophores, nucleic acid tags, quantum dots, fluorescent nanoparticles, fluorescent proteins). In some configurations, the nucleic acid-folded paper-based retention component may have a regular or symmetrical shape that allows for attachment of multiple binding components and/or multiple marking components at predetermined locations. For example, the components may be separated from each other by a predetermined distance, the components may be oriented to achieve a synergistic function, or the components may be oriented to reduce or prevent inhibition of each other's activity. The ability to adjust spacing and orientation can result in desirable detection characteristics such as reduced quenching between fluorophore labels or enhanced Forster Resonance Energy Transfer (FRET) between fluorophore labels. Alternatively or additionally, the spacing and orientation may be adjusted to adjust the affinity of the affinity reagent for one or more binding partners.

In particular configurations, the detectable probe may employ an optically detectable particle. The particles may have a modifiable surface to which a plurality of binding components may be attached. The optically detectable particles may include fluorescent or luminescent nanoparticles (e.g., fluoSphere ^TM Or quantum dots). The modifiable surface coating can include a hydrogel or a polymer. In such a configuration, the optically detectable particle may serve as both a retention component for attaching a binding component to the detectable probe and a labeling component for detecting the probe. In some configurations, the surface coating of the optically detectable particle may act as a retention component for attaching the binding component to the detectable probe.

In a further configuration, two or more detectable probes may be combined to form a complex of detectable probes. Similar complexes can be prepared between two affinity reagents or between an affinity reagent and a detectable probe. The complexes of probes and/or reagents can have binding members of similar affinity (e.g., monovalent probes or reagents) or different affinity (e.g., multivalent probes).

In some configurations, the detectable probe or affinity reagent is combined or coupled with a competing affinity reagent. The competing affinity reagent may be configured as a free molecule or as a binding component attached to the detectable probe or affinity reagent. Competing affinity reagents in some cases have reduced affinity or specificity for the binding partner of interest, resulting in higher levels of hybridization than non-competing affinity reagents. The presence of competing affinity reagents may enhance the affinity of the detectable probe or affinity reagent for a particular binding partner.

The present disclosure provides methods of detecting an analyte using a composition comprising a detectable probe or affinity reagent having enhanced affinity for a binding partner and enhanced observability. Methods for detecting polypeptides are exemplified herein. It will be appreciated that any of a variety of analytes may be used, such as those targeted by analytical chemistry assays, biochemical assays, molecular diagnostic assays, molecular forensic assays, quality control assays, and the like.

Characterization and quantification of heterogeneous polypeptide samples is often hampered by the coexistence of widely varying amounts of proteins and/or peptides. For example, in a quantitative characterization assay, signals from low copy number proteins may be overwhelmed by signals from high copy number proteins. The accuracy of polypeptide characterization assays performed at the proteome level (e.g., tens of thousands of unique protein species) can benefit from a combination of high sensitivity analysis techniques and high confidence prediction techniques.

Biological analysis can greatly benefit from advances in tools that can be used to examine molecular operations of biological systems. Advances in genomics technology, single cell analysis platforms, and high sensitivity chemical analysis systems have greatly improved the opinion of researchers and clinicians how biological systems operate and have further improved understanding, treatment, and prevention of disease and other human health conditions.

Many of these advanced tools benefit from the use of detectable reagents that are capable of binding or otherwise associating with different biomolecules, wherein the binding allows for the identification and/or quantification of the presence of such molecules in a given system, wherein the presence and/or amount provides insight into the function of the system. As an example, many of these tools provide for immobilization of different molecular species (e.g., proteins, nucleic acids, etc.) from a biological system on a support structure (e.g., immobilized substrates or beads). Molecules with a certain level of binding affinity for different molecular species (also known as affinity reagents) can then be used, and the bound molecules can be examined by contacting the two together to see if, where and/or for how long the affinity reagent binds, indicating the possible presence of a particular molecule in the biological sample.

Detection of binding can generally be accomplished by detecting the labeled component of the affinity reagent. While the labeling component may be an inherent part of the affinity reagent, in many cases the labeling component may be an exogenous chemical or structural moiety attached to the affinity reagent. Affinity reagents with labeling components can act as detectable probes.

A wide variety of exogenously detectable labels are commonly used for such purposes, including, for example, optically detectable labels such as fluorescent dye labels, enzymes (e.g., enzymes that catalyze reactions with coloring reagents or products), electrochemical labels such as highly charged label groups, or even groups detected by subsequent processing such as nucleic acid barcode labels that can be subsequently confirmed by nucleic acid amplification methods, sequencing methods, or hybridization assays. Fluorescent labels can be used to visualize the binding interactions between affinity reagents and other molecules such as proteins or peptides.

Depending on the application in which the affinity reagent or detectable probe will be used, different degrees of signal intensity may be desired or even required. For example, in many applications, amplification of the binding signal may be achieved by binding a number of affinity reagents or detectable probes. For example, the sample may be separated such that multiple molecules of a single detectable moiety are aggregated at a single location, and thus more easily observed or detected. Alternatively or additionally, a secondary binding system may be used to present a plurality of additional binding sites at which individual marker components may subsequently bind and be detected.

In some cases, however, it may be useful to visualize the binding of an individual molecule of an affinity reagent or a detectable probe to an individual molecule in a sample in a quantitative manner. As will be appreciated, detection of individual molecules presents a number of challenges in terms of detection. One such challenge is the ability to present a detectable signal from a single molecule of a reagent or probe bound to a single molecule that has sufficient signal strength (e.g., signal strength and duration) to be detected by an available analysis system, both in terms of raw signal strength and in terms of signal strength relative to the strength of the background noise of the system.

The signal strength challenges may be addressed by a variety of methods, alone or in combination. For example, the ability to detect extremely low level signals, including single molecule signals with low intensity and/or short duration, may be enhanced using highly sensitive microscopy techniques. In addition, the signal intensity may be increased by incorporating multiple marker components (e.g., multiple marker components on a single molecule such as an affinity reagent molecule or a detectable probe molecule) to significantly increase the signal associated with the single molecule, thereby improving its detectability. Also, for example, for fluorescent labels, increasing the signal intensity can be achieved by attaching several fluorescent moieties in a single affinity reagent or detectable probe. However, simply loading probes or reagents with fluorescent dye labels can present its own set of challenges. The probes or reagents may be carefully configured such that one fluorescent moiety on the probe or reagent does not quench another fluorescent moiety on the probe or reagent, the number of fluorescent moieties on the probe or reagent may be controlled, and the chemical process of attaching these labeling groups to the probe or reagent does not interfere with the binding affinity of the probe itself.

Particularly useful affinity reagents and detectable probes are capable of binding to a particular protein, metabolite, cell or cell interface. Examples of such affinity reagents and detectable probes may include protein-based probes consisting of naturally occurring amino acids, small molecule probes, nucleic acid-based probes consisting of naturally occurring bases, and probes consisting of non-natural nucleotides and amino acids. The agent may be configured to specifically bind to a given epitope or other target.

The exclusivity of binding is generally considered to be a desirable property in an affinity reagent or detectable probe. A great deal of effort has been made to ensure that affinity reagents or probes bind only one target substance, with minimal binding to the other targets. For example, the target substance may be a specific sequence of amino acids. If the sequence is unique to a protein present in the biological sample, then reagents or probes specific for the sequence will be specific for the protein in the environment of the sample. Thus, the reagent or probe may be used to confirm or even quantify the protein in a sample. As set forth in further detail herein, there are specific use cases in which it may be useful to have an affinity reagent or detectable probe that binds to one or more different proteins in a sample. For example, useful affinity reagents or detectable probes may bind to epitopes common to two or more different proteins. Alternatively or additionally, the probe or reagent may bind to two or more different epitopes present in different proteins or in different regions of the same protein. Affinity probes and detectable probes that bind to multiple targets in a sample can be used to produce useful effects in the compositions and methods set forth herein.

Unless otherwise indicated, terms used herein will be understood to have their ordinary meaning in the relevant art. Several terms used herein and their meanings are set forth below.

As used herein, the term "affinity reagent" refers to a molecule or other substance that is capable of binding specifically, reproducibly, or with a high probability to a binding partner. Specific binding may be characterized by a binding constant, such as less than 10 ^-4 M、10 ^-6 M、10 ^-8 M、10 ^-10 M、10 ^-12 M、10 ^-14 M or lower dissociation constant (K _D ). The high probability may be characterized as a probability (on a scale of 0 to 1) of at least 0.25, 0.5, 0.51, 0.75, 0.9, 0.99, or higher. The size of the affinity reagent may optionally be greater than, less than or equal toIts binding partner. The affinity reagent may form a reversible or irreversible interaction with the binding partner. The affinity reagent may be covalently or non-covalently bound to the binding partner. Affinity reagents are typically non-catalytic and chemically non-reactive and therefore do not permanently alter the chemical structure of the binding partner to which they bind in the methods set forth herein. Alternatively, the affinity reagent may be configured to catalyze or participate in a chemical modification (e.g., ligation, cleavage, tandem, etc.) that produces a detectable change in the binding partner to which it binds. Optionally, the product of the reaction may allow for detection of the interaction. Affinity reagents may include reactive affinity reagents (e.g., kinases, ligases, proteases, nucleases, etc.) or non-reactive affinity reagents (e.g., antibodies, antibody fragments, aptamers, DARPin, peptide aptamers (pepamer), etc.). Affinity reagents may include one or more known and/or characterized binding components or binding sites (e.g., complementarity defining regions) that mediate or promote binding to a binding partner. Thus, the affinity reagent may be monovalent (e.g., having only a single binding component), divalent (e.g., having only two binding components), trivalent (e.g., having only three binding components), tetravalent (e.g., having only four binding components), or multivalent (e.g., having two or more binding components). Exemplary affinity reagents include detectable probes and probes as set forth in U.S. provisional application No. 63/112,607, which is incorporated herein by reference.

As used herein, the term "antibody" refers to an immunoglobulin molecule or functional fragment thereof that specifically binds to a binding partner. Antibodies may be specific for one or more epitopes within the binding partner. Antibodies can be naturally occurring, engineered or evolved. Antibodies may include Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer a specific epitope for binding to the polypeptide. Linear antibodies are also included for the purposes described herein. Exemplary antibodies include immunoglobulin isotypes such as IgM, igA, igG, igD and IgE. Exemplary antibody fragments include F (ab ') 2 fragments, fab' fragments, fab fragments, fv fragments, scFV fragments, rlgG fragments, and Fc fragments.

As used herein, the term "substantially" when used in connection with a shape may mean a shape that is within 20% of the ideal shape with reference to two or more measures of the shape. For example, FIGS. 2A-2B show two-dimensional volumes 210 and 215 that are generally square and generally circular, respectively, with ideal boundaries of square 220 or circle 225 shown in dashed lines. As used herein, the term "about" when used in connection with a length, area, or volume may mean a length, area, or volume that is within 10% of a given measurement. For example, a length of 10 millimeters (mm) may refer to any length between 9mm and 11 mm.

As used herein, the term "array" refers to a population of analytes (e.g., proteins) associated with a unique identifier such that the analytes are distinguishable from one another. The unique identifier may be a solid support (e.g., a particle or bead), a structured nucleic acid particle, a retained component, a site on a solid support (e.g., a spatial address), a tag, a label (e.g., a luminophore), or a barcode (e.g., a nucleic acid barcode) that is associated with the analyte and that is different from the other identifiers in the array. Analytes may be associated with unique identifiers by attachment, for example, via covalent or non-covalent (e.g., ionic, hydrogen, van der waals, electrostatic, etc.) bonds. The array may include different analytes each attached to a different unique identifier. The array may include different unique identifiers attached to the same or similar analytes. The array may comprise separate solid supports or separate addresses each carrying a different analyte, wherein the different analytes may be identified based on the location of the solid support or address. The analyte or other molecule that may be included in the array may be, for example, a nucleic acid such as SNAP, a polypeptide, an enzyme, an affinity reagent, a binding partner, a ligand or a receptor.

As used herein, the term "attached" refers to a state in which two things are joined, fastened, adhered, connected, or joined to one another. For example, the binding component may be attached to the retention component by covalent or non-covalent bonds. Covalent bonds are characterized by sharing electron pairs between atoms. Noncovalent bonds are chemical bonds that do not involve sharing electron pairs, and may include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

As used herein, the term "binding affinity" or "affinity" refers to the strength or degree of binding between an affinity agent and a binding partner, epitope or target moiety. In some cases, the binding affinity of the affinity reagent to the binding partner, epitope, or target moiety may be zero or virtually zero. The binding affinity of an affinity agent to a binding partner, epitope or target moiety may be defined as "high affinity", "medium affinity" or "low affinity". If the equilibrium dissociation constant (K) _D ) Less than about 100nM, the binding affinity of the affinity reagent to the binding partner, epitope or target moiety may be quantified as "high affinity", if the dissociation constant of the interaction is between about 100nM and 1mM, as "medium affinity", and if the dissociation constant of the interaction is greater than about 1mM, as "low affinity". Binding affinity can be described in terms known in the biochemical arts, such as equilibrium dissociation constant, equilibrium association constant (K _A ) Association rate constant (k) _on ) Dissociation rate constant (k) _off ) Etc. See for example Segel (r) for example,Enzyme Kineticsjohn Wiley and Sons, new York (1975), which is incorporated herein by reference in its entirety.

As used herein, the term "binding component" refers to a portion of an affinity reagent that is capable of specifically, reproducibly or with high probability binding to a binding partner. Exemplary binding components include, but are not limited to, antibodies or functional fragments thereof (e.g., fab 'fragments, F (ab') ₂ Fragments, single chain variable fragment (scFv), di-scFv, tri-scFv, minibodies, intracellular antibodies, affinity antibodies, affilin, affimer, avidin (affitin), alpha antibodies, anti-icalin, affinity multimers (avimers), DARPin, kunitz domain peptides, mono-antibodies, nanomaterials, minipeptide binders, etc.), lectins or functional fragments thereof, avidin (avidins), streptavidin, aptamers, single or double stranded nucleic acids, or other affinity reagents set forth herein or known in the art. Exemplary binding components include, for example, U.S. temporaryThe probes described in application Ser. No. 63/112,607, which is incorporated herein by reference.

As used herein, the term "binding context" refers to the environmental conditions under which affinity reagent-binding partner interactions are observed. Environmental conditions may include any factor that may affect the interaction between the affinity reagent and the binding partner, such as temperature, fluid properties (e.g., ionic strength, pH), relative concentration, absolute concentration, fluid composition, binding partner conformation, affinity reagent conformation, and combinations thereof. The environmental conditions may include structural features of the binding partner that, although outside the epitope, affect the interaction of the epitope with the binding agent. Structural features may include, for example, amino acids or regions of a polypeptide that are proximal to epitopes in the primary, secondary, tertiary or quaternary structure of the polypeptide.

As used herein, the term "binding partner" refers to a molecule or other substance that is recognized by an affinity reagent or binding component. Affinity reagents can specifically or reproducibly recognize a particular binding partner relative to other molecules or substances in the sample. Alternatively, the affinity reagent may recognize a plurality of different binding partners in the sample, e.g. binding promiscuously to a different subset of polypeptide sequences among a larger set of different polypeptides. The binding partner may be capable of forming an interaction with the affinity reagent, whether or not such interaction occurs. The binding partner may comprise one or more epitopes. The binding partner may have a rigid structure, such as a nanoparticle or microparticle. The binding partner may have a fused or dynamic structure (e.g., globular proteins, globular polymers). The binding partner may be in solution or solid phase. For example, the binding partner may be free in a solution containing the affinity reagent or may be localized at a surface or interface accessible to the affinity reagent. The binding partner may be attached to a structured nucleic acid particle (e.g., nucleic acid folded paper) or a retention component.

As used herein, the term "binding probability" refers to the probability that interaction of an affinity reagent with a binding partner and/or epitope can be observed, e.g., within an immobilized binding context. The binding probability may be expressed as a discrete number (e.g., 0.4 or 40%), a matrix of discrete numbers, or as a mathematical model (e.g., a theoretical or empirical model). The probability of binding may include one or more factors including binding specificity, the likelihood of targeting the epitope, or the likelihood of binding for a sufficient amount of time to detect binding interactions. The total probability may include the probability of a combination when all factors have been weighted against the background of the combination.

As used herein, the term "binding specificity" refers to the tendency of an affinity agent to preferentially interact with a binding partner or epitope relative to other binding partners or epitopes. The affinity reagent may have a calculated, observed, known or predicted binding specificity for any possible binding partner or epitope. Binding specificity may refer to the selectivity of a single binding partner, epitope, or target moiety in a sample over all, some, or at least one other analyte in the sample. Furthermore, binding specificity may refer to the selectivity of a subset of binding partners, epitopes or target moieties in a sample over at least one other analyte in the sample.

As used herein, the term "bioorthogonal reaction" refers to a chemical reaction that can occur within a biological system (in vitro and/or in vivo) without interfering with some or all of the natural biological processes, functions, or activities of the biological system. Bioorthogonal reactions can be further characterized as inert to components of the biological system that are not targeted by the bioorthogonal reaction. Bio-orthogonal reactions may include click reactions. Bioorthogonal or click reactions may include Staudinger ligation, copper-free click reactions, nitrone dipolar cycloaddition, norbornene cycloaddition, oxanorbornadiene cycloaddition, tetrazine ligation, [4+1] cycloaddition, tetrazole click reactions, or tetracycloalkane ligation. The bioorthogonal reaction may utilize enzymatic methods, such as attachment between the first molecule and the second molecule by an enzyme such as sortase, ligase, or subtiligase (subtiligase). Bioorthogonal reactions may utilize irreversible peptide capture systems such as SpyCatcher/SpyTag, snoopCatcher/snootag or SdyCatcher/SdyTag.

As used herein, the term "click reaction" refers to a single step thermodynamically favored conjugation reaction utilizing a biocompatible reagent. Click reactions may be configured to not utilize toxic or biocompatible reagents (e.g., acids, bases, heavy metals) or to not produce toxic or biocompatible byproducts. Click reactions may utilize aqueous solvents or buffers (e.g., phosphate buffered solutions, tris buffered solutions, saline buffered solutions, MOPS, etc.). The click reaction may be thermodynamically favored if it has a negative Gibbs free energy, for example, less than about-5 kilojoules per mole (kJ/mol), -10kJ/mol, -25kJ/mol, -50kJ/mol, -100kJ/mol, -200kJ/mol, -300kJ/mol, -400kJ/mol, or less than-500 kJ/mol. Exemplary bio-orthogonal and click reactions are described in detail in WO2019/195633A1, which is incorporated herein by reference.

The term "comprising" is intended herein to be open-ended, including not only the recited elements, but also any additional elements.

As used herein, the term "each" when used in reference to a collection of items is intended to identify a single item in the collection, but does not necessarily refer to each item in the collection. An exception may be made if there is an explicit disclosure or if the context clearly dictates otherwise.

As used herein, the term "epitope" refers to a molecule or portion of a molecule that is recognized by or specifically binds to an affinity reagent. An epitope may include amino acid sequences that are sequentially adjacent in the primary structure of a polypeptide or amino acids that are structurally adjacent in the secondary, tertiary or quaternary structure of a polypeptide. The epitope may optionally be recognized by or bound to an antibody. However, the epitope need not necessarily be recognized by any antibody, for example, but rather by an aptamer, mini-protein, or other affinity agent. The epitope may optionally bind to an antibody to elicit an immune response. Epitopes need not necessarily be involved nor need they be able to elicit an immune response. The term "affinity target" is used synonymously herein with the term "epitope".

As used herein, the term "exogenous" when used in reference to a portion of a molecule means a portion that is not present in the natural analog of the molecule. For example, an exogenous tag for an amino acid is a tag that does not exist on a naturally occurring amino acid. Similarly, exogenous markers present on an antibody are not present on the antibody in its natural environment.

As used herein, the term "functional group" refers to a moiety or radical in a molecule that imparts chemical properties such as reactivity, polarity, hydrophobicity, hydrophilicity, solubility, binding affinity, and the like to the molecule. The functional group may comprise an organic moiety or may comprise an inorganic atom. Exemplary functional groups can include bioorthogonal reactants, click reactants, alkyl, alkenyl, alkynyl, phenyl, halide, hydroxyl, carbonyl, aldehyde, acyl halide, ester, carboxylate, carboxyl, carboalkoxy (carboalkoxy), methoxy, hydroperoxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, epoxide, carboxylic anhydride, carboxamide, amine, ketimine, aldimine, imide, azide, azo, cyanate, isocyanate, nitrate, nitrile, isonitrile, nitrooxy, nitro, nitroso, oxime, pyridyl, carbamate, mercapto, sulfide, disulfide, sulfinyl, sulfonyl, sulfonium (sulfoum), sulfo, thiocyanate, isothiocyanate, thiocarbonyl (carbothio), thioester, thiomonoester (thiosulfonate), phosphino, phosphonyl, phosphonate, phosphate, dihydroxyboron, borate, and dihydrocarbyl borate functions.

As used herein, the term "label" refers to a molecule or portion thereof that provides a detectable feature. The detectable feature may be, for example, an optical signal such as absorption of radiation, luminescence emission, luminescence lifetime, luminescence polarization, fluorescence emission, fluorescence lifetime, fluorescence polarization, etc.; rayleigh and/or mie scattering; binding affinity for ligand or receptor; magnetic properties; an electrical characteristic; a charge; quality; radioactivity, etc. Exemplary labels include, but are not limited to, fluorophores, chromophores, nanoparticles (e.g., gold, silver, carbon nanotubes), heavy atoms, radioisotopes, mass labels, charge labels, spin labels, receptors, ligands, and the like. The label may generate a signal (e.g., fluorescent, luminescent, radioactive) that is detected in real-time. The labeling component may generate a signal that is detected offline (e.g., nucleic acid bar code) or in a time-resolved manner (e.g., time-resolved fluorescence). The marker component may produce a signal having a characteristic frequency, intensity, polarity, duration, wavelength, sequence, or fingerprint.

As used herein, the term "labeling component" refers to a portion of an affinity reagent or other substance that provides a detectable characteristic. The detectable feature may be, for example, any of those set forth herein in the context of the marking. The labeling component may be attached or capable of being attached to another molecule or substance. Exemplary molecules that can be attached to the labeling component include affinity reagents or binding partners.

As used herein, the term "nucleic acid nanospheres" refers to spherical or spherical nucleic acid structures. Nucleic acid nanospheres can include concatemers of sequence regions arranged in a globular structure. The nucleic acid nanospheres may comprise DNA, RNA, PNA, modified or unnatural nucleic acids, or a combination thereof.

As used herein, the term "nucleic acid paper folding" refers to a nucleic acid construct that includes engineered tertiary or quaternary structures in addition to the naturally occurring helical structure of a nucleic acid. Nucleic acid folds may include DNA, RNA, PNA, modified or unnatural nucleic acids, or a combination thereof. Nucleic acid paper folding may include a plurality of oligonucleotides that hybridize via sequence complementarity to produce engineered structuring of paper folding particles. The nucleic acid fold may comprise segments of single-stranded or double-stranded nucleic acids, or a combination thereof. Exemplary nucleic acid paper folding structures may include nanotubes, nanowires, cages, tiles, nanospheres, blocks, and combinations thereof. The nucleic acid folding paper may optionally include a relatively long scaffold nucleic acid to which a plurality of smaller nucleic acids hybridize, thereby creating folds and bends in the scaffold, thereby creating a modified structure. The scaffold nucleic acid may be circular or linear. The scaffold nucleic acid may be single stranded but is used to hybridize to a smaller nucleic acid. Smaller nucleic acids (sometimes referred to as "staples") may 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 acids.

As used herein, the term "oligonucleotide" refers to a molecule comprising two or more nucleotides linked by phosphodiester linkages. The oligonucleotides may include DNA, RNA, PNA, modified nucleotides, non-natural nucleotides, or combinations thereof. An oligonucleotide may include a limited number of nucleotide subunits, for example, less than about 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, or less than 5 nucleotides.

As used herein, the term "offset" when used to refer to a molecular structure refers to the spatial difference in orientation between two lines (2 dimensions) or two planes (three dimensions). The plane may approximate the surface of a molecular structure, such as the face of a folded paper tile. The offset may include a distance offset and/or an angular offset. Fig. 1A and 1B depict examples of angular offsets of different two-dimensional shapes (which may be two-dimensional projections of a three-dimensional structure). The isosceles triangle 100 of fig. 1A has an angular offset of 120 ° between the first face 110 and the second face 120, the relative orientations of which are depicted by orthogonal vectors a and a'. Triangle 130 of fig. 1B has an angular offset of 180 ° between first face 110 and second face 120, the relative orientations of which are depicted by orthogonal vectors a and a'.

As used herein, the term "polypeptide" refers to a molecule comprising two or more amino acids linked by peptide bonds. Polypeptides may also be referred to as proteins, oligopeptides or peptides. Although the terms "protein", "polypeptide", "oligopeptide" and "peptide" may optionally be used to refer to molecules having different characteristics such as amino acid sequence composition or length, molecular weight, molecular origin, etc., the terms are not intended to inherently include such differences in all contexts. The polypeptide may be a naturally occurring molecule or a synthetic molecule. The polypeptide may include one or more unnatural amino acids, modified amino acids, or a non-amino acid linker. The polypeptide may contain D-amino acid enantiomers, L-amino acid enantiomers or both. The amino acids of the polypeptides may be modified naturally or synthetically, such as by post-translational modification.

As used herein, the term "promiscuous" when used in reference to an affinity reagent refers to a binding agent that binds to or has the ability to bind to two or more different binding partners. For example, the hybrid binder may: 1) Binding to a plurality of different binding partners due to the presence of a common epitope within the structure of the different binding partners, or 2) binding to a plurality of different epitopes; or 3) a combination of both properties. Due to the presence of a specific epitope or target moiety, a promiscuous binding agent can bind to multiple binding partners, regardless of the context of the binding of the epitope or target moiety. The binding context may include, for example, the local chemical environment surrounding an epitope or target portion, such as flanking, adjacent or neighboring chemical entities (e.g., for polypeptide epitopes, flanking amino acid sequences or adjacent or neighboring non-contiguous amino acid sequences relative to the epitope). The plurality of different epitopes bound by the cognate affinity reagent may comprise structurally or chemically related epitopes, although these epitopes have different amino acid content. For example, an affinity reagent may be considered promiscuous if it has binding affinity for a trimeric peptide sequence having the form WXK, where X is any possible amino acid. Additional concepts relating to binding confounds are discussed in WO2020/106889A1, which is incorporated herein by reference.

As used herein, the term "retention component" refers to the portion of an affinity reagent, detectable probe, or other substance that links at least two other components. The retaining component may keep the two other components within a certain distance of each other. For example, two other components may be kept at a distance of at most 1000nm, 500nm, 100nm, 50nm, 10nm, 5nm, 1nm or less. Alternatively or additionally, the retaining component may separate the two other portions by a minimum distance from each other. For example, two other components may be maintained at a distance of at least 1nm, 5nm, 10nm, 50nm, 100nm, 500nm, 1000nm or more. The retention component may include, for example, structured nucleic acid particles, nucleic acid nanospheres, nucleic acid folded papers, protein nucleic acids, polypeptides, synthetic polymers, polysaccharides, organic particles, inorganic particles, gels, hydrogels, coated particles, and the like. The retention component may optionally have a polymeric structure. Alternatively, the holding component need not have a polymeric structure. In some embodiments, the retention component has a composition similar to the other components to which it is attached. For example, a plurality of binding components consisting of polypeptide substances may be attached to the polypeptide retention component. Alternatively, the reserved component may have the same meaning as The composition of the other components to which it is attached is quite different. For example, a plurality of binding members composed of a polypeptide substance may be attached to a retention member composed partially or entirely of a substance other than the polypeptide, such as a nucleic acid substance or an organic or inorganic nanoparticle (e.g., carbon nanospheres, silica nanospheres, etc.). The retention component may include one or more attachment sites that allow another component, such as a labeling component or binding component, to be attached to the retention component. The attachment site may include a functional group, an active site, a binding ligand, a binding receptor, a nucleic acid sequence, or any other entity capable of forming a covalent or non-covalent attachment with a binding component, a labeling component, or other detectable probe component. The retention component may comprise organic or inorganic particles or nanoparticles. The stent may comprise a coating or surface layer allowing attachment of another component, e.g. a FluoSphere as in a polymer coating ^TM Or in polymer coated quantum dots. Examples of retention components include stents as set forth in U.S. provisional application No. 63/112,607, which is incorporated herein by reference.

As used herein, the term "site" when used in reference to an array means a location in the array occupied by or configured to be occupied by a particular molecule or analyte, such as a polypeptide, nucleic acid, structured nucleic acid, or functional group. The site may contain only a single molecule, or it may contain a population of several molecules of the same species (i.e. an aggregate of molecules). Alternatively, the sites may comprise a population of molecules of different species. The sites of the array are typically discrete. The discrete sites may be continuous or they may have interstitial spaces between each other. Arrays useful herein can have sites separated by, for example, less than 100 microns, 10 microns, 1 micron, or 0.5 microns, 0.1 microns, 0.01 microns, or less. Alternatively or additionally, the array may have sites separated by at least 0.01 microns, 0.1 microns, 0.5 microns, 1 micron, 10 microns, 100 microns, or more. The sites may each have an area of less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron, or less. The array may comprise at least about 1x10 ⁴ 、1x10 ⁵ 、1x10 ⁶ 、1x10 ⁷ 、1x10 ⁸ 、1x10 ⁹ 、1x10 ¹⁰ 、1x10 ¹¹ 、1x10 ¹² Or more sites.

As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate may be non-porous or porous. The substrate may optionally be capable of absorbing a liquid (e.g., due to porosity), but is generally sufficiently rigid such that the substrate does not substantially swell upon absorption of the liquid and does not substantially shrink upon removal of the liquid by drying. Non-porous solid supports are generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, teflon ^TM Cyclic olefin, polyimide, etc.), nylon, ceramic, resin, zeonor ^TM Silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glass, fiber bundles, and polymers.

As used herein, the term "species" is used to identify molecules or moieties sharing the same chemical structure. Separate epitope portions having the same amino acid sequence are the same kind of epitope, while epitope portions having different sequences are different kinds of epitopes. Proteins expressed from the same gene are the same class of gene products, and proteins expressed from the same gene and having the same post-translational modification are the same protein form or isoform. The two proteins may be expressed from the same gene but have different protein modifications, in which case they are the same species of gene product but differ in protein form or isotype.

As used herein, the term "structured nucleic acid particle" (or "SNAP") refers to a single-or multi-stranded polynucleotide molecule having a compressed three-dimensional structure. For nucleic acids having the same sequence length as SNAP, the compressed three-dimensional structure may optionally be characterized according to hydrodynamic radius or stokes radius of SNAP relative to a random coil or other unstructured state. The compressed three-dimensional structure may optionally be characterized with respect to tertiary structure. For example, SNAP may be configured to have an increased number of internal binding interactions between regions of a polynucleotide strand, a smaller distance between the regions, an increased number of bends in the strand, and/or a sharper bend in the strand, as compared to the same nucleic acid molecule in a random coil or other unstructured state. Alternatively or additionally, the compressed three-dimensional structure may optionally be characterized with respect to a quaternary structure. For example, SNAP may be configured to have an increased number of interactions in a polynucleotide strand, or a smaller distance between the strands, as compared to the same nucleic acid molecule in a random coil or other unstructured state. In some configurations, the secondary structure of SNAP (i.e., the helical twist or orientation of a polynucleotide strand) can be configured to be denser than the same nucleic acid molecule in a random coil or other unstructured state. SNAP may optionally be modified to allow additional molecules to attach to the SNAP. SNAP may comprise DNA, RNA, PNA, modified or unnatural nucleic acids, or a combination thereof. SNAP may comprise a plurality of oligonucleotides that hybridize to form a SNAP structure. The plurality of oligonucleotides in SNAP can include oligonucleotides attached to or configured to be attached to other molecules (e.g., through functional groups), such as affinity reagents, binding partners, functional groups, or detectable labels. SNAP may include engineered or rationally designed structures such as nucleic acid folds.

As used herein, the term "substantially identical" when used in reference to two or more structures refers to structures that perform substantially the same manner or are capable of performing substantially the same function to achieve the same result. For example, two structured nucleic acid particles that are substantially identical may have the same primary structure. Optionally, they may differ in primary structure as long as they do not differ in tertiary or quaternary structure. Optionally, they may differ in one or more of the primary, secondary, tertiary or quaternary structures, so long as they perform substantially the same function in substantially the same manner to achieve the same result in accordance with the methods set forth herein.

As used herein, the term "target moiety" refers to a specific chemical structure within an epitope that mediates or facilitates binding interactions. The target moiety may include a functional group, side chain, active site, or other chemical entity having a characterizable structure. For example, the target moiety may be one or more amino acids or side chains thereof that form part of a polypeptide epitope. The target moiety may specifically interact with a binding site of an affinity reagent to facilitate or mediate an interaction that results in binding of the affinity reagent to the binding partner.

As used herein, the term "tunable" refers to the specific, precise, and/or rational positional adjustability of a component or attachment site in the structure of a retained component, scaffold, or molecule. An adjustable retention component may refer to the ability to attach other components within or create attachment sites for attaching other components at specific sites or specific regions of a retention component structure. As used herein, "tunable" refers to the property of a probe or a retention component having an adjustable structure or architecture.

Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about". As used herein, the term "about" when used in connection with a percentage may mean ± 5% of the indicated value. For example, about 90% means 85% to 95%.

As used herein, the term "two-dimensional projection" refers to an area or shape that would be occupied by the projection of a three-dimensional structure onto a planar two-dimensional surface without significant geometric or spatial distortion. For example, a two-dimensional projection of a sphere onto a planar two-dimensional surface will produce a circular area on the surface with a diameter equal to the diameter of the sphere. The two-dimensional projection may be formed from any reference frame, including a reference frame orthogonal to any surface of the three-dimensional structure.

Structure and function of detectable probes and affinity reagents

Described herein are detectable probes or affinity reagents having increased binding affinity for a binding partner, increased observability, or both. The structural and functional features exemplified herein for the detectable probes may be present in other affinity reagents. Conversely, the structural and functional features exemplified herein for the affinity reagents may be present in the detectable probes. Thus, the compositions, structures, and methods set forth herein in the context of detectable probes may be applied to other affinity reagents, and vice versa.

The detectable probe or affinity reagent may comprise a plurality of binding components that collectively increase the overall binding affinity of the probe for the binding partner as compared to any individual binding component of the plurality contained within the detectable probe or affinity reagent structure. Any of a plurality of affinity reagents known in the art or set forth herein may act as binding components when attached to the detectable probes or affinity reagents of the present disclosure. The detectable probes or affinity reagents of the present disclosure may further comprise one or more labeling components that allow for visualization of the binding interaction between the detectable probe (or affinity reagent) and the binding partner. Any of a plurality of detectable labels known in the art or set forth herein may serve as a label component when attached to the detectable probes or affinity reagents of the present disclosure.

The detectable probes or affinity reagents of the present disclosure can comprise (a) a retention component; (b) A labeling component, and (c) one, two, or more binding components attached to the retention component. Of particular interest are retaining components that are spatially and/or in orientation adjustable to provide accurate, predetermined and/or rational presentation of other components attached thereto. For example, the binding component may be attached in a configuration that increases the binding affinity and/or observability of the detectable probe or affinity reagent. The detectable probe or affinity reagent may comprise a retention component that provides sufficient sites to attach at least one binding component and/or at least one labeling component. In some configurations, the retention component may be a natural, artificial, or synthetic particle that includes multiple functional groups, reactive sites, nucleic acids, or functional groups that allow other molecules to attach to the retention component. The retention component may have an amorphous, spherical, or irregular structure (e.g., DNA nanospheres, fluorescent nanoparticles such as FluoSphere ^TM Or quantum dots). The retained component may have a regular or symmetrical structure (e.gCarbon nanospheres, carbon nanotubes, metal nanotubes, ceramic nanoparticles). The retention component may comprise a shell or scaffold formed from template particles such as particles or nanoparticles. In some configurations, the template particles may include a labeling component, such as FluoSphere ^TM Or quantum dots. The retention component may include any suitable material including, but not limited to, polymers, metals, semiconductors, ceramics, glass, and biomolecules (e.g., nucleic acids such as DNA or RNA, proteins, polysaccharides).

In some cases, the retention component may include a nucleic acid that is partially or substantially fully double-stranded. The double stranded nucleic acid may impart additional structural rigidity to the retention components to provide better spacing between the labeling components. In such cases, the labeling component may be coupled to one or both strands of the double stranded nucleic acid. The retention component may comprise structured particles or rationally designed particles. The retention component may include a Structured Nucleic Acid Particle (SNAP) configured to attach one or more binding components and/or one or more labeling components. In some configurations, SNAP may comprise DNA origami particles or DNA nanospheres.

The retention component may include multiple sites for attaching other components (e.g., a binding component, a labeling component, or another retention component). The retention component may include unique or dedicated sites for coupling the binding component and/or the labeling component. For example, the retention component may comprise a nucleic acid having a first sequence complementary to a nucleic acid coupled to the binding component, and may further comprise a nucleic acid having a second sequence complementary to a nucleic acid coupled to the labeling component. The first sequence may be different from the second sequence such that different types of components are appropriately directed to a subset of coupling sites. Alternatively, the first and second sequences may be identical. Optionally, the retention component may include one or more first functional groups configured to form covalent bonds with functional groups coupled to the binding component, and may further include one or more second functional groups configured to form covalent bonds with functional groups coupled to the labeling component. The first functional group may be different from the second functional group such that different types of components are properly directed to a subset of coupling sites. For example, the first plurality of functional groups may participate in a bonding reaction orthogonal to the bonding reaction in which the second plurality of functional groups participate. Alternatively, the first and second functional groups may be the same. In some configurations, the retention component may include a mixture of attachment site types, such as nucleic acids and functional groups, wherein each type of attachment site is configured to attach a different type of component.

The retention component may include one or more other components (e.g., a binding component, a labeling component, or another retention component) attached to the retention component. The retention component may be attached to the other component by a covalent bond (e.g., via a click reaction), a coordination bond (e.g., a silane linker of a silicon nanoparticle), or a non-covalent bond (e.g., nucleic acid hybridization). The reserved component may be attached to the other component by a chemical reaction that forms a covalent bond between a functional group on the reserved component and a functional group on the other component. The reaction may be carried out by any suitable method, including nucleophilic substitution, electrophilic substitution, and elimination reactions. In some configurations, covalent bonds between the retained component and the other component may be formed by bio-orthogonal or click reactions. The retention component and/or other components may be modified to include functional groups configured to participate in bioorthogonal or click reactions. Exemplary functional groups and linkages useful for attaching a retention component to one or more other components are described in further detail herein, for example, in the context of preparing a detectable probe or affinity reagent.

In some configurations, the retention component may include a nucleic acid structure, such as SNAP. In some cases, the nucleic acid structure may include a region of single stranded nucleic acid that provides a targeted site-specific hybridization site for attaching other components (e.g., a binding component, a labeling component, or another retention component) to the retention component. In such a configuration, the other component attached to the complementary oligonucleotide may be annealed to a single stranded sequence on the retention component to form an attachment at a targeting site on the retention component. In other configurations, an oligonucleotide comprising a functional group may be annealed to a retained component, allowing for the subsequent attachment of another component via a chemical reaction (e.g., a click reaction) between the functional group on the oligonucleotide and the functional group on the other component.

The detectable probes or affinity reagents may comprise a retention component attached to a plurality of binding components. The plurality of binding members attached to the retention member may be selected to increase the affinity of the detectable probe or affinity reagent. In some configurations, the detectable probe or affinity reagent may contain multiple binding components that are homogenous in terms of species (e.g., antibody only, aptamer only, nanobody only, mini-peptide binding agent only, DARPin only, etc.). In other configurations, the detectable probe or affinity reagent may contain multiple binding components that are heterogeneous in species (e.g., a mixture of antibodies, nanobodies, minibodies, DARPin, and/or aptamers). In some configurations, the detectable probe or affinity reagent may contain a plurality of binding components that have substantially the same binding specificity for a particular binding partner, epitope, or target moiety. In other configurations, the detectable probe or affinity reagent may contain a plurality of binding components that have mixed binding specificities for a particular binding partner, epitope or target moiety.

The amount and/or type of binding component attached to the detectable probe or affinity reagent may be selected to increase the affinity of the detectable probe or affinity reagent. The detectable probes or affinity reagents can have a total of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more, whether different from each other (e.g., heterogeneous mixtures of binding components) or the same (e.g., homogeneous sets of binding components). Alternatively or additionally, the detectable probes or affinity reagents may have a total of no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less, whether different from each other or the same.

The detectable probes or affinity reagents may comprise a heterogeneous mixture of binding members in a selected ratio, the heterogeneity being based on the type of binding member and/or the binding specificity. For example, a detectable probe or affinity reagent for a particular epitope may include a high specific binding component and a medium specific binding component in a molar ratio of about 3:1. The detectable probe or affinity reagent can include a first binding component and a different second binding component in a ratio of at least about 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 25:1, 50:1, 100:1, 250:1, 500:1, 1000:1, 2500:1, 5000:1, 10000:1, 25000:1, 50000:1, 100000:1, 250000:1, 500000:1, 1000000:1 or more. Alternatively or additionally, the detectable probe or affinity reagent may comprise a first binding component and a different second binding component in a ratio of no greater than about 1000000:1, 500000:1, 250000:1, 100000:1, 50000:1, 25000:1, 10000:1, 5000:1, 2500:1, 1000:1, 500:1, 250:1, 100:1, 50:1, 25:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1 or less.

For binding partners, epitopes or target moieties, the affinity reagents, probes or binding components of the present disclosure may have a characteristic binding probability. For example, affinity reagents, probes, or binding members are known to bind to a polypeptide epitope and are known to exhibit a high probability of binding (e.g., 1 out of every 100 observations does not show evidence of binding). In another example, an affinity reagent, probe, or binding component can be characterized as binding to an epitope with a low but non-zero binding probability (e.g., 0.00001% chance of binding for a given observation). The probability characterization may include two aspects regarding the combined probability: 1) A structure-dependent probability of binding to a binding partner, epitope or target moiety; and 2) the environmental dependency potential of binding to a binding partner, epitope or target moiety.

The probability of binding of an affinity reagent, probe or binding component to a binding partner, epitope or target moiety can be extended beyond polypeptides to include non-polypeptides or heterogeneous systems (e.g., feces, crude cell lysate, body fluids). For example, the affinity reagent, probe, or binding component may have a characteristic binding probability for a component of the complex (e.g., metal nanoparticles embedded in a polymer matrix), or may preferentially bind to a structural subunit within the polysaccharide (e.g., glycosylation, hemicellulose, cellulose, lignin, pectin, etc.). The skilled artisan will recognize in light of the present disclosure that an affinity reagent, probe or binding component intended for use in a non-polypeptide or heterogeneous system may exhibit similar properties with a non-zero probability of binding to a non-polypeptide binding partner, epitope or target moiety for which the affinity reagent, probe or binding component has a low binding affinity.

The binding affinity or binding promiscuity of an affinity reagent, probe or binding component may be related to the effect of the primary, secondary, tertiary or quaternary structure (i.e., amino acid sequence) of a polypeptide on binding. For example, an affinity reagent, probe, or binding component can be characterized as binding preferentially that increases or decreases for a particular polypeptide epitope sequence (e.g., amino acid trimer, tetramer, pentamer, etc.). The structure-dependent probability of binding of an affinity reagent, probe or binding component to an epitope may also be influenced by the background of the sequence (e.g., amino acids flanking the amino-and/or carbonyl-terminus of the peptide epitope; amino acid residues in the secondary or tertiary structure of the polypeptide proximal to the peptide epitope, the presence or absence of post-translational modifications in or around the peptide epitope, etc.). The affinity reagents, probes, or binding components of the present disclosure can have significant affinity or promiscuity for a family of amino acid epitopes (e.g., AXA, where a represents alanine and X represents any of 20 naturally occurring amino acids). The structure-dependent probability of binding may be calculated for each affinity reagent, probe or binding component used for polypeptide characterization, such as by empirical binding models or databases of binding probabilities. The sequence-specific likelihood of binding of an affinity reagent, probe, or binding component to a binding partner, epitope, or target moiety may be at least about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, 99.999999% or greater. Alternatively or additionally, the sequence-specific likelihood of binding of an affinity reagent, probe, or binding component to a binding partner, epitope, or target moiety may be no greater than about 99.999999%, 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%, or less.

The environmental dependency potential of binding of an affinity reagent, probe or binding component to an epitope in a polypeptide may be related to the effect of a variable other than the epitope and/or the structure of the polypeptide on binding. For example, the binding of an affinity reagent, probe, or binding component to a particular epitope may vary depending on the solvent chemistry (e.g., solvent characteristics, solvent polarity, ionic strength of the solvent, buffer concentration, pH, presence of surfactant or denaturant, etc.). Other non-polypeptide variables may include duration of binding; concentration of affinity reagent, probe or binding component; concentration of binding partner, epitope or target moiety; and the presence of externally applied fields such as heat, electric fields, magnetic fields, and fluid velocity fields. The environmental dependency probability of binding of an affinity reagent, probe, or binding component to a binding partner, epitope, or target moiety may be at least about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, 99.999999% or more. Alternatively or additionally, the environmental dependency probability of binding of an affinity reagent, probe, or binding component to a binding partner, epitope, or target moiety may be no greater than about 99.999999%, 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or less.

In some configurations, the structure-dependent binding potential and the environment-dependent binding potential may be combined to determine the total likelihood or probability of binding of an affinity reagent, probe, or binding component to a binding partner, epitope, or target moiety. The total likelihood or probability of some or all of the known binding partners, epitopes or target moieties may be pooled to create a probabilistic binding profile for the affinity reagent, probe or binding component. In some configurations, an affinity agent, probe, or binding component can be characterized as binding to a set of N binding partners, epitopes, or target moieties with a total binding probability of at least about 20%, and binding to a set of M binding partners, epitopes, or target moieties with a total binding probability of no greater than 0.1%, where N.gtoreq.1, M.gtoreq.1, and M.gtoreq.10N. The total likelihood or probability of binding of an affinity reagent, probe, or binding component to a binding partner, epitope, or target moiety may be at least about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, 99.999999% or more. Alternatively or additionally, the total likelihood or probability of binding of an affinity reagent, probe, or binding component to a binding partner, epitope, or target moiety may be no greater than about 99.999999%, 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%, or less.

The retention component may include one or more marker components associated with the retention component. In some configurations, the one or more labeling components may be attached to a retention component of the detectable probe or affinity reagent. In some configurations, the one or more label components can be non-covalently associated with the detectable probe (e.g., a nucleic acid intercalating dye) or an affinity reagent. In some configurations, the retention component can surround or enclose the labeling component (e.g., fluoSphere ^TM Or a polymer coating on the quantum dots). The labeling component can be attached to the retention component by a covalent bond (e.g., via a click reaction), a coordination bond (e.g., a silane linker of a silicon nanoparticle), or a non-covalent bond (e.g., nucleic acid hybridization). The labeling component may be attached to the retention component by a chemical reaction that forms a covalent bond between a functional group on the retention component and a functional group on the labeling component. The reaction may be carried out by any suitable method, including those set forth herein or known in the art.

The retention component may include particles (e.g., microparticles or nanoparticles) that provide one or more attachment sites for other components (e.g., binding component, labeling component, or another retention component). In some configurations, the particles may include a surface that is functionalized, may be functionalized, or may be otherwise modified to provide attachment sites for other components. In some configurations, the particles may provide a template for a shell or coating (e.g., a polymer or hydrogel coating) that contains or may be modified to contain attachment sites for the components. In some configurations, the retention component can effectively act as a labeling component (e.g., fluoSphere ^TM Or quantum dots). The particles may include a surrounding or concentric shell, layer or coating that provides attachment sites or modifiable sites for attaching components. The shell, coating or layer may comprise a polymer or hydrogel that has been covalently or non-covalently attached to the surface of the particle. The shell, coating or layer may include a plurality of functional groups that may form covalent bonds with another molecule or may be modified toForming a covalent bond with another molecule. The shell, coating, or layer may be modified with additional groups that provide attachment sites or otherwise modify the surface (e.g., providing steric hindrance through pegylation of the surface).

Any of a plurality of detectable moieties may be used as the labeling component of the affinity reagents or detectable probes described herein. For example, in some cases, the label may be an electrochemical label and may be detected by electrochemical detection. These may be charged moieties such as large nucleic acids, polylysine, and other highly charged chemical structures, and the detection zone may be a ChemFET-type sensor. Alternatively or additionally, in some cases the detectable moiety may be an optically detectable moiety, i.e. detectable based on observing the differential light energy from the label.

The detectable probes can include one or more retention components attached to one or more labeling components. The one or more labeling components attached to the retention component may be selected to increase the observability of the detectable probe. In some configurations, the detectable probe may contain multiple label components that are homogeneous in the type of label (e.g., a single type of fluorophore, a homogeneous nucleic acid barcode, etc.). In other configurations, the detectable probe may contain a heterogeneous plurality of label component species (e.g., a mixture of fluorophores having different emission wavelengths, a mixture of fluorophores and nucleic acid barcodes). Optionally, the detectable probe may contain a plurality of labeling components that produce overlapping signals or signals that are indistinguishable when detected by the methods or devices set forth herein. For example, multiple fluorophores present in the detectable probe can emit fluorescence at a common wavelength, whether the fluorophores excite at the same wavelength as each other or at different wavelengths from each other. Alternatively, the detectable probe may contain a plurality of label components that produce signals that are different from one another, such as distinguishable or distinguishable signals when detected by the methods or devices set forth herein.

The detectable label or label component may produce a detectable signal that allows for the confirmation of the interaction between the affinity reagent (e.g., a detectable probe) and the binding partner, epitope, or target moiety. The detectable label or label component may be configured to provide spatial information, such as providing a detectable signal at a spatially resolved location. The detectable label or label component may be configured to provide temporal information, such as providing an evanescent or decaying signal, optionally at a spatially resolved position. The detectable label or label component may emit a detectable signal in the presence of an excitation source (e.g., radiation, heat, chemical substrate). The detectable label or label component can emit a detectable signal in the absence of an excitation source (e.g., a radiolabel or chemiluminescent label). The detectable label or label component may contain a coded signal, such as a nucleic acid or polypeptide barcode.

In some configurations, the labeling component may include an attached enzyme, protein, or sequence of an enzyme that produces a detectable chemical signal. Exemplary enzymes may include horseradish peroxidase (HRP) or alkaline phosphatase. Enzymes or proteins that convert a substrate molecule into a detectable molecule (e.g., a fluorescent compound) or bind to a matrix molecule, which in turn produces a fluorescent or luminescent effect in the enzyme or protein, may be selected. For example, HRP can convert a substrate molecule such as ABTS, OPD, amplexRed, DAB, AEC, ^TM B. Homovanillic acid or luminol is converted into fluorescent or luminescent molecules. In some configurations, the detectable probe or affinity reagent may interact with the binding partner, epitope, or target moiety in the presence of the substrate molecule to produce a deposited fluorescent or luminescent molecule that provides a spatial signal of the location of the detectable probe or affinity reagent binding interaction. In alternative configurations, the detectable probe or affinity reagent binding interaction may be detected by a reaction pathway or reaction sequence that produces a detectable signal through a series of reactions of the matrix molecule. For example, an enzyme such as HRP or alkaline phosphatase may be co-localized with the binding partner, epitope, or target moiety. The detectable probe or affinity reagent may include one or more enzymes that convert a substrate molecule from a substrate that cannot be processed or altered by the co-localized enzyme to a product that is a substrate for the co-localized enzyme. The detectable probe or affinity reagent may comprise converting a substrate into a detectable productA plurality of different enzymes or enzyme complexes (e.g., polyketide synthases).

The detectable label or label component may be detected by a signal detection source appropriate to the type of label selected. Optical markers and optical detectors are particularly useful. Examples of optical detection devices and components thereof that may be used herein include those commercially available for nucleic acid sequencing, such as those described by Illumina ^TM Inc (e.g. HiSeq ^TM 、MiSeq ^TM 、NextSeq ^TM Or NovaSeq ^TM System), life Technologies ^TM (e.g. ABI PRISM ^TM Or SOLID ^TM System), pacific Biosciences (e.g., using SMRT ^TM Systems of technology, e.g. sequence ^TM Or RS II ^TM System) or Qiagen (e.g. Genereader) ^TM System) or U.S. patent application publication No. 2010/011768 A1 or U.S. patent No. 7,329,860;8,951,781 or 9,193,996, each of which is incorporated herein by reference. Other useful detectors are described in U.S. patent 5,888,737;6,175,002;5,695,934;6,140,489; or 5,863,722; or U.S. patent publication No. 2007/007991 A1, 2009/0247114 A1 or 2010/011768; or WO2007/123744, each of which is incorporated herein by reference in its entirety.

Other detection techniques that may be used in the methods set forth herein include, for example, mass spectrometry that may be used to perceive mass; can be used to sense surface plasmon resonance associated with a surface; absorbance at a wavelength that is useful for sensing the energy absorbed by the label; calorimetric methods useful for sensing temperature changes due to the presence of a label; conductivity or impedance, or other known analytical techniques, that may be used to sense the electrical characteristics of the tag. For example, charged labels may be detected using an electronic detector, such as a chemFET detector for detecting protons or pyrophosphates (see, e.g., U.S. patent application publication nos. 2009/0026082 A1, 2009/012589 A1, 2010/01375143 A1, or 2010/0282617 A1, each of which is incorporated herein by reference in its entirety, or at Ion Torrent commercially available from ThermoFisher, waltham, mass ^TM Commercial detectors in systems). Can be used forTo use FET detectors as described in U.S. patent application publication nos. 2017/0240962 A1, 2018/0051316 A1, 2018/0110265 A1, 2018/0155773 A1, or 2018/0305727 A1; or one or more of those described in U.S. patent nos. 9,164,053, 9,829,456, 10,036,064, or 10,125,391, each of which is incorporated herein by reference.

The labeling component may comprise a luminescent moiety or molecule (e.g., a luminophore or fluorophore). Luminophores that emit light of one wavelength in response to excitation by light of another wavelength are particularly useful as optically detectable labels because they are capable of providing readily detectable optical signals and they are capable of accommodating a variety of different excitation and emission spectra, providing great flexibility in their deployment and use.

Any of a variety of luminophores may be used herein. In some cases, the luminophore may be a small molecule. In some cases, the luminophore may be a protein. The luminophore may comprise a label that emits in the ultraviolet spectrum, visible spectrum or infrared spectrum. In some cases, the luminophore may be selected from FITC, alexa350、Alexa/>405、Alexa488、Alexa/>532、Alexa/>546、Alexa/>555、Alexa/>568、Alexa/>594、Alexa/>647、Alexa/>680、Alexa/>750. Pacific blue, coumarin, BODIPY FL, pacific green, oregon green, cy3, cy5, pacific orange, TRITC, texas red, R-phycoerythrin, phycocyanin (APC). In some cases, the label may be an Atto dye, such as Atto 390, atto 425, atto 430, atto 465, atto 488, atto 490, atto 495, atto 514, atto 520, atto 532, 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, atto MB2, atto Oxa12, atto Rho101, atto Rho12, atto Rho13, atto Rho14, atto Rho3B, atto Rho6G, or Atto Rho 12. In some cases, the luminophore may be a fluorescent protein, for example a fluorescent protein selected from the group consisting of 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 variety of available chromophores are commercially available, for example from the Molecular Probes department of ThermoFisher Scientific, and/or are generally described in Molecular Probes Handbook(11 th edition), which is hereby incorporated by reference. The labeling component may also include an intercalating dye, such as ethidium bromide, propidium bromide, crystal violet, 4', 6-diamidino-2-phenylindole (DAPI), 7-amino actinomycin D (7-AAD), hoescht 33258, hoescht 33342, hoescht 34580, YOYO-1, diYO-1, TOTO-1, diTO-1, or combinations thereof.

The particular fluorescent label or other luminophore may be selected according to the desired use and in some cases based on their absorption/emission spectra, e.g. to optimise multiplex detection. Also, in some cases, the luminophore may include a fluorescent dye pair that interacts to provide favorable fluorescent properties. For example, a paired dye may act as a Forster Resonance Energy Transfer (FRET) pair, wherein excitation of one member of the pair (i.e., the "donor") results in energy transfer that excites or transfers to the other member (i.e., the "acceptor"). The acceptor then emits light at a wavelength offset from that emitted by the donor alone.

In some cases, the optically detectable label may include other types of detectable moieties. For example, in some cases, luminescent particles (e.g., microparticles or nanoparticles) such as semiconductor nanoparticles, "quantum dots" or FluoSphere may be included ^TM The particles serve as a marker component.

Luminophores may be characterized by a characteristic excitation or absorption wavelength. The excitation source may include a light source tuned to a characteristic excitation or absorption wavelength of the luminophore. The luminophore may absorb light in a range of wavelengths, wherein the maximum absorption of light occurs at the peak wavelength. The luminophore may be excited at or near the peak excitation. In particular configurations of the methods set forth herein, the luminophores may be excited by radiation in the Ultraviolet (UV), visible (VIS), or Infrared (IR) regions of the spectrum. Excitation in the VIS region can occur in one or more of the red, orange, yellow, green, blue, or violet regions of the spectrum. The luminophore may be characterized by a characteristic emission wavelength. The luminophore may emit light in a range of wavelengths, wherein the maximum emission of light occurs at the peak wavelength. The luminophore may be detected at or near the peak emission. In particular configurations of the methods set forth herein, emissions from luminophores may be detected in the Ultraviolet (UV), visible (VIS), or Infrared (IR) regions of the spectrum. Detection in the VIS region can be in one or more of the red, orange, yellow, green, blue, or violet regions of the spectrum.

The presence of the plurality of label components in the detectable probe may increase the observability of the detectable probe, for example, by 1) increasing the likelihood of detection; 2) Redundancy is provided in the event of label loss (e.g., photobleaching, chemical damage, label cutting, etc.); and/or 3) increasing the intensity of the signal generated by the detectable probe. The amount of label component associated with the detectable probe can be determined by a variety of factors including probe size, desired label spacing, signal strength, measurement length, measurement environment, and label size.

The detectable probe can have a selected number of associative tag components. The detectable probe can have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more, whether different from each other (e.g., a heterogeneous mixture of labels that produce distinguishable signals in a method or apparatus set forth herein) or the same (e.g., a mixture of labels that produce indistinguishable signals in a method or apparatus set forth herein). Alternatively or additionally, the detectable probes can have no greater than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less, whether different from each other (e.g., a heterogeneous mixture of labels that produce distinguishable signals in a method or apparatus set forth herein) or the same (e.g., a mixture of labels that produce indistinguishable signals in a method or apparatus set forth herein).

The type or amount of the selected label component on the detectable probe may provide a signal that exceeds the background signal. For example, probes detected by fluorescence may produce fluorescent signals that exceed the existing background from sources such as autofluorescence, signal crosstalk, and impingement on external sources. The detectable probes can include a plurality or amount of a label component configured to produce a detectable signal intensity that exceeds the background intensity (e.g., maximum, minimum, or average) of the signal by at least 2x, 5x, 10x, 25x, 50x, 100x, or more. Additionally or alternatively, the detectable probes may include an amount of a labeling component configured to produce a detectable signal intensity that exceeds the background intensity of the signal (e.g., maximum, minimum, or average) by no more than about 100x, 50x, 25x, 10x, 5x, 2x, or less.

The detectable probe may comprise more than one type and/or kind of label component. For example, the detectable probe may comprise at least one luminophore and at least one nucleic acid barcode sequence. In another example, the detectable probe may comprise two or more different luminophores (e.g.,488 and->647). The different luminophores may differ in one or more signal properties, such as their excitation spectrum, emission spectrum, luminescence lifetime or luminescence polarity. In some configurations, the different luminophores may be similar in one or more signal properties. For example, two emitters may be excited at the same wavelength but emit at different wavelengths. In this way, a single excitation source can be used to excite two different luminophores, which are however distinguished based on the differences in their emission characteristics. The detectable probes may include more than one type and/or kind of label component for a variety of purposes, including generating unique signal fingerprints, implementing multi-label detection methods (e.g., FRET or luminescence quenching), and/or generating signal redundancy to reduce false positive or false negative detection.

Based on the type and/or kind of label, the detectable probe may comprise a heterogeneous mixture of label components. The detectable probe can have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more labeling components, whether the same as one another or different from one another. Alternatively or additionally, the detectable probe may have no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less labeling components, whether the same as or different from each other.

Based on the detectable label species, the detectable probe can comprise a heterogeneous mixture of label components in a selected ratio. For example, detectable probes for a particular epitope may each comprise a molar ratio of about 3:1 488 and the like-647 dye. The detectable probe can include a first label component and a different second label component in a ratio of at least about 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 25:1, 50:1, 100:1, 250:1, 500:1, 1000:1, 2500:1, 5000:1, 10000:1, 25000:1, 50000:1, 100000:1, 250000:1, 500000:1, 1000000:1, or greater. Alternatively or additionally, the detectable probe may comprise a first label component and a different second label component in a ratio of no greater than about 1000000:1, 500000:1, 250000:1, 100000:1, 50000:1, 25000:1, 10000:1, 5000:1, 2500:1, 1000:1, 500:1, 250:1, 100:1, 50:1, 25:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1 or less.

FIG. 49A provides a schematic diagram of a detectable probe. Each detectable probe consists of two parts: a labeled affinity probe (200) and a template (201). As shown, the labeled affinity probes 200 include a binding partner 200a, an annealing zone 200b that provides a retention partner, and an enzymatically extended labeling partner 200c. Template 201 includes a complementary sequence 201a of annealing region 200b and template 201b for enzymatic extension of 200c. FIG. 49B shows a step-wise display of enzymatically extended aptamer probes to form a tag component.

In some cases, the marker component may include or be attached to a retention component (e.g., a scaffold as set forth in U.S. provisional application No. 63/112,607). Multiple marker components may be randomly spaced along the retention component or, in some cases, may be positioned at regular or semi-regular intervals along the length of the retention component. In other cases, a single labeling component may be attached to a retention component, e.g., at the 5' end of the nucleic acid, see fig. 50A. In yet other cases, the component-retaining nucleic acid may be designed to include an annealing region, and may have a single labeling component (e.g., a fluorophore) at the end of the annealing region. The second strand of nucleic acid with an additional single fluorophore may anneal to this region, see fig. 50B.

It will be appreciated that the affinity reagents of the present disclosure need not include any labeling components. Thus, the affinity reagent may be configured to omit or lack one or more of the labeling components or labeling component species set forth herein. Furthermore, exemplary affinity reagents or detectable probes set forth herein as having one or more labeling components may be reconfigured to omit one or more of the exemplified labeling components. It will also be appreciated that the affinity reagents or detectable probes of the present disclosure may be configured to omit or lack one or more of the binding components or binding component species set forth herein.

The retained component may form a central structural element for the detectable probe or affinity reagent. In some configurations, the retention component may be structured to provide a limit or control over the physical positioning of other components, such as binding components, labeling components, or other retention components. For example, the detectable probes or affinity reagents may include binding members that attach at sufficiently spaced locations on the retention member to prevent contact or cross-reaction between the binding members. In another example, the detectable probe may include luminophores attached at sufficiently spaced apart locations on the retention component to reduce or prevent quenching between adjacent luminophores. In a third example, the detectable probe may include a luminophore attached at a spaced and oriented position on the retention component to facilitate FRET. The retained components may be adapted or rationally designed to control the location and/or positioning of components attached thereto. Factors that may affect the design of the retained components include: 1) The nature of the possible interactions between adjacent components; 2) The possibility of interactions between adjacent components; 3) Critical dimensions (e.g., distance, volume, time) of possible interactions between adjacent components; 4) Retaining physical properties (e.g., shape, conformation, size, rigidity, etc.) of the components; 5) Physical properties (shape, conformation, size, hydrodynamic radius, etc.) of the attached probe components; 6) The nature and characteristics of the optional linker that binds the component to the retained component; and 7) the nature of the possible interactions between the probes or affinity reagents and the binding partners can be detected.

In some configurations, the detectable probe or affinity reagent can include a retention component that includes one or more nucleic acids. For example, the one or more nucleic acids forming the retention component may take the form of a nucleic acid paper-break structure, a nucleic acid nanosphere structure, or other structured nucleic acid particles. The nucleic acid retaining component may contain a single nucleic acid strand or multiple nucleic acid strands, such as multiple oligonucleotide strands. The retention component comprising a plurality of nucleic acid strands may be formed by hybridization between the plurality of strands to form a nucleic acid structure having increased structural complexity beyond the native helical structure. The nucleic acid may include a nucleic acid such as DNA, RNA, PNA or a combination thereof. The nucleic acid may include non-natural nucleotides, such as light-emitting modified nucleotides. The nucleic acid may include non-nucleotide residues within its structure, such as a photocleavable linker (e.g., a nitrobenzyl, carbonyl, or benzyl-based photocleavable linker). The retained components, including nucleic acids, may be structured to decompose or destabilize under certain conditions. The retention component may be broken down or destabilized, for example, to facilitate the removal of the detectable probe or affinity reagent from the binding partner. The retention component comprising the nucleic acid may comprise a plurality of restriction sites that may be cleaved by a restriction enzyme, thereby facilitating the decomposition or destabilization of the nucleic acid retention component. The retention component comprising the nucleic acid may comprise a plurality of photocleavable, chemically cleavable, or otherwise reactive bonds, thereby facilitating decomposition or destabilization of the retention component.

The retention component may comprise a nucleic acid nanosphere. The nanospheres may contain a single strand of nucleic acid that folds upon itself to form a compressed structure. Optionally, the nanospheres may be crosslinked to confine the nanospheres to a relatively compressed structure. Exemplary cross-links include chemical cross-links, such as psoralens or oligonucleotides that hybridize to different regions of the single strand. Nucleic acid nanospheres can be produced by rolling circle amplification of a circular template to produce concatemer amplification products, wherein each unit of sequence in the concatemer has a sequence complementary to the circular template. Exemplary sequence elements that may be incorporated into a nucleic acid nanosphere include tag sequences that provide information about the nanosphere, such as its source or history of use, sequences that are complementary to oligonucleotides used as intrachain cross-linkers, sequences that are complementary to oligonucleotides attached to functional groups or components, such as binding or labeling components, and the like. Exemplary nucleic acid nanospheres and methods of making and using the same are set forth in U.S. patent No. 8,445,194, which is incorporated herein by reference.

Nucleic acid nanospheres can be formed by methods such as Rolling Circle Amplification (RCA) or ligation of repeat concatemer units. The nucleic acid nanospheres can include a specific number of repeating concatemer units. The nucleic acid nanospheres can comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more concatemer units. Alternatively or additionally, the nucleic acid nanospheres can include no greater than about 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or fewer concatemer units. The nucleotide sequence length of the concatemer units in the nucleic acid nanospheres can be at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more nucleotides. Alternatively or additionally, the nucleotide sequence length of the concatemer units in the nucleic acid nanospheres may be no greater than about 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20 or fewer nucleotides.

The retention component may comprise nucleic acid folded paper. Thus, the retention component may comprise one or more nucleic acids having a tertiary or quaternary structure, such as spheres, cages, tubules, cassettes, tiles, blocks, trees, cones, wheels, combinations thereof, and any other possible structure. Examples of such structures formed with DNA origami are set forth in Zhao et al, nano Lett.11,2997-3002 (2011), incorporated herein by reference. In some configurations, a nucleic acid fold may include a scaffold and a plurality of staples, wherein the scaffold is a single continuous nucleic acid strand and the staples are oligonucleotides configured to fully or partially hybridize to the scaffold nucleic acid. Examples of nucleic acid paper folding structures formed using a continuous stent chain and several staple chains are set forth in Rothemund Nature 440:297-302 (2006) or U.S. patent No. 8,501,923 or 9,340,416, each of which is incorporated herein by reference. The retention component comprising one or more nucleic acids (e.g., as found in a folded paper or nanosphere structure) may comprise a single stranded nucleic acid region, a double stranded nucleic acid region, or a combination thereof.

In some embodiments, the nucleic acid paper comprises a scaffold having closed nucleic acid strands and a plurality of oligonucleotides hybridized to the scaffold. A nucleic acid scaffold can comprise a continuous strand of nucleic acid that is circular or linked (i.e., without 5 'or 3' ends) without complementary oligonucleotides. In some configurations, the nucleic acid scaffold is derived from a natural source, such as a viral genome or a bacterial plasmid. In other configurations, the nucleic acid scaffold may be engineered, rationally designed, or synthesized. The scaffold may comprise one or more modified nucleotides. The modified nucleotide may provide a functional group or attachment site for attaching an additional component (e.g., a binding component, a labeling component, or another retention component) before, during, or after assembly of the detectable probe or affinity reagent. The modified nucleotide may include a linking group or functional group (e.g., a functional group configured to perform a click reaction). In some configurations, the nucleic acid scaffold can comprise a single strand of the M13 viral genome. The size of the nucleic acid scaffold can vary depending on the desired size of the retention component. The nucleic acid scaffold can comprise at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more nucleotides. Alternatively or additionally, the nucleic acid scaffold can comprise up to about 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, or fewer nucleotides.

A retaining component such as a nucleic acid fold may include a plurality of oligonucleotides (e.g., staples). The staples may include oligonucleotides configured to hybridize to a nucleic acid scaffold, other staples, or a combination thereof. The staples may include one or more modified nucleotides. Staples may be modified to include additional chemical entities such as binding components, labeling components, chemically reactive groups (e.g., functional groups or handles), or other groups (e.g., polyethylene glycol (PEG) moieties). Staples may include linear or circular nucleic acids. The staple may include regions of single-stranded nucleic acid, double-stranded nucleic acid, or a combination thereof. Staples may be configured to bind other nucleic acids by complementary base pair hybridization or ligation. The staple can be configured to act as a primer for the complementary nucleic acid strand, and the priming staple can be extended by an enzyme (e.g., a polymerase, such as a template-directed polymerase or a non-template-directed polymerase, such as a terminal transferase) to form an extension of the double-stranded nucleic acid.

The staples may be of any length, depending on the design of the retained components. Staples may be designed by a software package, such as CADNANO, ATHENA or DAEDALUS. Staples can be at least about 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more nucleotides in length. Alternatively or additionally, the staple may be no greater than about 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 25, 10 or fewer nucleotides in length.

The staples may include one or more modified nucleotides. The modified nucleotide may provide an attachment site for attaching additional components, such as a binding component or a labeling component. The modified nucleotides may be used as attachment sites for additional components before, during or after assembly of the detectable probes or affinity reagents. Staples can include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, or more modified nucleotides. Alternatively or additionally, the staple may include no more than about 100, 75, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 modified nucleotide.

Staples may be designed or modified to achieve the desired stability of the detectable probes, affinity reagents, or nucleic acid fold. Stability may be affected by dissociation of individual oligonucleotides from assembled paper breaks. The loss of an oligonucleotide may have various destabilizing effects, including loss of function of the detectable probe or affinity reagent (e.g., loss of component attachment site, loss of binding component, loss of labeling component, etc.) or destabilization of secondary or tertiary structure, thereby facilitating further destabilization of other oligonucleotides. The scaffold or staple in the nucleic acid retaining component can include one or more modified nucleotides configured to form covalent or non-covalent bonds that promote stability of the nucleic acid retaining component. For example, an oligonucleotide may include one or more modified nucleotides that form covalent bonds or cross-links with other oligonucleotides or modified nucleotides in the scaffold strand. Alternatively or additionally, the oligonucleotides may be designed to have a minimum hybridization length or a minimum melting temperature to reduce the likelihood of dissociation.

The oligonucleotides in the detectable probes or affinity reagents can hybridize to another oligonucleotide or to the scaffold strand, forming a specific number of base pairs. The oligonucleotides may form a hybridization region of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous or total base pairs. Alternatively or additionally, the oligonucleotides may form a hybridization region of no greater than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer consecutive or total base pairs.

The oligonucleotides in the detectable probes, affinity reagents, or nucleic acid break paper may have a characteristic melting temperature. Melting temperature may refer to the temperature at which the binding interaction of the nucleotide base pair is interrupted, resulting in dissociation of the oligonucleotide. The oligonucleotides in the nucleic acid retaining component may have a characteristic melting temperature of at least about 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃ or more. Alternatively or additionally, the oligonucleotides in the nucleic acid retaining component may have a characteristic melting temperature of no greater than about 90 ℃, 89 ℃, 88 ℃, 87 ℃, 86 ℃, 85 ℃, 84 ℃, 83 ℃, 82 ℃, 81 ℃, 80 ℃, 79 ℃, 78 ℃, 77 ℃, 76 ℃, 75 ℃, 74 ℃, 73 ℃, 72 ℃, 71 ℃, 70 ℃, 69 ℃, 68 ℃, 67 ℃, 66 ℃, 65 ℃, 64 ℃, 63 ℃, 62 ℃, 61 ℃, 60 ℃, 59 ℃, 58 ℃, 57 ℃, 56 ℃, 55 ℃, 54 ℃, 53 ℃, 52 ℃, 51 ℃, 50 ℃ or less.

The detectable probes or affinity reagents may include a retention component configured to localize a plurality of binding components at specific locations on the retention component. The relative positioning may be determined in part by the likelihood of positive or negative interactions between adjacent binding members. For example, some adjacent binding components (e.g., aptamers, peptide aptamers) may be susceptible to misfolding or conformational changes when in close proximity to a particular class of binding components. Such binding components may benefit from sufficient separation to minimize the likelihood of such negative interactions. In another example, when multiple binding components are in close proximity, some of the binding components may experience an increase in affinity. In some configurations, the location of a specific location of a binding component on a retention component can be determined by optimizing the balance between the interaction between the positive affinity effect and the negative affinity reagent.

The localization of the binding component to the detectable probe or affinity reagent may be determined in whole or in part by preserving the structural properties of the component. In some configurations, the retention component may include an inherently rigid, inelastic, or non-deformable material (e.g., carbon or metal nanoparticles) that is not easily deformed when used in a particular composition or method, such as in solution or on a solid support. In other configurations, the retention component may include a flexible or deformable material (e.g., polymer, nucleic acid, etc.) that is susceptible to a degree of deformation, such as stretching, compression, or bending (e.g., twisting or lateral bending). The natural deformation of the retention component may produce a conformational change that increases or decreases the relative proximity of adjacent binding components attached to the retention component.

The binding component can be positioned on the retention component sufficiently separated relative to the other binding components that the affinity reagents do not have overlapping effective occupied volumes. Fig. 3A depicts a schematic of a first binding component 310 and a second binding component 320 attached to a retention component 300. The first bonding component 310 has an effective occupied volume 315 and the second bonding component 320 has an effective occupied volume 325. The first binding component 310 and the second binding component 320 are attached to the retention component 300 at a separation distance deltas that is the minimum distance required to ensure that the effective occupied volumes 315 and 325 do not overlap.

In some configurations, the binding component can be positioned on the retention component sufficiently separated relative to the other binding components such that the binding component has a separation gap. Fig. 3B depicts a schematic view of a first binding component 310 and a second binding component 320 attached to a retention component 300 with a separation gap deltas. The first bonding component 310 has an effective occupied volume 315 and the second bonding component 320 has an effective occupied volume 325. The first binding component 310 and the second binding component 320 are attached to the retention component 300 at a separation distance deltas, which is the minimum distance required to ensure a separation gap having a length deltag for the effective occupied volumes 315 and 325.

In some configurations, the binding component may be positioned on the retention component in a spaced apart manner relative to the other binding components such that the binding components have overlapping effective footprints. Fig. 3C depicts a schematic of a first binding component 310 and a second binding component 320 attached to a retention component 300. The first bonding component 310 has an effective occupied volume 315 and the second bonding component 320 has an effective occupied volume 325. The first binding component 310 and the second binding component 320 are attached to the retention component 300 by a separation distance deltas that results in the overlapping of the effective occupied volumes 315 and 325, thereby creating an overlapping volume deltav.

The localization of the binding component to the detectable probe or affinity reagent may be determined in whole or in part by the structural characteristics of the optional linker that connects the binding component to the retention component. The linker may be used for various purposes, such as to provide separation between the retention component and the binding component, to localize the binding component, to provide attachment sites for other chemical entities, to minimize the possibility of unwanted retention component-binding component interactions, or to create desired chemical properties (e.g., hydrophobicity) between the retention component and the binding component. The linker may include a rigid or conformationally constrained chemical group (e.g., alkene, alkyne, cyclic compound). The linker may include a flexible, dynamic, or mobile chemical group (e.g., polyethylene glycol (PEG), polyethylene oxide (PEO), or an alkane chain). A linker may include a polynucleotide that is not configured to bind to other nucleic acids present in the method or apparatus in which it is present. A linker comprising a nucleic acid (e.g., RNA, DNA, PNA) can comprise a polynucleotide that forms a region of secondary structure (e.g., hairpin, stem, and/or loop structure) with itself. The linker may provide additional degrees of freedom for movement of the binding component attached to the retention component.

Fig. 4A-4D depict an exemplary method of controlling the volume that a binding component may occupy when attached to a retention component by a linker. FIG. 4A depicts respectivelySchematic representations of the first binding component 410 and the second binding component 412 attached to the substantially planar retention component surface 400 by joints 420 and 422. The first binding component 410 and the second binding component 412 have static effective occupied volumes 415 and 417, respectively (wherein the static effective occupied volume is the maximum volume occupied by the binding component without movement caused by the linker). Joints 420 and 422 provide additional degrees of freedom for movement of first bonding component 410 and second bonding component 412, resulting in dynamic effective occupied volumes 425 and 427, respectively. The first binding component 410 and the second binding component 412 are attached to the planar retention component surface 400 by the joints 420 and 422 by a distance deltas ₁ At the spaced apart locations, the distance creates a separation gap Δg between the dynamic effective occupied volumes 425 and 427 ₁ Thereby ensuring that the binding components cannot contact or interact with each other. Alternatively, fig. 4B depicts the use of an intermediate chemical moiety to disrupt the interaction between two adjacent binding components. Fig. 4B depicts a schematic of a first binding component 410 and a second binding component 412 and an intermediate chemical moiety or blocking group 430 (e.g., PEG, PEO, alkane chain, dextran) attached to a substantially planar retention component surface 400 by linkers 420 and 422, respectively. The first binding component 410, the second binding component 412, and the blocking group 430 have static effective occupied volumes 415, 417, and 435, respectively (where the static effective occupied volume is the maximum volume occupied by the binding component without movement caused by the linker). Joints 420 and 422 provide additional degrees of freedom for movement of first bonding component 410 and second bonding component 412, resulting in dynamic effective occupied volumes 425 and 427, respectively. The first binding component 410 and the second binding component 412 are attached to the planar retention component surface 400 by the distances deltas by means of the joints 420 and 422 ₂ At spaced apart locations, and due to obstruction (e.g., by steric repulsion) of the intermediate chemical moiety or blocking group 430, an effective separation gap Δg is created between the dynamic effective occupied volumes 425 and 427 ₂ . The use of intermediate chemical moieties or blocking groups 430 may reduce the necessary distance between binding components on a substantially planar surface due to the blocking of interactions between the binding componentsΔs ₂ 。

Fig. 4C depicts a schematic diagram that minimizes binding component interactions by changing the conformation of the retention component. Fig. 4C depicts a schematic view of a first binding component 410 and a second binding component 412 attached to a non-planar retention component surface 450 by joints 420 and 422, respectively. The first binding component 410 and the second binding component 412 have static effective occupied volumes 415 and 417, respectively (wherein the static effective occupied volume is the maximum volume occupied by the binding component without movement caused by the linker). Joints 420 and 422 provide additional degrees of freedom for movement of first bonding component 410 and second bonding component 412, resulting in dynamic effective occupied volumes 425 and 427, respectively. The first binding component 410 and the second binding component 412 are attached to the non-planar retaining component surface 450 by the distances deltas by the joints 420 and 422 ₃ At the spaced apart locations, the distance creates a separation gap Δg between the dynamic effective occupied volumes 425 and 427 ₃ Thereby ensuring that the binding components cannot contact or interact with each other. Alternatively, fig. 4D depicts the use of an intermediate chemical moiety to disrupt the interaction between two adjacent binding components. Fig. 4D depicts a schematic of a first binding component 410 and a second binding component 412 and an intermediate chemical moiety or blocking group 430 (e.g., PEG) attached to a non-planar retention component surface 450 by linkers 420 and 422, respectively. The first binding component 410, the second binding component 412, and the intermediate chemical moiety or blocking group 430 have static effective occupied volumes 415, 417, and 435, respectively (where the static effective occupied volumes are the largest volumes occupied by the binding components without movement caused by the linker). Joints 420 and 422 provide additional degrees of freedom for movement of first bonding component 410 and second bonding component 412, resulting in dynamic effective occupied volumes 425 and 427, respectively. The first binding component 410 and the second binding component 412 are attached to the planar retaining component surface 450 by the joints 420 and 422 by a distance deltas ₄ At the location of the separation, and due to obstruction (e.g., by spatial repulsion) of the intermediate chemical portion 430, an effective separation gap Δg is created between the dynamic effective occupied volumes 425 and 427 ₄ . Due to interactions between binding componentsThe use of intermediate chemical moieties or blocking groups 430 may reduce the necessary distance deltas between binding moieties on non-planar surfaces.

Fig. 5A and 5B illustrate alternative configurations for controlling the relative position and/or orientation of a marker component on a retention component comprising nucleic acid. Fig. 5A shows a short region of a helical nucleic acid formed by hybridization between a continuous scaffold strand 510 and shorter staple oligonucleotides (e.g., oligonucleotides 520 and 522). The oligonucleotide is occasionally interrupted by short strand break 525. Staple oligonucleotides 520 and 522 further include fluorophores 530 and 532, respectively. Due to the location of attachment of fluorophores 530 and 532 to oligonucleotides 520 and 522, fluorophores 530 and 532 are separated by a distance Δs _f1 Positioned on the same side of the retained component. The separation distance can be increased or decreased as desired by changing the attachment positions of fluorophores 530 and 532 or by changing the oligonucleotide to which the fluorophore is attached. Alternatively, fig. 5B shows a short region of a helical nucleic acid formed by hybridization between a continuous scaffold strand 510 and shorter staple oligonucleotides (e.g., oligonucleotides 520 and 522). The oligonucleotide is occasionally interrupted by short strand break 525. Staple oligonucleotides 520 and 522 further include fluorophores 530 and 532, respectively. Due to the location of attachment of fluorophores 530 and 532 to oligonucleotides 520 and 522, fluorophores 530 and 532 are separated by a distance Δs _f2 Positioned on the opposite side of the retained component. Fig. 5A and 5B illustrate how similar separation distances between marking components are achieved by different marking orientations.

The detectable probes or affinity reagents of the present disclosure may be configured to provide a highly tunable platform for displaying the binding component and/or the labeling component. The adjustability of the detectable probe or affinity reagent may be manifested as the ability to customize and/or optimize the affinity of the probe and/or the intensity of the detectable signal produced. The adjustability may result from customizable retention components that may be attached to one or more binding components and/or to one or more labeling components at specific locations on the probe.

The detectable probes or affinity reagents of the present disclosure can be characterized as having significantly increased affinity. Without wishing to be bound by theory, it is possible toThe increased affinity of the detection probe or affinity reagent may result from the presence of multiple binding components that collectively increase the binding rate of the detectable probe or affinity reagent to the binding partner (e.g., as by an increased dissociation rate constant k _on Shown), decreasing the dissociation rate of the detectable probe or affinity reagent from the binding partner (e.g., as by increasing the dissociation rate constant k _off As shown) or reduce the likelihood that the probe will diffuse away from the binding partner before the binding component can re-bind to the binding partner. Unexpectedly, it has been found that the detectable probes or affinity reagent compositions of the present disclosure can exhibit an affinity for a binding partner that is at least an order of magnitude greater than the affinity from any single binding component of the plurality of binding components attached to the probe, as characterized by a dissociation constant or the binding or dissociation rate of the binding.

The tunable nature of the detectable probe or affinity reagent may result in part from the ability to customize the attachment of the binding component to the retention component of the probe. Several factors can influence the intensity of the affinity effect, including: 1) Total number of binding components; 2) The position and/or orientation of the binding component; 3) Area density or bulk density of the binding component; 4) Affinity of the plurality of binding components; 5) The structure of the components is preserved; and 6) detecting the overall size of the probe or affinity reagent. Furthermore, the design flexibility of the described probe structures allows for the inclusion of additional components that may increase affinity, such as overhanging tails that may weakly interact with other neighboring entities to temporarily localize the detectable probe or affinity agent near the binding partner.

The affinity and/or observability of the detectable probe or affinity reagent can be tuned by exploiting the design flexibility of the retained components therein. The retention component may be selected if it provides: 1) Providing a three-dimensional structure for a wide range of potential positions and orientations for presentation of the binding components; and 2) the ability to attach the binding component to the three-dimensional structure at the desired location with high specificity. Of particular interest are retention components comprising nucleic acids that exploit the specificity of nucleic acid hybridization to create complex three-dimensional structures with precisely located binding sites for binding components (and labeling components).

In some configurations, a nucleic acid structure (e.g., a DNA nanosphere or a nucleic acid fold) may be utilized as a retention component. Nucleic acid structures can have the advantage of providing a high degree of spatial control over the position and orientation of components added to the retained components. The nucleic acid structure may typically have about 10 to 11 base pairs per turn of the helix structure, meaning that each unique physical position within the double-stranded nucleic acid has an associated orientation angle. This property of nucleic acids facilitates adjustability of the structure of the retention component based on nucleic acids by providing rich positional and orientation changes to tailor the amount of separation between probe components, such as binding components and/or labeling components.

In other configurations, the retention component may have controlled spatial and/or orientation control of the probe component by rational control or modification of the retention component structure and/or chemistry. The non-nucleic acid retaining component can be manipulated or modified to produce a retaining component having a controlled or varying probe component position and/or orientation. In some cases, the position or orientation of the probe component can be controlled by the shape or conformation of the non-nucleic acid particle, nanoparticle, or host. For example, the shell-like or plate-like structure may provide a plurality of surfaces with varying properties that allow for different positions of the binding component relative to the labeling component. Furthermore, the non-nucleic acid retaining components may be provided with a coating or formed as a complex in a spatially controlled manner, allowing for increased control of attachment location and orientation.

Fig. 6A-6F depict various simplified configurations of detectable probe compositions to demonstrate the flexibility of arrangement resulting from three-dimensional retention component structures (e.g., DNA origami, carbon nanoparticles, silicon nanoparticles, etc.). Fig. 6A depicts a top view of a rectangular or tile-shaped detectable probe. The probe contains a retention component 610 having a width and height and a depth (not shown) forming a top surface (not shown) plus side and bottom surfaces (not shown). A plurality of binding members 620 are attached to the retention member 610 at specific locations along the sides of the retention member. The labeling component 630 is located at a specific location on the top (and optionally bottom) surface of the detectable probe. Fig. 6B depicts a top view of a rectangular or tile-shaped probe with an attachment scheme that is inverted from the probe shown in fig. 6A. The probe contains a retention component 610 having a width and height and a depth (not shown) forming a top surface (not shown) plus side and bottom surfaces (not shown). A plurality of binding members 620 are attached to the retention members 610 at specific locations on the top (and optionally bottom) surface of the detectable probes. The marking component 630 is located at a specific location along the sides of the retaining component. The probe configuration depicted in fig. 6A may be preferred for systems requiring strong detection signals or detection signals distributed over a larger area or volume, as the locations for the labels on the top surface of the large area increase. The probe configuration depicted in fig. 6B may be preferred for increasing the probe affinity of the detectable probes or affinity reagents, as the density of binding components on the top surface of a large area may be increased.

Fig. 6C and 6D depict side view configurations of rectangular or tile-shaped detectable probes, demonstrating the orientation of the probe components relative to the thinner depth dimension of the probes. Fig. 6C shows a detectable probe containing a retention component 610 having a width and height (not shown) and a depth, forming sides and top and bottom surfaces (not shown). A plurality of binding members 620 are attached to the retention members 610, the binding members being attached at specific locations on the top surface of the detectable probes. The labeling component 630 is located at a specific location on the bottom surface of the detectable probe. Fig. 6D shows a detectable probe containing a retention component 610 having a width and height (not shown) and a depth, forming sides and top and bottom surfaces (not shown). A plurality of binding components 620 and labeling components 630 are attached to the retention components 610, the binding components being attached at specific locations on the top and bottom surfaces of the detectable probes. The probe configuration in fig. 6C may be advantageous for fluorescent detection systems because large probes may shield some or all of the binding partners from interacting with the label component, potentially reducing or alleviating any quenching of the label by the binding partners or their immediate environment. The probe configuration in fig. 6D can be advantageous to maximize the amount of binding component present for the detectable probe or affinity reagent by increasing the likelihood of the detectable probe coming into contact with the binding partner.

Fig. 6E and 6F depict alternative retained component geometries. Fig. 6E depicts a detectable probe comprising a circular or spherical configuration. The probe contains a retention component 610, which optionally includes internal structures that provide additional locations for attachment of probe components. A plurality of binding components 620 are attached to the outer perimeter or surface of the retention component 610. The interior region of the retention component 610 contains a plurality of marker components 630. The configuration shown in fig. 6E may increase the likelihood of probes localizing binding partners due to the high coverage of the binding component 620 on the retention component 610, and may further provide a highly detectable signal due to the possibility of concentrating the plurality of label components 630 in the interior space of the retention component 610. Fig. 6F depicts a side or cross-sectional view of a probe having a retention component 610 featuring an angular offset on one or more faces. One side of the retention component 610 contains an attached binding component 620. The opposite side of the retention component 610 contains an attached marker component 630. The configuration shown in fig. 6F may be advantageous for increasing the affinity because the bulk density of the binding component 620 attached to the bottom surface of the retention component 610 increases. More complex shapes may increase contact of the binding component 620 with the binding partner and increase resistance of the probe to diffusion away from the binding partner. Those skilled in the art will recognize that there may be countless variations to the geometries depicted in figures 6A-6F, taking into account many possible designs of retained components.

The retention component may include a body (e.g., a particle, nanoparticle, or microparticle) that is not primarily composed of nucleic acid. Can be used as a host for preparation or synthesis such as silicon or silica nanoparticles, carbon nanoparticles, cellulose nanoparticles, PEG nanoparticles, polymer nanoparticles (e.g., polyacrylate particles, polystyrene-based particles, fluoSpheres) ^TM Etc.) or a reserved component of the quantum dot to form a reserved component. The particles, nanoparticles, or bodies may include solid materials and shell-like materials (e.g., carbon nanospheres, silica nanoshells, iron oxide nanospheres, polymethyl methacrylate nanospheres, etc.). The retention component comprising particles, nanoparticles or bodies may comprise different surfaces, such as platesOr a shell. In some configurations, different surfaces on the retained component may be utilized to isolate the component (e.g., binding component on the first surface, labeling component on the second surface). The retention component may include a material that may be directly functionalized or modified to allow for attachment of the component (e.g., silanization of silicon or silica nanoparticles). The retention component may comprise a commercially available particle, nanoparticle, or host. The retained component may be prepared by modification of commercially available particles, nanoparticles or hosts. Methods and chemicals for modifying structures such as particles and nanoparticles are widely described in the art.

Fig. 31A-31D illustrate various configurations for forming a retained component from particles or nanoparticles. Fig. 31A depicts solid spherical particles 3110 having a surface directly functionalized with a plurality of attachment sites 3120. The attachment site may be configured to form a covalent or non-covalent attachment with a detectable probe component, such as a binding component and/or a labeling component. Fig. 31B depicts solid spherical particles 3110 comprising heterogeneous first and second pluralities of attachment sites 3120, 3125. In some configurations, there may be an equal number of first attachment sites 3120 and second attachment sites 3125. In other configurations, there may be a different number of first attachment sites 3120 and second attachment sites 3125. The first attachment site 3120 and/or the second attachment site 3125 can be configured to form a covalent or non-covalent attachment with a detectable probe component, such as a binding component and/or a labeling component. The solid spherical particles of fig. 31A and 31B can be easily replaced with hollow particles. The hollow particles may be provided with attachment sites 3120 or 3125 on the inner surface. In some configurations, the hollow particles may include a first plurality of attachment sites 3120 on the outer surface and a second plurality of attachment sites 3125 on the inner surface to allow for isolation of the detectable probe components onto different surfaces.

Fig. 31C and 31D illustrate a retained component formed from solid spherical particles comprising a surface coating, shell, or layer (e.g., a polymer, hydrogel coating, or monolayer functionality). Fig. 31C shows solid spherical particles 3110 including a surface coating or layer 3130. The coating or layer 3130 is provided with a plurality of attachment sites 3120. The attachment site may be fittedIs placed in covalent or non-covalent attachment with a detectable probe component, such as a binding component and/or a labeling component. Fig. 31D shows spherical detectable particles 3115 (e.g., fluoSphere) including a surface coating 3130 ^TM Quantum dots). The coating or layer 3130 is provided with a plurality of attachment sites 3120. The attachment site may be configured to form a covalent or non-covalent attachment with a detectable probe component, such as a binding component and/or a labeling component. The coating or layer may act as a scaffold allowing binding or other components to attach to the particle, nanoparticle or host. The skilled artisan will readily recognize that the described retention components can be readily adapted to non-spherical particles, nanoparticles, or bodies, such as nanotubes, plates, bowls, rods, and cones.

FIGS. 34A-34C illustrate various configurations of non-nucleic acid retaining components with spatially controlled or isolated probe components. Fig. 34A shows a spherical particle, nanoparticle, or body 3410 having a spatially separated binding component 3420 and a labeling component 3430. Fig. 34B shows a hollow or shell-like particle or body 3412 that utilizes the hollow interior region of the particle or body 3412 to isolate the marker component 3430 from the externally attached binding component 3420. Fig. 34C shows a plate-like particle or body 3414 having two different surfaces with an angular offset of about 180 °. The upper surface may be used to attach a plurality of marker components 3430 and the bottom surface may be used to attach a plurality of binding components 3420.

The two binding components may be attached to the retention component in such a way that they have an angular offset in terms of their relative positions. For example, two binding components attached to the planar face of the retention component will have an angular offset of about 0 degrees (°). In another example, two binding components attached to opposite sides of a cube-like retention component will have an angular offset of about 180 °. Angular offset may be utilized to limit contact or other interactions between adjacent binding components. The two binding components can have a relative angular offset of at least about 0 °, 5 °, 10 °, 15 °, 20 °, 25 °, 30 °, 35 °, 40 °, 45 °, 50 °, 55 °, 60 °, 65 °, 70 °, 75 °, 80 °, 85 °, 90 °, 95 °, 100 °, 105 °, 110 °, 115 °, 120 °, 125 °, 130 °, 135 °, 140 °, 145 °, 150 °, 155 °, 160 °, 165 °, 170 °, 175 °, or more. Alternatively or additionally, the two binding components may have a relative angular offset of no more than about 180 °, 175 °, 170 °, 165 °, 160 °, 155 °, 150 °, 145 °, 140 °, 135 °, 130 °, 125 °, 120 °, 115 °, 110 °, 105 °, 100 °, 95 °, 90 °, 85 °, 80 °, 75 °, 70 °, 65 °, 60 °, 55 °, 50 °, 45 °, 40 °, 35 °, 30 °, 25 °, 20 °, 15 °, 10 °, 5 °, or less.

The detectable probe or affinity reagent may be further modified to increase the overall affinity of the probe. Figures 7A and 7B depict the use of linkers to increase the spatial freedom of exhibiting binding components. Fig. 7A depicts a detectable probe comprising a retention component 710 having a plurality of attached binding components 720 and a plurality of labeling components 730. A subset of the plurality of binding members 720 are attached to the retention member 710 by a linker 740 (e.g., PEG, PEO, alkane chain, etc.) that allows the subset of binding members 720 to extend away from the retention member 710. Fig. 7B depicts a probe configuration similar to that of fig. 7A, however the probe includes two different kinds of binding components. The probe contains a retention component 710 having a plurality of attached labeling components 730. The probe further includes a first plurality of attached binding components 720 (e.g., antibodies or antibody fragments) and a second plurality of attached binding components 722 (e.g., aptamers) attached to the retention components 710 via linkers 740. The linker may facilitate increasing probe affinity by facilitating increased sensing of the binding partner, epitope or target moiety over a larger volume per unit time. In addition, linkers can provide flexibility or separation of components, which can accelerate binding of the probe to a binding partner, epitope, or target moiety, and slow diffusion of the probe possibly away from the binding partner, epitope, or target moiety.

The size of the detectable probe or affinity reagent may be configured to suit the intended mode of use. The mode of use (e.g., polypeptide characterization, non-polypeptide characterization, therapeutics, diagnostics) can indicate the level of avidity and/or observability of the detectable probe or affinity reagent. The size of the detectable probes or affinity reagents can be sufficiently adjusted to allow for the attachment of a sufficient number of binding and/or labeling components for the intended mode of use. In some configurations, the size of the detectable probe or affinity reagent may refer to the approximate length, area, or volume of the retained component. Components attached to the retention component (e.g., binding component, labeling component, linker, blocking group) may have increased spatial freedom, which makes it more difficult to characterize their length, area size, or volume size. The retention component may be configured to be more regular in size or to vary less in size, such that the size of the retention component is a viable representation of the size of the detectable probe or affinity reagent.

The detectable probe, affinity reagent, or a retained component thereof may have a characteristic length. For width, height, radius, diameter, circumference, or other dimensions, the characteristic length may include a maximum, average, or minimum length. The detectable probe, affinity reagent, or a retained component thereof may have a characteristic length of at least about 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, or more. Alternatively or additionally, the detectable probe, affinity reagent, or a retained component thereof may have a characteristic length of no greater than about 1000nm, 900nm, 800nm, 700nm, 600nm, 500nm, 450nm, 400nm, 350nm, 300nm, 250nm, 200nm, 180nm, 160nm, 140nm, 120nm, 100nm, 95nm, 90nm, 85nm, 80nm, 75nm, 70nm, 65nm, 60nm, 55nm, 50nm, 45nm, 40nm, 35nm, 30nm, 25nm, 20nm, 15nm, 10nm, 5nm, or less.

The detectable probes, affinity reagents, or retained components thereof may have a characteristic footprint (e.g., an occupied area on a surface). The blot may constitute the area where a two-dimensional projection of the detectable probe, affinity reagent or a retained component thereof will be produced on a planar surface. The two-dimensional projection may have a regular shape or an approximately regular shape, such as a triangle, square, rectangle, circle, pentagon, hexagon, octagon, or oval. Fig. 2A-2B show examples of 2-dimensional projections of the retained components 210 or 215 having approximate shapes, where the ideal shapes are shown as dashed lines (220 and 215, respectively225). The detectable probe, affinity reagent, or a retained component thereof may have a particle size of at least about 25nm ² 、100nm ² 、500nm ² 、1000nm ² 、2000nm ² 、3000nm ² 、4000nm ² 、5000nm ² 、5500nm ² 、6000nm ² 、6500nm ² 、7000nm ² 、7500nm ² 、8000nm ² 、8500nm ² 、9000nm ² 、10000nm ² 、15000nm ² 、20000nm ² 、25000nm ² 、50000nm ² 、100000nm ² 、1000000nm ² Or a larger footprint. Alternatively or additionally, the detectable probe, affinity reagent, or retained component thereof may have a wavelength of no greater than about 1000000nm ² 、100000nm ² 、50000nm ² 、25000nm ² 、20000nm ² 、15000nm ² 、10000nm ² 、9000nm ² 、8500nm ² 、8000nm ² 、7500nm ² 、7000nm ² 、6500nm ² 、6000nm ² 、5500nm ² 、5000nm ² 、4000nm ² 、3000nm ² 、2000nm ² 、1000nm ² 、500nm ² 、100nm ² 、25nm ² Or a smaller footprint. The above-mentioned ranges of footprints may refer to all sides of a detectable probe, affinity reagent, or a retained component thereof, as desired; a minimal surface of the detectable probe, affinity reagent, or a retained component thereof; or the average of the largest faces of the detectable probes, affinity reagents, or retained components thereof.

The retention component may be configured with other components (e.g., binding components, labeling components, or other retention components) attached at a desired or optimal spacing. The spacing of the binding components can be based on a minimum spacing to reduce or eliminate unwanted or adverse interactions (e.g., aptamer misfolding; fluorophore self-quenching). The spacing of the binding components may be based on the maximum spacing to obtain a desired characteristic, such as affinity or detectable signal strength. For components that are connected to a retained component (i.e., components having additional degrees of freedom of movement) by a linker or other flexible attachment method, the spacing of the attached components may be measured as the spacing between attachment sites on the retained component. Two adjacent attachment components (e.g., binding component, labeling component, blocking group) can have a feature spacing of at least about 0.1nm, 0.2nm, 0.3nm, 0.4nm, 0.5nm, 0.6nm, 0.7nm, 0.8nm, 0.9nm, 1.0nm, 1.1nm, 1.2nm, 1.3nm, 1.4nm, 1.5nm, 1.6nm, 1.7nm, 1.8nm, 1.9nm, 2nm, 3nm, 4nm, 5nm, 6nm, 7nm, 8nm, 9nm, 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm, 20nm, 21nm, 22nm, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, 29nm, 30nm, 35nm, 40nm or greater. Alternatively or additionally, two adjacent attachment components may have a characteristic spacing of no greater than about 40nm, 35nm, 30nm, 29nm, 28nm, 27nm, 26nm, 25nm, 24nm, 23nm, 22nm, 21nm, 20nm, 19nm, 18nm, 17nm, 16nm, 15nm, 14nm, 13nm, 12nm, 11nm, 10nm, 9nm, 8nm, 7nm, 6nm, 5nm, 4nm, 3nm, 2nm, 1.9nm, 1.8nm, 1.7nm, 1.6nm, 1.5nm, 1.4nm, 1.3nm, 1.2nm, 1.1nm, 1.0nm, 0.9nm, 0.8nm, 0.7nm, 0.6nm, 0.5nm, 0.4nm, 0.3nm, 0.2nm, 0.1nm or less.

The detectable probes or affinity reagents may comprise a plurality of binding members attached to a surface or face of the retention member. The number of binding members displayed on the surface of the detectable probe or affinity reagent can be configured to increase the affinity or sufficiently space the binding members to avoid unwanted interactions. The number of binding components on the surface of the detectable probe or affinity reagent can be characterized as the average number density (number per surface) or the areal density (number per unit area). The two different surfaces or faces of the detectable probe or affinity reagent may have different number densities or areal densities of binding components. The average binding component density of the detectable probe or affinity reagent can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more per surface. Alternatively or additionally, the average binding component density of the detectable probes or affinity reagents may be no greater than about 50, 49, 48, 4746, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The average binding member surface density of the detectable probe or affinity reagent may be at least about 0.00001, 0.0001, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1/nm ² Or larger. Alternatively or additionally, the average binding member areal density of the detectable probes or affinity reagents may be no greater than about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001, 0.0001, 0.00001/nm ² Or smaller.

The detectable probe or affinity reagent may comprise a plurality of label components attached to the surface or face of the retention component. The number of label components displayed on the surface of the detectable probe or affinity reagent can be optimized to increase observability or to sufficiently space the label components to avoid unwanted interactions. The number of label components on the surface of the detectable probe or affinity reagent can be characterized as the average number density (number per surface) or the areal density (number per unit area). The two different surfaces or faces of the detectable probe or affinity reagent may have different number densities or areal densities of the labeling components. The average labeling element number density of the detectable probe or affinity reagent can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more per surface. Alternatively or additionally, the average labeling component density of the detectable probe or affinity reagent may be no greater than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27. 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2. The average areal density of the labeling elements of the detectable probes or affinity reagents can be at least about 0.00001, 0.0001, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1/nm ² Or larger. Alternatively or additionally, the average areal density of the labeling elements of the detectable probes or affinity reagents can be no greater than about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001, 0.0001, 0.00001/nm ² Or smaller.

The detectable probe or affinity reagent may comprise a heterogeneous mixture of binding components. The heterogeneous mixture of binding components may include a mixture of different types and/or kinds of binding components. For example, the detectable probe or affinity reagent may comprise a mixture of antibodies having different affinities for the binding partner. In another example, the detectable probe or affinity reagent may comprise a mixture of antibodies and aptamers having affinity for the same binding partner. In another example, the detectable probe or affinity reagent may comprise a mixture of antibodies and antibody fragments having affinity for the same binding partner. Heterogeneous mixtures of binding components may be advantageous for controlling the affinity of the detectable probes or affinity reagents. The mixture of binding components having different binding affinities may facilitate optimizing the binding rate and/or dissociation rate of the binding of the detectable probe or affinity agent relative to the binding of the particular binding partner, epitope or target moiety.

In some configurations, the detectable probe or affinity reagent can include a plurality of first binding components having affinity for a binding partner, epitope, or target moiety and a second plurality of competing binding components. Competing binding components can be characterized as having a reduced affinity (e.g., having increased binding promiscuity, e.g., binding to multiple different binding partners, epitopes, or target moieties) and an increased rate of dissociation (e.g., binding components that bind to but readily dissociate from a number of targets). Without wishing to be bound by theory, competing binding components may be identified as binding components whose displacement by another binding component is beneficial in terms of energy and/or entropy. For example, a plurality of small low affinity aptamers or minipeptide conjugates can be easily replaced with large high affinity antibodies, thereby facilitating antibody binding due to increased entropy of replacement of multiple binding components. The competing binding members may further increase the affinity of the detectable probe or affinity reagent by forming a short weak interaction with the target moiety to which the first plurality of binding members lack affinity. Such short weak interactions may promote an increase in the duration of association between the detectable probe or affinity agent and the binding partner, epitope or target moiety.

The detectable probe or affinity reagent may be structurally stable under certain environmental conditions. Of particular interest are detectable probes or affinity reagents that are structurally stable under conditions intended to move the bound probe away from the binding partner. For example, the detectable probe or affinity reagent may be stable in the presence of heat or chemical compositions (e.g., surfactants or denaturants) that interfere with intermolecular interactions. Structural stability may refer to the composition of the detectable probe or affinity agent that retains its integrity and optionally its shape or conformation, i.e., without loss of binding, labeling, or other components; there is no loss or degradation of the components (e.g., the nucleic acid from the DNA fold retains dehybridization of the components).

The detectable probe or affinity reagent may be structurally stable at a given temperature. The temperature may be changed during the process of using the detectable probe or the affinity reagent. For example, each step of binding, observing and removing the detectable probe or affinity reagent may be performed at a unique and/or optimal temperature. Furthermore, the detectable probe or affinity reagent may be structurally stable at a given storage temperature. The detectable probe or affinity reagent may be structurally stable at a temperature of at least about-100 ℃, -90 ℃, -80 ℃, -70 ℃, -60 ℃, -50 ℃, -40 ℃, -30 ℃, -20 ℃, -10 ℃, -5 ℃, 0 ℃, 4 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 90 ℃ or more. Alternatively or additionally, the detectable probe or affinity reagent may be structurally stable at temperatures no greater than about 90 ℃, 80 ℃, 75 ℃, 70 ℃, 65 ℃, 60 ℃, 55 ℃, 50 ℃, 45 ℃, 40 ℃, 35 ℃, 30 ℃, 25 ℃, 20 ℃, 15 ℃, 10 ℃, 4 ℃, 0 ℃, -10 ℃, -20 ℃, -30 ℃, -40 ℃, -50 ℃, -60 ℃, -70 ℃, -80 ℃, -90 ℃, 100 ℃ or less.

The detectable probe or affinity reagent may be structurally stable in the presence of a particular solution or solvent. The detectable probe or affinity reagent may be contacted with one or more solutions or solvents, depending on the mode of use of the detectable probe or affinity reagent. The solution or solvent may have different compositions depending on the mode of use of the detectable probe or affinity reagent. Solutions or solvents may be used in processes such as probe formulation, probe storage, probe-partner binding, washing, rinsing, interaction detection, probe-partner separation, probe capture (e.g., with post-recovery probes), and probe analysis (e.g., post-processing sequencing of nucleic acid barcodes). The solution or solvent may be formulated to maintain stability of the detectable probe or affinity reagent structure. Solutions or solvents may be formulated according to chemical composition, pH and ionic strength to ensure stability of the detectable probe or affinity reagent. A detectable probe or affinity reagent comprising a nucleic acid may be present in a solution or solvent comprising a magnesium salt to stabilize the nucleic acid.

The solution or solvent in contact with the detectable probe or affinity reagent may comprise one or more detectable probes or affinity reagents in solution or suspension. The solution or solvent in contact with the detectable probe or affinity reagent may be formulated as a homogeneous liquid medium. The solution or solvent in contact with the detectable probe or affinity reagent may be formulated as a single phase liquid medium. The solution or solvent in contact with the detectable probe or affinity reagent may be formulated as a multi-phase liquid medium, such as an oil-in-water emulsion or a water-in-oil emulsion.

With detectable probes or affinitiesThe solution or solvent in contact with the reagent may include a solvent material, a pH buffering material, a cationic material, an anionic material, a surfactant material, a denaturing material, or a combination thereof. The solvent material may include water, acetic acid, methanol, ethanol, n-propanol, isopropanol, n-butanol, formic acid, ammonia, propylene carbonate, nitromethane, dimethyl sulfoxide, acetonitrile, dimethylformamide, acetone, ethyl acetate, tetrahydrofuran, dichloromethane, chloroform, carbon tetrachloride, dimethyl ether, diethyl ether, 1, 4-dioxane, toluene, benzene, cyclohexane, hexane, cyclopentane, pentane, or combinations thereof. Solvents or solutions may include buffer substances including, but not limited to, MES, tris, bis-tris, bis-tris propane, ADA, ACES, PIPES, MOPSO, MOPS, BES, TES, HEPES, HEPBS, HEPPSO, DIPSO, MOBS, TAPSO, TAPS, TABS, POPSO, TEA, EPPS, trimethylglycine (Tricine), gly-Gly, dihydroxyethyl glycine (Bicine), AMPD, AMPSO, AMP, CHES, CAPSO, CAPS, and CABS. The solvent or solution may include a cationic species, such as Na ⁺ 、K ⁺ 、Ag ⁺ 、Cu ⁺ 、NH ₄ ⁺ 、Mg ²⁺ 、Ca ²⁺ 、Cu ²⁺ 、Cd ²⁺ 、Zn ²⁺ 、Fe ²⁺ 、Co ²⁺ 、Ni ²⁺ 、Cr ²⁺ 、Mn ²⁺ 、Ge ²⁺ 、Sn ²⁺ 、Al ³⁺ 、Cr ³⁺ 、Fe ³⁺ 、Co ³⁺ 、Ni ³⁺ 、Ti ³⁺ 、Mn ³⁺ 、Si ⁴⁺ 、V ⁴⁺ 、Ti ⁴⁺ 、Mn ⁴⁺ 、Ge ⁴⁺ 、Se ⁴⁺ 、V ⁵⁺ 、Mn ⁵⁺ 、Mn ⁶⁺ 、Se ⁶⁺ And combinations thereof. The solvent or solution may include anionic species such as F ^- 、Cl ^- 、Br ^- 、ClO ₃ ^- 、H ₂ PO ₄ ^- 、HCO ₃ ^- 、HSO ₄ ^- 、OH ^- 、I ^- 、NO ₃ ^- 、NO ₂ ^- 、MnO ₄ ^- 、SCN ^- 、CO ₃ ^2- 、CrO ₄ ^2- 、Cr ₂ O ₇ ^2- 、HPO ₄ ^2- 、SO ₄ ^2- 、SO ₃ ^2- 、PO ₄ ^3- And combinations thereof. Solvents or solutions may include surfactant materials including, but not limited to, stearic acid, lauric acid, oleic acid, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, dodecylamine hydrochloride, cetyltrimethylammonium bromide, polyethylene oxide, nonylphenyl ethoxylate, triton X, pentapropylene glycol monolodecyl ether, octapropylene glycol monolodecyl ether, pentaethylene glycol monolodecyl ether, octaethylene glycol monolodecyl ether, lauramide monoethyl amine, lauramide diethylamine, octylglucoside, decyl glucoside, lauryl glucoside, tween 20, tween 80, N-dodecyl-beta-D-maltoside, nonylphenol ether 9, glycerol monolaurate, polyethoxylated tallow amine, poloxamer, digitonin, zonyl FSO, 2, 5-dimethyl-3-hexyne-2, 5-diol, igepal CA630, aerosol-OT, triethylene amine hydrochloride, cetrimide, benzethonium chloride, tenidine dihydrochloride, cetylpyridinium chloride, methyltriaben ammonium chloride (aden), dioctadecyl ammonium chloride, octodecyl ammonium chloride, cocoyl chloride, 16-amidobetaine, 16-methyl-sodium lauryl chloride, amidopropyl chloride, 16-amidobetaine, N- (dimethylammonium) butyrate, lauryl-N, N- (dimethyl) -glycine betaine, cetyl phosphorylcholine, lauryldimethylamine N-oxide, lauryl-N, N- (dimethyl) -propane sulfonate, 3- (1-pyrazinyl) -1-propane sulfonate, 3- (4-tert-butyl-1-pyrazinyl) -1-propane sulfonate, N-lauryl sarcosine and combinations thereof. Solvents or solutions may include denatured substances including, but not limited to, acetic acid, trichloroacetic acid, sulfosalicylic acid, sodium bicarbonate, ethanol, ethylenediamine tetraacetic acid (EDTA), urea, guanidine chloride, lithium perchlorate, sodium lauryl sulfate, 2-mercaptoethanol, dithiothreitol, and tris (2-carboxyethyl) phosphine (TCEP).

The pH buffering substance may be formulated in any amount in a solvent or solution. The pH buffering substance can be in the form of at least about 0.0001M, 0.001M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 1.7M, 1.8M, 1.7M, 0.8M concentrations of 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6M, 7M, 8M, 9M or more are present in the detectable probe or the affinity reagent solvent composition. Alternatively or in addition to this, the pH buffering substance can be no greater than about 10M, 9M, 8M, 7M, 6M, 5.9M, 5.8M, 5.7M, 5.6M, 5.5M, 5.4M, 5.3M, 5.2M, 5.1M, 5.0M, 4.9M, 4.8M, 4.7M, 4.6M, 4.5M, 4.4M, 4.3M, 4.2M, 4.1M, 4.0M, 3.9M, 3.8M, 3.7M, 3.6M, 3.5M, 3.4M, 3.3M, 3.2M, 3.1M, 3.0M, 2.9M, 2.8M, 2.7M, 2.6M, 2.5M a concentration of 2.4M, 2.3M, 2.2M, 2.1M, 2.0M, 1.9M, 1.8M, 1.7M, 1.6M, 1.5M, 1.4M, 1.3M, 1.2M, 1.1M, 1.0M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, 0.001M or less is present in the solvent or solution.

The cationic species may be formulated in any amount in a solvent or solution. The cationic species can be present in an amount of at least about 0.0001M, 0.001M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 1.7M concentrations of 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6M, 7M, 8M, 9M or more are present in the detectable probe or the affinity reagent solvent composition. Alternatively or in addition to this, the cationic species may be in a range of no greater than about 10M, 9M, 8M, 7M, 6M, 5.9M, 5.8M, 5.7M, 5.6M, 5.5M, 5.4M, 5.3M, 5.2M, 5.1M, 5.0M, 4.9M, 4.8M, 4.7M, 4.6M, 4.5M, 4.4M, 4.3M, 4.2M, 4.1M, 4.0M, 3.9M, 3.8M, 3.7M, 3.6M, 3.5M, 3.4M, 3.3M, 3.2M, 3.1M, 3.0M, 2.9M, 2.8M, 2.7M, 2.6M, 2.1M, 4.1M, 4.2.1M, 3.5M, 3.2.8M a concentration of 2.5M, 2.4M, 2.3M, 2.2M, 2.1M, 2.0M, 1.9M, 1.8M, 1.7M, 1.6M, 1.5M, 1.4M, 1.3M, 1.2M, 1.1M, 1.0M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, 0.001M or less is present in the solvent or solution.

The anionic species may be formulated in any amount in a solvent or solution. The anionic species may be present in an amount of at least about 0.0001M, 0.001M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M concentrations of 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6M, 7M, 8M, 9M or higher are present in the solvent or solution. Alternatively or in addition to this, the anionic species may be present in an amount of no greater than about 10M, 9M, 8M, 7M, 6M, 5.9M, 5.8M, 5.7M, 5.6M, 5.5M, 5.4M, 5.3M, 5.2M, 5.1M, 5.0M, 4.9M, 4.8M, 4.7M, 4.6M, 4.5M, 4.4M, 4.3M, 4.2M, 4.1M, 4.0M, 3.9M, 3.8M, 3.7M, 3.6M, 3.5M, 3.4M, 3.3M, 3.2M, 3.1M, 3.0M, 2.9M, 2.8M, 2.7M, 2.6M, 2.1M, 4.1M, 4.2.1M, 3.8M a concentration of 2.5M, 2.4M, 2.3M, 2.2M, 2.1M, 2.0M, 1.9M, 1.8M, 1.7M, 1.6M, 1.5M, 1.4M, 1.3M, 1.2M, 1.1M, 1.0M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, 0.001M or less is present in the solvent or solution.

The surfactant material may be formulated in any amount in a solvent or solution. The surfactant material can be in an amount of at least about 0.0001M, 0.001M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M concentrations of 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6M, 7M, 8M, 9M or higher are present in the solvent or solution. Alternatively or in addition to this, the surfactant material may be in a range of no greater than about 10M, 9M, 8M, 7M, 6M, 5.9M, 5.8M, 5.7M, 5.6M, 5.5M, 5.4M, 5.3M, 5.2M, 5.1M, 5.0M, 4.9M, 4.8M, 4.7M, 4.6M, 4.5M, 4.4M, 4.3M, 4.2M, 4.1M, 4.0M, 3.9M, 3.8M, 3.7M, 3.6M, 3.5M, 3.4M, 3.3M, 3.2M, 3.1M, 3.0M, 2.9M, 2.8M, 2.7M, 2.6M, 2.3.1M, 4.1M, 3.5M, 3.6M a concentration of 2.5M, 2.4M, 2.3M, 2.2M, 2.1M, 2.0M, 1.9M, 1.8M, 1.7M, 1.6M, 1.5M, 1.4M, 1.3M, 1.2M, 1.1M, 1.0M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, 0.001M or less is present in the solvent or solution.

The denaturing substances may be formulated in any suitable amount in a solvent or solution. The denaturing substances may be present in an amount of at least about 0.0001M, 0.001M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M concentrations of 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6M, 7M, 8M, 9M or higher are present in the solvent or solution. The denatured material may be in a range of no greater than about 10M, 9M, 8M, 7M, 6M, 5.9M, 5.8M, 5.7M, 5.6M, 5.5M, 5.4M, 5.3M, 5.2M, 5.1M, 5.0M, 4.9M, 4.8M, 4.7M, 4.6M, 4.5M, 4.4M, 4.3M, 4.2M, 4.1M, 4.0M, 3.9M, 3.8M, 3.7M, 3.6M, 3.5M, 3.4M, 3.3M, 3.2M, 3.1M, 3.0M, 2.9M, 2.8M, 2.7M, 2.6M, 2.5M a concentration of 2.4M, 2.3M, 2.2M, 2.1M, 2.0M, 1.9M, 1.8M, 1.7M, 1.6M, 1.5M, 1.4M, 1.3M, 1.2M, 1.1M, 1.0M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, 0.001M or less is present in the solvent or solution.

The solvent or solution may be formulated to have a pH of a certain value or within a certain range of values. The solvent or solution may have at least about 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 1.3, 2.4, 2.6 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.6, 12.7, 13.3, 13.0, 13.13.0, 13.3, 13.0, 13.4 or 13.0. Alternatively or in addition to this, the solvent or solution may have a concentration of no greater than about 14.0, 13.9, 13.8, 13.7, 13.6, 13.5, 13.4, 13.3, 13.2, 13.1, 13.0, 12.9, 12.8, 12.7, 12.6, 12.5, 12.4, 12.3, 12.2, 12.1, 12.0, 11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11.0, 10.9, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, 10.0, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8.8, 8.7, 8.6, 8.5, 8.4.3, 8.7, 7.1, 8.7.7, 8.7, 8.5, 7.1, 7.7, 8.2, 7.1. 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0 or lower pH.

The detectable probe or affinity reagent may comprise one or more additional modifying groups. Modification groups may be included to alter the chemical or physical properties of the detectable probes or affinity reagents. The modifying group may be included on the detectable probe or affinity reagent to alter a property such as hydrophobicity, hydrophilicity, amphiphilicity, charge, susceptibility, or any other physical property. In some cases, the modifying group may be used to increase, decrease, or alter the solubility or solution stability of the detectable probe or affinity reagent. In some cases, the modifying group may be used to maintain separation of the detectable probes or affinity reagents by mechanisms such as electrical repulsion or steric obstruction. In some cases, modifying groups may be used to increase the attractive force between particles. For example, the modifying groups may form a short attractive interaction between the detectable probes and/or the affinity reagents to create a multiprobe complex without causing probe aggregation or deposition.

The surface of the retention component may include one or more modifying groups. Modification groups may be added to the surface to alter the characteristics of the surface while mediating association between the detectable probe or affinity reagent and the surface or interface. For example, a hydrophobic modification group may be added to the detectable probe or affinity reagent to allow the probe to interact with the oil droplets in the oil-in-water emulsion. The modifying groups may be attached covalently or non-covalently. The modifying group may be coupled to the retention component before, during, or after assembly of the retention component. The surface modifying groups may include charged moieties, magnetic moieties, steric moieties, amphiphilic moieties, hydrophobic moieties, and hydrophilic moieties. The charged moiety may include a functional group (e.g., carboxylic acid, nitrate, sulfone, phosphate, phosphonate, etc.) that may carry an inherent positive or negative charge or may carry a charge under dissociative conditions. The magnetic portion may include paramagnetic, diamagnetic, and ferromagnetic particles, such as nanoparticles (e.g., gadolinium, manganese, iron oxide, bismuth, gold, silver, cobalt nanoparticles, etc.). The steric moiety may include polymers and biopolymers (e.g., PEG, PEO, alkane chains, dextran, sheared nucleic acids). The amphiphilic moiety can include phospholipids (e.g., phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol phosphate, phosphatidylinositol diphosphate, phosphatidylinositol triphosphate, ceramide phosphorylcholine, ceramide phosphorylethanolamine, ceramide phosphoryl lipid), glycolipids (e.g., glyceroglycolipid, sphingoglycolipid, rhamnolipid, etc.), and sterols (e.g., cholesterol, campesterol, sitosterol, stigmasterol, ergosterol, etc.). Hydrophobic moieties may include sterols (e.g., cholesterol), saturated fatty acids (e.g., caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachic acid, behenic acid, lignoceric acid, cerotic acid, etc.), and unsaturated fatty acids (e.g., myristoleic acid, palmitoleic acid, saponaric acid, oleic acid, elaidic acid, isooleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexenoic acid, etc.). Hydrophilic compounds may include charged molecules and polar molecules (e.g., diols, cyclodextrins, celluloses, polyacrylamides, and the like).

The surface of the detectable probe or affinity reagent may include one or more modifying groups. The surface of the detectable probe or affinity reagent may comprise at least about 10, 50, 100, 500, 1000, 5000, 10000, 50000, 100000, 50000, 1000000 or more modifying groups. Alternatively or additionally, the surface of the detectable probe or affinity reagent may comprise no more than about 1000000, 500000, 100000, 50000, 10000, 5000, 1000, 500, 100, 50, 10 or less modifying groups.

Multiple detectable probes, affinity reagents, or both may be assembled into a multi-probe complex. The multi-probe complex may include, for example, a plurality of detectable probes, a plurality of affinity reagents, an affinity reagent and a detectable probe, an affinity reagent and a plurality of detectable probes, or a plurality of affinity reagents and a detectable probe. The multi-probe complex may be prepared to further enhance desired properties of the detectable probe or affinity reagent, such as enhanced affinity and/or enhanced observability. In some configurations, a multi-probe complex may include multiple detectable probes, affinity reagents, or both, formed as a single complex, having similar or identical configurations. A multiprobe complex comprising a plurality of similar or identical probes may be formed to increase the overall brightness of the probes upon interaction with the binding partner, or an increased number of affinity reagents may be provided to increase the affinity beyond that observed by the detectable probes or affinity reagents alone. In other configurations, the multi-probe complex may include multiple detectable probes, affinity reagents, or both, of different or dissimilar configurations attached to a single complex. The multi-probe complex may comprise a plurality of detectable probes, affinity reagents, or both, having different or dissimilar configurations, and may be formed to bind to a plurality of binding partners, epitopes, or target moieties simultaneously, or to increase the likelihood of forming a binding interaction by increasing the number of binding partners, epitopes, or target moieties that are recognizable by the detectable probes or affinity reagents. The multi-probe complex may appear as a monovalent complex (e.g., configured to bind only a single species of binding partner, epitope, or target moiety) or a multivalent complex (e.g., configured to bind to multiple different binding partners, epitopes, or target moieties). The multiprobe complexes may be attached by covalent or non-covalent interactions. Covalent bonds between two detectable probes, between two affinity reagents, or between a probe and a reagent may be formed by, for example, a click reaction. Non-covalent interactions between probes and/or reagents may be formed, for example, by nucleic acid hybridization or streptavidin-biotin coupling.

The multi-probe complex may be formed by coupling together two or more detectable probes, affinity reagents, or both. The multi-probe complex can be formed by direct attachment of the detectable probes and/or affinity reagents (e.g., by nucleic acid hybridization, cross-linking, etc.). In some configurations, the multi-probe complex may be formed by the attachment between one or more secondary retention components, such as structured nucleic acid particles or nanoparticles. Secondary retention components may be of particular interest if they provide for adjustable positions and/or orientations of the detectable probes or affinity reagents. In some configurations, the secondary retention component may include additional components, such as a modifying group, a binding component, or a labeling component. 39A-39B show an exemplary configuration of a multi-probe complex including a secondary retention component. Fig. 39A shows a secondary retention component 3910 (e.g., structured nucleic acid particles, nanoparticles) that includes two or more attached detectable probes 3920 attached in an inward orientation. The secondary retention component 3910 further includes an attached marker component 3930. Fig. 39B shows a secondary retention component comprising two or more separate particles 3910 (e.g., SNAP, nanoparticles) comprising a plurality of detectable probes 3920 coupled to form a plurality of pockets of inwardly oriented binding components. The secondary retention component comprising two or more particles 3910 further comprises a plurality of marker components 3930 attached to the secondary retention component. In other configurations, the multi-probe complex may be formed by coupling or attaching a plurality of detectable probes or affinity reagents to a non-structured material or group such as a polymer, metal, ceramic, semiconductor, glass, fiber, resin, or combination thereof. The unstructured material may be amorphous, spherical, porous, or some combination thereof. The unstructured material may comprise a plurality of attachment sites allowing attachment of a plurality of detectable probes or affinity reagents.

Fig. 35A-35C depict various configurations of multi-probe complexes. Fig. 35A depicts two identical or similar tile-shaped nucleic acid detectable probes 3510 linked by a linking group 3520 (e.g., a covalent or non-covalent linking group). The detectable probes 3510 can have affinity for a single target or a group of targets (e.g., binding partners, epitopes, or target moieties). Fig. 35B depicts a first tile-shaped nucleic acid detectable probe having an antibody binding component 3510 linked by a linking group 3520 (e.g., a covalent or non-covalent linking group) to a second tile-shaped detectable probe having an aptamer binding component 3515. Detectable probes 3510 and 3515 can have different affinities, allowing the detectable probes to bind more than one binding partner, epitope, or target moiety simultaneously. Fig. 35C depicts a pair of non-nucleic acid based detectable probes 3530 (e.g., particles, nanoparticles, etc.) that are linked by a non-covalent attractive interaction such as electrostatic or magnetic interaction. Non-nucleic acid based detectable probes 3530 can include binding members with similar or dissimilar binding partners, epitopes, or target moieties.

In some configurations, the multi-probe complex may be configured to form a complex having a controlled geometry. Controlled geometries can be employed to more advantageously orient the binding component to increase affinity. The multiprobe complex may be configured to change shape or conformation upon formation of a binding interaction with a binding partner, epitope or target moiety. The multi-probe complex may be configured to resist a change in shape or conformation to increase contact or proximity of the binding component with the binding partner, epitope, or target moiety. Individual probes or reagents included in a multiprobe complex may have modifying groups that form interactions with other probes or reagents in the multiprobe complex that affect the relative orientation of the probes within the complex. 36A-36B depict configurations of detectable probes having designed or structured geometries. FIG. 36A depicts two tile-shaped nucleic acid-detectable probes 3610 connected by a linker 3620. Each probe 3610 includes one or more modifying groups 3630 (e.g., same charge, same magnetic polarity, spatial groups, etc.) that repel or retard the free movement of the linker 3620. Modifying groups 3630 orient the probes inward, increasing the density of binding members of the probe complex. FIG. 36B depicts two tile-shaped nucleic acid-detectable probes 3610 connected by a linker 3620. Each probe 3610 includes one or more modifying groups 3640 (e.g., hydrophobic groups, hydrophilic groups, etc.) that create attractive forces between the probes that create an inward orientation of the probes 3610 that increases the binding composition density of the probe complex. These configurations may resist deformation of the complex during binding interaction with the binding partner.

The multiplex complex may include two or more detectable probes and/or affinity reagents attached to a single complex. The multi-probe complex can include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more detectable probes and/or affinity reagents. Alternatively or additionally, the multi-probe complex may include no greater than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less detectable probes and/or affinity reagents.

The affinity reagents or detectable probes described herein may include a binding component (also referred to as a binding component or probe component) that is capable of binding or associating with a polypeptide or other analyte in a manner that allows such binding to be interpreted in order to confirm the polypeptide or other analyte with which it is associated (or with which it is not). The affinity reagent or detectable probe may also include a labeling component that allows for the observation or detection of binding or association of the affinity reagent or detectable probe with a target analyte, such as a polypeptide or other analyte. The label component may be separate from the probe component, for example when the label component is an exogenous label attached via a synthetic linker or attachment moiety. Alternatively, the labeling component may be an inherent feature of the probe, such as a detectable mass, charge, or optical feature. By identifying which affinity reagents or detectable probes bind to different polypeptides or other analytes within a sample, the presence and potential amounts of such proteins or polypeptides within the sample can be identified.

In some embodiments, the affinity reagent or detectable probe intended to confirm the target amino acid sequence may actually comprise a set of different components that are indistinguishable or distinguishable from each other when used in the methods described herein. In particular, different components that can be used to confirm the same target amino acid sequence can use the same tag moiety to confirm the same target amino acid sequence. For example, an affinity reagent or detectable probe that binds to a trimeric amino acid sequence (AAA) regardless of the flanking sequence may comprise a single binding component that binds to the trimeric AAA sequence without any effect of the flanking sequence, or a set of binding components (e.g., 400 binding components), each of which binds to a different 5 amino acid epitope of the αaaa β form, where α and β may be any amino acid. In an example of the second case, the binding components may have a combined effect.

Binding and affinity

For a single monovalent affinity reagent, the strength of the binding interaction between the affinity reagent and the binding target (e.g., binding partner, epitope, or target moiety) can be characterized by affinity. Affinity can be expressed quantitatively in terms of dissociation constants or association constants. Without wishing to be bound by theory, dissociation constant K _D Can result from equilibrium analysis of binding between affinity reagent a and binding partner B to form bound complex AB. The rate of association and dissociation between A and B depends on the relative concentrations of A, B and AB, expressed as [ A]、[B]And [ AB]. Association rate (or binding rate) r _on Can be expressed as:

r _on ＝k _on [A][B] (1)

wherein k is _on Is the rate constant of binding. Dissociation rate (or dissociation rate) r _off Can be expressed as:

r _off ＝k _o ff[AB] (2)

wherein k is _off Is the rate constant of dissociation. Based on the balance between the binding rate (1) and the dissociation rate (2), the dissociation constant can be calculated as:

/>

for a given affinity reagent, a smaller K _D Indicating stronger binding and higher values indicate weaker binding.

Apparent dissociation constants can be determined for detectable probes or affinity reagents having multiple binding members. The apparent dissociation constant can be calculated similarly to the dissociation constant of the monovalent affinity reagent. Without wishing to be bound by theory, apparent dissociation constant K _D,a Can result from an equilibrium assay in which the detectable probe (or affinity reagent) P binds to the binding partner B to form a bound complex PB. The apparent rate of association and dissociation between P and B can depend on the relative concentrations of P, B and PB, expressed as [ P]、[B]And [ PB]. Apparent rate (or apparent binding rate) r of association _on,a Can be expressed as:

r _on，a ＝k _on，a [P][B] (4)

wherein k is _on,a Is the apparent rate constant of the binding. Apparent rate of dissociation (or apparent dissociation rate) r _off,a Can be expressed as:

r _off，a ＝k _off，a [PB] (5)

wherein k is _off,a Is the apparent rate constant of dissociation. Based on the balance between apparent binding rate (1) and apparent dissociation rate (2), the apparent dissociation constant can be calculated as:

for multivalent detectable probes or multivalent affinity reagents, a smaller K _D,a A value indicates a stronger bond and a higher value indicates a weaker bond.

The affinity of a detectable probe or affinity reagent can generally be described as a decrease in the apparent dissociation constant of the probe relative to the dissociation constant of its constituent binding members. For example, for a probe comprising N binding members, each individual binding member may have an individual dissociation constant as described in equations 1-3 above, e.g., { K } _D,1 、K _D,2 ……K _D,N }. Apparent dissociation constant K when the detectable probe or affinity reagent has N binding members as a whole _D,a Less than the respective dissociation constant of each of the N binding members, e.g., K _D,a <{K _D,1 、K _D,2 ……K _D,N The detectable probe or affinity reagent may be considered to have increased affinity. Where the detectable probe or affinity reagent comprises a plurality of dissociation constants, this is simply K _D (i.e., K _D,1 ＝K _D,2 ＝……＝K _D,N ＝K _D ) In the case of N binding components of the same type and kind, the affinity can be defined simply as K _D,a <K _D . Where the detectable probe or affinity reagent comprises a plurality of dissociation constants { K } _D,1 、K _D,2 ……K _D,N And is most probableStrong binding of K _D,min In the case of N binding components of different types and/or kinds, the affinity can be defined simply as K _D,a <K _D,min 。

K displayed if the detectable probes or affinity reagents of the present disclosure display _D,a Significantly less than K _D，min For example K _D,a <K _D,min Up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 orders of magnitude, it may be preferred. If K _D,a <K _D,min Up to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 orders of magnitude, a detectable probe or affinity reagent may be selected for a particular binding target. Alternatively or additionally, if K _D,a <K _D,min No more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than 1 order of magnitude, a detectable probe or affinity reagent may be selected for a particular binding target. If K _D,a <K _D,min If the amount of detectable probe or affinity reagent is too large, it may be less advantageous as this may indicate excessive binding, as indicated by a very small dissociation rate.

The dissociation constant of the detectable probe, affinity reagent, or binding component thereof may be at least about 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or more. Alternatively or additionally, the dissociation constant of the detectable probe, affinity reagent, or binding component thereof may be no greater than about 10. Mu.M, 9. Mu.M, 8. Mu.M, 7. Mu.M, 6. Mu.M, 5. Mu.M, 4. Mu.M, 3. Mu.M, 2. Mu.M, 1. Mu.M, 950nM, 900nM, 850nM, 800nM, 750nM, 700nM, 650nM, 600nM, 550nM, 500nM, 450nM, 400nM, 350nM, 300nM, 250nM, 200nM, 150nM, 100nM, 95nM, 90nM, 85nM, 80nM, 75nM, 70nM, 65nM, 60nM, 55nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 15nM, 10nM, 5nM, 1nM, 0.5nM, 0.1nM or less.

The detectable probe or affinity reagent may have an apparent or measured dissociation constant that is less than the dissociation constant of the binding member attached to the detectable probe or affinity reagent. The lower dissociation constant of the detectable probe or affinity reagent may be attributed to the increased binding rate, k, of binding _on Increased, reduced dissociation rate of binding, k _off Reduced, or a combination thereof. The decrease in apparent or measured dissociation constant of the detectable probe or affinity reagent relative to any of the plurality of binding components attached to the detectable probe or affinity reagent may be a decrease of 1/N (i.e., K _{D, probe} ＝(1/N)K _{D, binding component} ). The decrease in apparent or measured dissociation rate constant of the detectable probe or affinity reagent relative to any of the plurality of binding components attached to the detectable probe or affinity reagent may be at least about 1/2, 2/3, 3/4, 4/5, 5/6, 6/7, 7/8, 8/9, 9/10, 14/15, 19/20, 24/25, 49/50, 99/100, 249/250, 499/500, 999/1000, or greater. Alternatively or additionally, the decrease in apparent or measured dissociation constant of the detectable probe or affinity reagent relative to any of the plurality of binding components attached to the detectable probe or affinity reagent may be no greater than about 999/1000, 499/500, 249/250, 99/100, 49/50, 24/25, 19/20, 14/15, 9/10, 8/9, 7/8, 6/7, 5/6, 4/5, 3/4, 2/3, 1/2, or less.

In some cases, it may be useful to quantitatively describe the affinity. Having dissociation constant K for the strongest conjugate against the binding target _D,min Affinity A of a set of N binding members _N Can be defined as:

wherein K is _D,a Is the apparent dissociation constant of a detectable probe or affinity reagent comprising a set of N binding members. In some configurations, a detectable probe or affinity reagent of the present disclosure can have an a greater than 1 _N . A detectable Probe or affinity reagent _N May be at least about 2, 3,4. 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, 10000, 25000, 50000, 100000, 1000000, 10000000, 100000000, 1000000000, 10000000000, or more. Alternatively or additionally, a of the probe or affinity reagent may be detected _N May be no greater than about 10000000000, 1000000000, 100000000, 10000000, 1000000, 100000, 50000, 25000, 10000, 5000, 2500, 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less.

Affinity may refer to the ability of an affinity reagent or a detectable probe to exhibit a change in apparent affinity for a binding partner when the reagent or probe has multiple pathways for binding interaction with the binding partner. For example, if a detectable probe or affinity reagent comprises ten binding components, and each binding component has affinity for binding to target X, then the detectable probe or affinity reagent has ten possible pathways to form binding interactions with the target X. The avidity may be an amplified effect in which the apparent affinity of a detectable probe or affinity reagent comprising a plurality of binding components to a binding partner, epitope or target moiety may be stronger than the affinity of any of the plurality of binding components. For example, a detectable probe or affinity reagent having 10 binding members may have a measured dissociation constant of 50 nanomolar (nM) when the strongest binding member attached to the detectable probe or affinity reagent has a dissociation constant of only 800 nM.

Affinity may occur due to the propensity of the binding component to form new binding interactions shortly after the previous binding interactions are disrupted in the presence of the binding component. Thus, the binding interaction between the detectable probe (or affinity reagent) and the binding partner may be observed to occur on a longer time scale than the binding interaction between the individual binding components and the binding partner observed. Without wishing to be bound by theory, detectable probes or affinity reagent binding may be tuned by altering probe design to alter thermodynamic, kinetic, and/or mass transfer characteristics of the probe. The affinity of the detectable probe or affinity reagent may be affected by factors that alter the likelihood of binding interactions, including: 1) Binding component density on the probe; 2) The total number of binding components on the probe; 3) The type and/or kind of binding component; 4) Binding component binding thermodynamics; 6) Binding component binding kinetics; 5) Probe size and/or weight; 6) Probe shape and/or conformation; and 7) a secondary binding interaction mediator.

Depending on the thermodynamic and kinetic binding behavior of the detectable probe or affinity reagent pool, increasing concentrations of the detectable probe or affinity reagent may facilitate binding of the detectable probe or affinity reagent to the binding partner, epitope or target moiety. The effective or target concentration of detectable probes or affinity reagents utilized in the binding assays can be calculated with reference to the probes themselves, the total number of binding members on the reference probes, or, in the case of probes having a heterogeneous mixture of binding members, the number of binding members of a particular species. For example, a solution provided at 0.1 molar (M) of detectable probe or affinity reagent made to contain a total of 20 binding components per probe at a 1:1 antibody/aptamer ratio will have a total concentration of 2M binding components or 1M concentration of aptamer or antibody. The effective or target concentration of detectable probes or affinity reagents can be determined on a mass or molar basis. The detectable probes, affinity reagents, or binding members thereof may be provided in a preferred concentration (e.g., a concentration that optimizes affinity) for the intended purpose.

May be at least about 1x10 ^-15 M、5x10 ^-15 M、1x10 ^-14 M、5x10 ^-14 M、1x10 ^-13 M、5x10 ^-13 M、1x10 ^-12 M、5x10 ^-12 M、1x10 ^-11 M、5x10 ^-11 M、1x10 ^-10 M、5x10 ^-10 M、1x10 ^-9 M、5x10 ^-9 M、1x10 ^-8 M、5x10 ^-8 M、1x10 ^-7 M、5x10 ^-7 M、1x10 ^-6 M、5x10 ^-6 M、1x10 ^-5 M、5x10 ^-5 M、1x10 ^-4 M、5x10 ^-4 M、1x10 ^-3 M、5x10 ^-3 M、1x10 ^-2 M、5x10 ^-2 M、1x10 ^-1 M、5x10 ^-1 M, 1M or higher provides a detectable probe, an affinity reagent or binding component thereof. Alternatively or additionally, it may be no greater than about 1M, 5x10 ^-1 M、1x10 ^-1 M、5x10 ^-2 M、1x10 ^-2 M、5x10 ^-3 M、1x10 ^-3 M、5x10 ^-4 M、1x10 ^-4 M、5x10 ^-5 M、1x10 ^-5 M、5x10 ^-6 M、1x10 ^-6 M、5x10 ^-7 M、1x10 ^-7 M、5x10 ^-8 M、1x10 ^-8 M、5x10 ^-9 M、1x10 ^-9 M、5x10 ^-10 M、1x10 ^-10 M、5x10 ^-11 M、1x10 ^-11 M、5x10 ^-12 M、1x10 ^-12 M、5x10 ^-13 M、1x10 ^-13 M、5x10 ^-14 M、1x10 ^-14 M、5x10 ^-15 M、1x10 ^-15 M or less provides a detectable probe, an affinity reagent, or a binding component thereof.

The size of the probe may have an effect on the affinity properties of the detectable probe or affinity reagent. Without wishing to be bound by theory, the mass transfer properties of the detectable probe or affinity reagent may be affected by the size and/or weight of the probe. The mass transfer physical properties (e.g., diffusion coefficient) of the detectable probe or affinity reagent may have a scaling relationship of the mass transfer physical properties that depends on the size and/or weight of the probe. For example, the detectable probe or affinity reagent can have a power law relationship with the probe molecular weight (i.e., D-MW ^-2 ) Proportional one-component diffusion coefficient. Larger and/or heavier affinity reagents or detectable probes may experience a decrease in diffusion rate in a solution or solvent. A decrease in the diffusion rate of the detectable probe or affinity reagent may be observed as a decrease in the apparent affinity of the detectable probe or affinity reagent due to an increased likelihood of binding interactions occurring before the probe can diffuse away from the binding partner, epitope or target moiety.

The probe can have a characteristic molecular weight of at least about 1kDa, 10kDa, 50kDa, 100kDa, 500kDa, 1000kDa, 1500kDa, 2000kDa, 2500kDa, 3000kDa, 3500kDa, 4000kDa, 4500kDa, 5000kDa, 5500kDa, 6000kDa, 6500kDa, 7000kDa, 7500kDa, 8000kDa, 8500kDa, 9000kDa, 9500kDa, 10000kDa or more. Alternatively or additionally, the probe may have a characteristic molecular weight of no greater than about 10000kDa, 9500kDa, 9000kDa, 8500kDa, 8000kDa, 7500kDa, 7000kDa, 6500kDa, 6000kDa, 5500kDa, 5000kDa, 4500kDa, 4000kDa, 3500kDa, 3000kDa, 2500kDa, 2000kDa, 1500kDa, 1000kDa, 500kDa, 100kDa, 50kDa, 10kDa, 1kDa or less.

In some cases, the affinity of the detectable probe or affinity reagent may be affected by the secondary binding interaction. The secondary binding interactions may be weak or short term interactions involving secondary molecules that the designed detectable probes or affinity reagents have. Secondary binding interactions may include weak or short term interactions of secondary molecules that involve a decrease in apparent rate of diffusion of a detectable probe or affinity agent away from a binding partner, epitope, or target moiety. Secondary binding interactions may increase the likelihood that a detectable probe or affinity reagent will be observed to associate with a binding partner for which the detectable probe or affinity reagent has affinity. In some configurations, the secondary molecule may include one or more ligands and/or moieties that may form a weak interaction with complementary ligands and/or moieties on the detectable probe or affinity reagent. For example, the secondary molecule may have a nucleic acid sequence that is at least partially complementary to a nucleic acid sequence in the detectable probe or affinity reagent. As such, the secondary molecule and probe may be configured to form a weak interaction via hybridization of the two sequences. For example, weak interactions may result from incomplete or discontinuous hybridization, such as interactions resulting from the use of hairpin or loop structures in the sequence of the detectable probe or affinity reagent or secondary molecule, mismatched nucleotides in otherwise complementary regions of the detectable probe (or affinity reagent) and secondary molecule, or modified or unnatural nucleotides in the detectable probe (or affinity reagent) or secondary molecule that result in binding of a weaker base pair. In other configurations, the secondary molecule may include one or more ligands and/or moieties that may form a weak interaction with the binding partner. In some configurations, the detectable probe (or affinity reagent) or secondary molecule may comprise a plurality of molecules configured to form a weak binding interaction.

In some configurations, a secondary molecule that interacts with a detectable probe or affinity reagent may be attached to a solid support. For example, the secondary molecule may be SNAP attached to a solid support, such as a nucleic acid fold or a nucleic acid nanosphere. The SNAP may optionally be attached to a binding partner of one or more binding components of a detectable probe or affinity reagent. Thus, the secondary molecule may mediate association of the detectable probe or affinity reagent with the solid support via binding of one or more binding components to the binding partner, and the association may be enhanced by weak interaction between the secondary molecule and the SNAP. In some configurations, the plurality of SNAP are attached to the solid support in an array. Each site in the array may have SNAP attached to a spatially resolved binding partner, and each of the binding partners may be recognized by one or more detectable probes or affinity reagents. Exemplary SNAP and solid supports that may be usefully employed in the compositions or methods of the present disclosure are set forth in U.S. patent application serial No. 17/062,405 (published as U.S. patent application publication No. 2021/0101930 A1) and 2019/195633A1, each of which is incorporated herein by reference.

Fig. 8 depicts a detectable probe 810 configured to interact with a binding partner 830 or an epitope or target moiety within binding partner 830. The detectable probe further includes a pendent ligand 820 (e.g., a nucleic acid) configured to form a weak interaction with the complementary molecule 850 at the interaction region 860. In some configurations, the secondary molecule 850 may be bound or associated with the anchor group 840, the binding partner 830, or the solid support 870. In some configurations, the interaction between the overhanging ligand 820 and the complementary molecule 850 at the interaction region 860 may be naturally unstable, thereby facilitating the eventual dissociation of the detectable probe 810 from the binding partner 830. In other configurations, the interaction between the pendent ligand 820 and the complementary molecule 850 at the interaction region 860 may be configured to be disrupted (e.g., in the presence of a denaturing agent).

Figures 9A-9B depict possible configurations for weak interactions between a detectable probe and a secondary molecule using a pair of complementary nucleic acids. Fig. 9A shows a first nucleic acid 910 that can be coupled to a detectable probe or a secondary molecule, and a second nucleic acid 920 that can be coupled to the opposite molecule of the detectable probe-secondary molecule pair. The first nucleic acid 910 is configured to hybridize to the second nucleic acid 920 in a incomplete or discontinuous manner, thereby forming a weak interaction region 930. In some configurations, the incomplete or discontinuous hybridization may include hairpin or loop structures, mismatched nucleotides, or modified or unnatural nucleotides that result in binding of a weaker base pair. FIG. 9B shows the weak interaction between a detectable probe and a secondary molecule formed between two nucleic acids by a short hybridization region. The detectable probe 940 may include a first nucleic acid 910 configured to hybridize to a second nucleic acid 920. The first nucleic acid 910 and the second nucleic acid 920 hybridize at a weak interaction region 930 that contains a limited number of base pairs that bind, such that the base pair binding has a degree of reversibility or dissociation. The weak interaction region may comprise a limited number of nucleotides, such as no greater than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than about 2 nucleotides, for example. The weak base pairing interaction region can be located in a terminal portion of the nucleic acid or an internal portion of the nucleic acid sequence.

In some embodiments, the detectable probe further comprises a partially non-hybridizing nucleic acid having a single stranded region. In some embodiments, the single stranded region binds to a complementary nucleic acid associated with a binding partner. In some embodiments, the single stranded region comprises a primer sequence configured to bind to a complementary primer sequence. In some embodiments, the primer sequences are configured to cause a foothold displacement reaction. In some embodiments, the primer sequence is configured to form a hairpin or loop structure upon binding to the complementary primer sequence.

FIGS. 10A-10C depict a foothold displacement strategy for creating a weak binding interaction between a secondary molecule and a detectable probe. Figure 10A shows the first step of the foothold displacement reaction. A detectable probe 1010 comprising a first nucleic acid 1020 is in proximity to a second nucleic acid 1050 associated with a secondary molecule. The first nucleic acid molecule 1020 includes a hybridization sequence comprising a foothold sequence 1030 and a priming sequence 1040. The second nucleic acid 1050 includes complementary hybridization sequences including a complementary foothold sequence 1060 and a complementary priming sequence 1070. The second nucleic acid 1050 can hybridize to the setback primer 1080 that hybridizes only to the complementary setback sequence 1060 of the hybridization sequence. The priming sequence 1040 of the first nucleic acid 1020 may hybridize to the complementary priming sequence 1070 of the second nucleic acid 1050, thereby forming an initial weak binding interaction between the detectable probe 1010 and the secondary molecule. The weak binding interactions between the detectable probe 1010 and the secondary molecule may have a degree of reversibility or dissociation. Figure 10B shows the second step of the foothold displacement reaction. The footer sequence 1030 of the first nucleic acid 1020 replaces the footer primer 1080, allowing the footer sequence to complete hybridization with the complementary footer sequence 1060 of the second nucleic acid 1050. The continued presence of the foothold primer 1080 in a solution or solvent adjacent to the weak binding interaction may cause reversal of the foothold displacement reaction by the foothold primer 1080 displacing the first nucleic acid 1020 from the second nucleic acid 1050.

FIG. 10C depicts an alternative method of forming a weak binding interaction between a detectable probe and a secondary molecule. The detectable probe 1010 includes a first nucleic acid strand 1020 that contains a priming sequence 1040 that binds to a complementary priming sequence 1070 on a second nucleic acid strand 1050. The second nucleic acid strand 1050 also contains a complementary set-point sequence 1060 that is not complementary to the sequence of the first nucleic acid 1020. The detectable probe-secondary molecule complex can be contacted with a solution or solvent comprising a set-point primer comprising a set-point sequence 1030 and a priming sequence 1040. The foothold primer may reversibly displace the first nucleic acid strand 1020 by disrupting the base binding of the priming sequence 1040 to the complementary priming sequence 1070. In some configurations, the balance between binding and displacement of the detectable probe can be controlled by adjusting the concentration of one or more of the foothold displacement reactants (e.g., the foothold primer, the detectable probe).

The affinity of the detectable probe or affinity reagent may also be increased by using another binding molecule having a different affinity. In some configurations, the affinity reagent may include one or more binding components, e.g., a retention component attached to a plurality of binding components. In some configurations, the affinity reagent has a single binding component. In some configurations, the binding molecule is a detectable probe or an affinity reagent. The binding molecule may have the property of binding to the binding partner, epitope or target moiety with a lower affinity and/or avidity than the detectable probe or affinity reagent. The binding molecule may be coupled to a detectable probe or affinity reagent such that simultaneous binding of the detectable probe or affinity reagent to the binding molecule results in a substantial increase in the observed affinity or avidity of the detectable probe or affinity reagent.

FIGS. 11A-11C depict systems that employ secondary binding interactions of binding molecules to increase the avidity of a detectable probe. As shown in FIG. 11A, the detectable probe 1110 is linked to a binding molecule 1130 (e.g., a second detectable probe) through a linker 1190 (e.g., a covalent heterobifunctional linker, a hybridized nucleic acid, etc.). The linker may be flexible, e.g., allowing the detectable probe 1110 to interact directly with the binding molecule 1130. Alternatively, the linker may be relatively rigid, e.g., limiting the detectable probes 1110 to not directly interact with the binding molecules 1130. The detectable probes 1110 comprise a first plurality of binding members 1120 and the binding molecules 1130 comprise a second plurality of binding members 1140. A composition comprising a complex formed by ligating a detectable probe 1110 and a binding molecule 1130 is contacted with a binding partner 1150 comprising an epitope or target moiety 1160 and a secondary binding site 1170. As shown in fig. 11B, the binding interaction may be initiated by initial binding of the binding component of the first plurality of binding components 1120 to the epitope or target moiety 1160. Upon initial binding, the binding molecule may not have formed a secondary interaction with the binding partner 1150. As shown in fig. 11C, when a binding component of the second plurality of binding components 1140 forms a secondary binding interaction with the secondary binding site 1170, the binding process can be completed, thereby binding the detectable probes 1110 and binding molecules 1130 to the binding partners 1150. The weak secondary binding interaction between the binding molecule 1130 and the secondary binding target 1170 may increase the likelihood that the detectable probe 1110 will be observed to bind to the binding partner 1150. The skilled artisan will readily recognize that the order of binding of the binding complexes may be reversed, e.g., binding molecule 1130 first binds to secondary binding site 1170, followed by binding of detectable probe 1110 to epitope or target moiety 1160.

FIGS. 11D-11E depict alternative systems that employ secondary binding interactions of binding molecules to increase the avidity of a detectable probe. As shown in fig. 11D, a detectable probe 1110 comprising a first plurality of binding components 1120 and a first linking group 1191 comprising a linking region 1192 binds to an epitope or target portion 1160 of binding partner 1150. Unbound binding molecules 1130 comprising a second plurality of binding components 1140 and a second linking group 1193 comprising a complementary linking region 1194 are contacted with binding partner 1150. As shown in fig. 11E, the binding component of the plurality of binding components 1140 binds the binding molecule 1130 to the secondary binding site 1170 of the binding partner, thereby forming a weak secondary binding interaction. Upon binding of binding molecule 1130, the linking region 1192 and complementary linking region 1194 may be linked, thereby forming a linker between the detectable probe 1110 and the binding molecule 1130. The weak secondary binding interaction between the binding molecule 1130 and the secondary binding target 1170 may increase the likelihood that the detectable probe 1110 will be observed to bind to the binding partner 1150. The skilled artisan will readily recognize that the order of binding of the binding complexes may be reversed, e.g., binding molecule 1130 first binds to secondary binding site 1170, followed by binding of detectable probe 1110 to epitope or target moiety 1160. The first linking group 1191 and the second linking group 1193 may be configured to form covalent or non-covalent interactions. For example, the first linking group 1191 can include a nucleic acid linking region 1192 and the second linking group 1193 can include a complementary nucleic acid linking region 1194 configured to hybridize to the nucleic acid linking region 1192. In another example, the linking region 1192 may include a functional group (e.g., a click-reaction group) configured to chemically bind (e.g., by a click reaction) with a functional group in the complementary linking region 1194.

In many cases, an affinity reagent, detectable probe, or binding component thereof may have a non-random probability of binding to a selected binding partner (e.g., an amino acid epitope within a polypeptide molecule or larger polypeptide structure), meaning that it exhibits a certain level of increased affinity for a given epitope or group of epitopes as compared to other epitopes or groups of epitopes, also referred to as "specificity. In many cases, the specificity may not be binary, meaning that the affinity reagent or detectable probe may sometimes appear not to bind to a given epitope for which it exhibits increased specificity, and may sometimes appear to bind to an epitope for which it does not exhibit specificity. In many cases, whether a given affinity reagent or detectable probe is specific for a given epitope or binding partner as described above will correlate with the likelihood that the affinity reagent or detectable probe will bind to the given epitope or binding partner under the conditions of the binding assay. For example, an affinity reagent or detectable probe may be considered specific for a particular epitope or binding partner if the probability of binding is greater than a probability threshold. The probability threshold may be known (i.e. the threshold is predetermined) prior to performing the binding assay, or it may be determined by the binding assay itself (i.e. the threshold is empirically determined). In some cases, the probability threshold associated with a specific binding may be greater than 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater. In some cases, the probability threshold associated with specific binding may be greater than 1%, 5%, 10%, 20%, 30%, or 40%. Conversely, in some cases, the probability that a given affinity reagent or detectable probe does not bind to a given epitope or binding partner (e.g., provides false negative binding results) under a given set of circumstances may range from less than 1% to greater than 99%, meaning that there may be a significant likelihood that the affinity reagent or detectable probe does not bind to its complementary epitope or polypeptide. While in some cases the probability threshold associated with non-specific or non-binding will be less than 50%, 40%, 30%, 20%, 10%, 5% or less. In some cases, the probability threshold associated with non-specific or non-binding may be less than 60%, 70%, 80%, 90%, or 95%.

In some embodiments, the detectable probe or affinity reagent may have one or more of the following properties: may specifically bind to a particular amino acid species and may not bind to more than nineteen other amino acid species. For example, the detectable probe or affinity reagent may bind to one of the 20 essential amino acids, but not the other 19 essential amino acids. The detectable probe or affinity reagent can bind at least 10% of the sequence in the form of alpha X beta, where X is the desired epitope and alpha and beta are any amino acid residues. In some cases, the detectable probe or affinity reagent can bind at least 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30%, 40%, 50%, 75%, or 90% of the sequence in the αxβ form. In some cases, the detectable probe or affinity reagent may bind at least 10% of the sequence in the alpha X beta form, where K _D Value ratio of the average K of the detectable probe or affinity reagent to the pool of random sequences _D The value is at least 9/10 lower. In some cases, the detectable probe or affinity reagent can bind at least 10% of the sequence in the alpha X beta form, where K _D Value ratio of the average K of the detectable probe or affinity reagent to the pool of random sequences _D The value is at least 99/100 lower. In some cases, the detectable probe or affinity reagent can bind at least 10% of the sequence in the alpha X beta form, where K _D Value ratio of the average K of the detectable probe or affinity reagent to the pool of random sequences _D The value is at least 999/1000 lower.

In some embodiments, the detectable probe or affinity reagent may have one or more of the following properties: may specifically bind to a desired epitope having a particular three amino acid sequence, may not bind to any other three amino acid sequences, and may bind to the desired epitope with substantially similar affinity, regardless of the flanking sequences surrounding the desired epitope. In some cases, the detectable probe or affinity reagent may have some, more, or all of these properties. In some cases, the detectable probes or affinity reagents may not bind to a subset of the epitopes. Another aspect of the present disclosure provides a detectable probe or affinity reagent that preferentially binds to a known set of three amino acid epitopes whose preference over other epitopes and influence by flanking amino acid residues has been determined.

In some cases, the detectable probe or affinity reagent may comprise a switchable aptamer that binds to between 5% and 10% of all proteins in the human proteome. In some cases, the switchable aptamer may include two or more fluorescent moieties. The switchable aptamer may be a binding component of a detectable probe or affinity reagent of the present disclosure.

In some cases, a detectable probe or affinity reagent is configured to bind to a given sequence, wherein the detectable probe or affinity reagent and the K of the given sequence _D Value ratio of the average K of the detectable probe or affinity reagent to a pool of proteins having random sequences _D The value is at least 9/10, 99/100 or 999/1000 lower.

In some cases, the detectable probe or affinity reagent may have the ability to bind to a desired epitope in one or more structural environments. In some embodiments, the detectable probe or affinity reagent can bind to a desired epitope when the polypeptide having the epitope is in a denatured environment, a native environment, or both. In some embodiments, the detectable probe or affinity reagent may have the ability to bind a desired epitope in a polypeptide within a folded or unfolded environment. In some embodiments, a polypeptide that has been denatured may contain or create one or more micro-folding regions within the polypeptide. The detectable probe or affinity reagent may be designed to bind to a desired epitope in the polypeptide that has been allowed to fold after denaturation. The resulting polypeptide may be renatured to its active folded state, partially refolded, or folded into a different state. The detectable probe or affinity reagent may bind to an epitope in a folded or unfolded region of the polypeptide that has been denatured and/or allowed to fold from a denatured state.

In some embodiments, a detectable probe or affinity reagent that recognizes a desired epitope sequence (e.g., AAA) may bind equally well or nearly so well to all polypeptides containing the epitope sequence. In some cases, the detectable probe or affinity reagent may bind to the desired epitope with different affinities depending on the differences in the sequence context of the epitope. In some cases, the detectable probe or affinity reagent may bind several different epitopes, regardless of the sequence context. In some cases, the detectable probes or affinity reagents of the present disclosure may bind several different epitopes with different affinities depending on the sequence context. Detectable probes or affinity reagents having such properties can be identified by a combination of screening methods to determine the binding characteristics of the detectable probes or affinity reagents. For example, detectable probes or affinity reagents may be screened for their ability to bind to a set of polypeptide sequences that differ from each other except for a common core sequence (e.g., an amino acid epitope in the form of αaaa β, where AAA is the common core sequence and α and β may be any amino acid).

In some cases, the desired epitope of the detectable probe or affinity reagent may be a peptide sequence. In some cases, several different epitopes may be required. In this case, a detectable probe or affinity reagent that binds to multiple desired epitopes may be selected. In some cases, the desired epitope or epitopes may be referred to as X. In some cases, the epitope comprises a discontinuous amino acid sequence. For example, an epitope may comprise a specified amino acid per second, third or fourth amino acid residue in a region of the primary sequence of a polypeptide. An epitope may include a 3 amino acid sequence in which two specified amino acids are separated by a variable amino acid (e.g., aαa, where a is alanine and α is any amino acid), a 4 amino acid sequence in which two specified amino acids are separated by two variable amino acids (e.g., aαβa, where a is alanine and α and β are any amino acids), a 5 amino acid sequence in which two specified amino acids are separated by three variable amino acids, and the like. In some embodiments, an epitope may comprise a sequence of two or more non-contiguous epitopes. In another example An epitope may include a number of amino acid residues that are located adjacent to each other in the secondary or tertiary structure of a protein, even if the residues are not adjacent in the protein sequence (i.e., are not adjacent in the primary structure of the protein). In some cases, the epitope comprises a contiguous sequence of specified amino acids. In some embodiments, the desired epitope X is a short amino acid sequence of 2, 3, 4, 5, 6 or 7 amino acids. In some cases, X comprises several different short amino acid sequences. In some embodiments, the desired epitope X is a three amino acid sequence X ₁ X ₂ X ₃ . Detectable probes or affinity reagents that bind a desired epitope in a variety of sequence contexts can be identified by screening for binding to a target polypeptide that includes the desired epitope.

The target may comprise a plurality of polypeptides, including the desired sequence X. The plurality may have any of a variety of configurations, such as a pool of polypeptides in a solution phase, on a solid support, in a container, on a solid support, an array of polypeptides in a collection of containers each containing one or more polypeptides in the plurality, and the like. In some cases, the target is a plurality of polypeptides that are all sequence X. In some embodiments, the target may comprise a plurality of polypeptides of the sequence αxβ, where X is a desired epitope, and α and β may be any sequence of zero, one, or more amino acids. For example, if the desired epitope X is AAA, examples of sequences that may be present in the target polypeptide may include: AAAAA, AAAAC, CAAAA, CAAAC and CAAAD. In some cases, α and β may each be any single amino acid. The amino acid of α may be the same as or different from the amino acid of β. In some cases, at least one of α and β may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. The sequence of α may be the same as or different from the sequence of β. In some cases, at least one of α and β may include a linker or spacer. The linker or spacer may be any linker or spacer described herein or known in the art. In some cases, the linker has a peptide backbone, e.g., an amino acid linker. In some cases, the linker is a polyethylene glycol (PEG) or a PEG polymer chain. The PEG chain may be composed of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 48, 50 or more ethylene glycol monomers. In some cases, the linker may be a carbon chain. The polypeptide may also include an N-terminal or C-terminal modification, such as capping. In some cases, the polypeptide may be modified to remove charge, such as terminal amidation (C-terminal) or acetylation (N-terminal). In some cases, the αxβ peptide may contain non-naturally occurring amino acids. In some cases, the αxβ peptide may be modified with linkers and functional groups. For example, the molecule may have the structure F-L- αxβ, where F is a functional group and L is a linker. In other cases, the molecule may have the structure αxβ -L-F, where F is a functional group and L is a linker. The functional group F may optionally be capable of forming a covalent bond with a reactive moiety or binding to a receptor. In some cases, α and β may each be glycine, or may each be one or more glycine residues. In some embodiments, amino acid residues may be modified to alter their aptamer (aptamer). For example, residues may be altered by: adding positive charges; adding negative charges; adding a hydrophobic group; modifying to add sugar; or other modifications to increase chemical diversity.

The polypeptides may be synthesized by any method known in the art. There are several commercial platforms for polypeptide synthesis, such as the MultiPep RSi synthesizer (Intavis, germany). The polypeptide may be synthesized by liquid phase or solid phase methods. The synthesized polypeptides may be validated using any known polypeptide analysis method. For example, mass spectrometry, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), matrix assisted laser desorption/ionization, AMS (accelerator mass spectrometry), gas chromatography-MS, liquid chromatography-MS, inductively coupled plasma-mass spectrometry (ICP-MS), isotope Ratio Mass Spectrometry (IRMS), ion mobility spectrometry-MS, tandem MS, thermal ionization-mass spectrometry (TIMS), or Spark Source Mass Spectrometry (SSMS) may be used to verify polypeptides. The concentration of synthetic peptides can also be assessed by spectroscopy.

FIG. 48 shows an exemplary list of immobilized targets for selection or characterization of detectable probes or affinity reagents, and polypeptides comprising the targets. In the example of fig. 48, the desired epitope is AAA and the polypeptide of the target includes the sequence αaaa β, where α and β are each a single amino acid. In this example, the target comprises 400 different polypeptides representing each possible sequence of αaaa β, where α and β are each a single amino acid.

Thus, for any given 3 mer surface, the target comprising a 5 mer pool may contain 400 different sequences (20 possibilities for a and 20 possibilities for β, where each of a and β is a single amino acid). In some cases, the target may comprise a pool of polypeptides longer than 5 amino acids, where each or both of α and β may comprise two or more amino acids. In some cases, one of α and β may comprise zero amino acids, and the other of α or β may comprise one or more amino acids. In some cases, the target may comprise a polypeptide of sequence X without additional amino acids.

In some cases, the target sequence X may be embedded in a longer sequence. For example, the target sequence X may be a core sequence of less than 15 amino acids, which is embedded in a 15-mer polypeptide molecule. The target sequence X may be embedded at any position within the 15 mer, for example in the case of a three amino acid target sequence X, the target sequence X may start at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the 15 mer. The polypeptides comprising the embedded target sequences may be synthesized in solution or may be synthesized on a chip, such as a PEPperPRINT chip or other peptide array. In some embodiments, polypeptides comprising an embedded target sequence may be bound or synthesized onto a single molecule protein array. Longer sequences may be selected to form secondary structures, or to lack secondary structures. Examples of such secondary structures include alpha helices, beta sheets, proline bends, turns, loops, and cysteine bridges. In some cases, the longer sequence may include non-naturally occurring amino acids or other groups.

The initial selection step may comprise screening a library of affinity reagents or detectable probes for a target comprising the desired epitope. The affinity reagent or detectable probe or library of probes may comprise DNA, RNA or peptide aptamers having random sequences or having sequences similar to known protein binding aptamers.

In some cases, the library may be an aptamer library. In some cases, the aptamer library can be a commercial library. In some cases, the aptamer library may be obtained from a research institute, university, academic center, or research center. In some cases, the library may comprise a library of aptamers attached to beads or other particles. In some cases, the library of aptamers may be generated from a library of known sequences or random sequences. In some cases, the library of aptamers may include aptamers having a specific structure, such as a stem-loop library. In some cases, the library of aptamers may include switchable aptamers—aptamers that can be switched between two conformations. For example, an aptamer may form a first conformation in the presence of a metal ion cofactor and a second conformation in the absence of the cofactor. Thus, the addition of chelators such as EDTA or EGTA will chelate metal ions and adapt the aptamer to different conformations. Other factors that may be used to induce aptamer switching include light, pH, temperature, magnetic field, and current.

Screening of the aptamer library against the target may be performed by any method known in the art. In one aspect, the target may be immobilized on a solid support and the aptamer may be added under conditions that allow binding of the aptamer with low specificity. Unbound aptamer can be washed from the target with a series of washes of increasing stringency. The aptamer that remains bound to the target by the washing step can be sequenced and amplified for further rounds of selection or for designing additional aptamers with high sequence similarity. Several rounds of target binding, washing, sequencing and amplification or design of new aptamers can be repeated until an aptamer with the desired specificity and binding affinity is produced. Libraries of aptamers can also be screened using bead-based methods that utilize beads that each include multiple copies of an aptamer. An array-based approach can also be used to screen an aptamer library, for example by spotting multiple copies of each aptamer of the library onto an array, and then evaluating the points of binding to the target. Particle display methods can also be used to screen libraries of aptamers. For example, beads or other particles attached to the aptamer may be arranged on a support to form an array. In some embodiments, a single molecule protein array may be used to screen an aptamer library.

In some cases, the fraction or percentage of targets that bind to the identified detectable probes or affinity reagents may be measured, for example, by comparing the binding copy number of the probes to the number of polypeptides available for binding. In some embodiments, the detectable probe or affinity reagent can bind to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the polypeptide comprising the target. In addition, once a particular detectable probe or affinity reagent is identified and selected, it can be validated. In some embodiments, selected detectable probes or affinity reagents can be validated against sequences containing an epitope to which they are characterized as binding. In some embodiments, the selected detectable probes or affinity reagents can be validated by evaluating the selected detectable probes or affinity reagents against a plurality of protein sequences on a single molecule protein array.

The detectable probes or affinity reagents may be attached to a detectable label either before or after the selection and characterization steps. In some cases, the detectable probe or affinity reagent may be attached to the label prior to the initial selection step. In some cases, the detectable probe or affinity reagent may be attached to the label after the initial selection step and before the characterization step. In some cases, the detectable probe or affinity reagent may be attached to the label after the selection and characterization steps. In some cases, the detectable probe or affinity reagent may be attached to a first label for the selection step and to a second label for the characterization step. The second marker may be added together with the first marker or in place of the first marker. In some cases, a first label may be used for the selection and characterization steps and a second label is used to generate the final affinity reagent or detectable probe.

Detection and observability

The detectable probes of the present disclosure may be configured to provide a detectable signal that imparts observability to the probe. Observability may refer to the property of generating a physical signal that can be easily sensed by a detection device at a level exceeding the background or noise of the detection system. For example, a fluorescent probe may be considered observable if the fluorescent signal intensity produced by the fluorescent probe exceeds the background fluorescent signal intensity by a threshold amount, e.g., 10%, 50%, 100%, 2x, 3x, 4x, 5x, or more. In another example, a nucleic acid barcode signal may be considered observable if it produces a threshold amount of sequence count (e.g., by next generation sequencing or array-based hybridization) with or without amplification, e.g., 1000 or more after 10 rounds of signal amplification. In general, observability may be affected by several factors, including: 1) The likelihood of the detectable probe binding to the target; 2) The intensity of the background signal; and 3) a signal damping mechanism. Factors that may affect the likelihood of the detectable probe binding to the target may include those set forth above.

The background signal may refer to a detectable signal having the same or similar characteristics as the desired signal. For example, in a fluorescent labeling system, the background signal may include radiation at the same wavelength or detected in a range of radiation wavelengths as the expected fluorescent signal from the fluorescent label, e.g., when attempting to observe At 488 dye, a background fluorescent signal may be detected between 485nm and 495 nm. For radiation signals, the background signal may be generated due to natural fluorescence, natural luminescence or autofluorescence of the material, transmission, reflection or refraction of external radiation at the detection wavelength, or residual signals left by the fluorescent probe from a previous detection process.

The background radiation signal may be spatially uniform or spatially non-uniform. For some characterization assays, the radiation background signal may be measured before, during, or after the assay. For example, the spatial composition of a material may be characterized by applying one or more detectable probes to the material over multiple cycles. Over successive cycles, residual detectable probes may remain randomly on the material, creating a non-uniform background signal during successive detection cycles. Residual detectable probes may eventually dissociate, meaning that the background signal may have spatial and temporal non-uniformities. In another example, the same above-described material characterization assay may be performed in an optically unsealed detection system, allowing some external radiation to be detected in the system. The external radiation does not have a uniform distribution over the material, resulting in a non-uniform gradient or distribution of radiation being detected in the background signal. The external radiation may not change over time, resulting in a spatially non-uniform but temporally uniform background.

The detectable probe may be characterized as producing a detectable signal that exceeds a background signal threshold. The fluorescently labeled detectable probe can be characterized as having a fluorescent signal (or other type of luminescent signal) that exceeds the background fluorescent signal. The luminescence signal may be quantified, for example, by total photon count over a fixed period of time, to obtain a background signal or a detected probe signal. The quantized luminescence signal may be measured at a point, location or address or over an area comprising a plurality of points, locations or addresses. The detectable probe may be configured to have a detectable signal exceeding a threshold background signal level, such as an average signal over the entire area or a maximum signal within the area. The total intensity of the signal from the detectable probe may be controlled by the total number of signaling markers (e.g., fluorophores) attached to the detectable probe. The spatial intensity of the probe (i.e., the signal intensity at a particular location) can be controlled by the density of the signaling tag (e.g., fluorophore) attached to the probe.

The detectable probe may be configured to produce a detectable signal that exceeds an expected or observed background signal. For example, a detectable probe configured to bind to a material or a binding partner on the material may produce a detectable signal that exceeds the measured background signal produced by the material. The detectable probe may generate a detectable signal that exceeds the background signal by a specific amount. Instead, the detectable probe may generate a detectable signal that is less than a detectable limit, depending on the mode of signal sensing. The detectable probe can produce a detectable signal strength that exceeds the background signal strength by at least about 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 40x, 50x, 75x, 100x or more. Alternatively or additionally, the detectable probe may have a detectable signal strength of no more than about 100x, 75x, 50x, 40x, 30x, 25x, 20x, 19x, 18x, 17x, 16x, 15x, 14x, 13x, 12x, 11x, 10x, 9x, 8x, 7x, 6x, 5x, 4x, 3x, 2x, 1.5x or less above background signal strength.

For a radiated signal (e.g., fluorescence), signal damping may occur due to one or more mechanisms that cancel or interfere with the radiated signal. Exemplary signal damping mechanisms may include quenching, self-quenching, photobleaching, and label loss. Quenching may occur due to the presence of chemicals that inhibit or absorb the emitted photons. Some substances in the characterization system may inherently absorb the emitted photons, resulting in a reduction or quenching of the radiation signal. In some configurations, a particular substance that inhibits the formation of a substance that quenches the radiation signal may be added to the detection system (e.g., an oxygen scavenger such as ascorbate). One particular form of quenching is self-quenching, in which the luminescence signal from a luminophore may be quenched by a second luminophore of the same or similar species. Self-quenching may be related to inter-label configuration, such as spacing between adjacent luminophores and relative orientation of luminophores. In some configurations, the luminescent signal may decrease as the spacing between adjacent luminophores decreases. In other configurations, the luminescence signal may increase as the spacing between adjacent luminophores decreases (e.g., forster resonance energy transfer).

Quenching, self-quenching and related optical phenomena can be characterized by effective quantum yields. Individual fluorophores can have characteristic or measured quantum yields. The effective quantum yield of a detectable probe composition comprising a plurality of fluorophores may be the quantum yield measured or characterized under assay conditions. The detectable probe composition can have a quantum yield of at least about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or more. Alternatively or additionally, the detectable probe composition can have a quantum yield of no greater than about 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, 0.90, 0.89, 0.88, 0.87, 0.86, 0.85, 0.84, 0.83, 0.82, 0.81, 0.80, 0.79, 0.78, 0.77, 0.76, 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.69, 0.68, 0.67, 0.66, 0.65, 0.64, 0.63, 0.62, 0.61, 0.60, 0.59, 0.58, 0.57, 0.56, 0.55, 0.54, 0.53, 0.52, 0.51, 0.50, 0.45, 0.4, 0.35, 0.3, 0.25, 0.15, or more effective.

The radiation signal may also be damped or otherwise attenuated by chemical mechanisms such as photobleaching or label loss. Without wishing to be bound by theory, photobleaching may occur due to irreversible chemical changes in the label, such as a fluorescent label. Photobleaching may be due to any known mechanism including photon interactions with fluorophores, photon interactions with species that may damage fluorophores (e.g., free radicals such as triplet oxygen), and interactions between chemical species and fluorophores that alter or damage fluorophores. Photobleaching and other forms of signal damping may be transient or time-dependent phenomena. For example, the photobleaching due to irradiation may increase in proportion to the total time or total irradiation time. Also, the total amount or concentration of destructive chemical modifying substances may affect the rate of photobleaching. For example, the severity of photobleaching can increase with increasing photon count or photon density. The rate of signal loss due to photobleaching or other chemical mechanism may be constant or variable over time (e.g., exponential, logarithmic).

The detectable probe composition may experience variable or increased signal loss over time due to quenching, self-quenching, photobleaching, label loss, or other mechanisms. The detectable probe composition can have a signal loss rate of at least about 0.001%/min, 0.01%/min, 0.1%/min, 0.5%/min, 1%/min, 2%/min, 3%/min, 4%/min, 5%/min, 6%/min, 7%/min, 8%/min, 9%/min, 10%/min, 15%/min, 20%/min, 25%/min, 30%/min, 35%/min, 40%/min, 45%/min, 50%/min, 55%/min, 60%/min, 65%/min, 70%/min, 75%/min, 80%/min, 85%/min, 90%/min, 95%/min, or greater. Alternatively or additionally, the detectable probe composition can have a signal loss rate of no greater than about 95%/min, 90%/min, 85%/min, 80%/min, 75%/min, 70%/min, 65%/min, 60%/min, 55%/min, 50%/min, 45%/min, 40%/min, 35%/min, 30%/min, 25%/min, 20%/min, 15%/min, 10%/min, 9%/min, 8%/min, 7%/min, 6%/min, 5%/min, 4%/min, 3%/min, 2%/min, 1%/min, 0.5%/min, 0.1%/min, 0.01%/min, 0.001%/min, or less.

The detectable probe may generate a detectable signal distinguishable from the background, for example by having an intensity exceeding that of the background signal or having a detectable feature distinguishable from the background signal. The detectable probe may be characterized as producing a signal intensity that exceeds the background signal for a given amount of time, although there are signal reduction mechanisms such as photobleaching and label loss. For example, a fluorescent detectable probe may have a continuously or discontinuously excited fluorescent signal that exceeds the background fluorescent signal for at least 10 minutes. The observable time may be defined as the shortest time length for which the detectable probe produces a detectable signal that is distinguishable from the local or average background signal. In some configurations, the observable time can be increased by increasing the total number of labeled components on the detectable probe. The observable time may be scaled in proportion to the number of labeled components attached to the detectable probe. The observable time of the detectable probes may be increased or even maximized to ensure detectability throughout the assay. The observable time of the detectable probe may be limited to a value that enables the removal of the desired signal, such as by photobleaching. The detectable probe can be observed for at least about 1s, 15s, 30s, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, or longer, or have an observable time as described above. Alternatively or additionally, the detectable probe may be observed for no more than about 120 minutes, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 30s, 15s, 1s, or less, or have an observable time as described above.

Detectable probes can improve the observability of binding interactions by generating a detectable signal far above background signal. Furthermore, the detectable probes may increase the observability of the binding interactions by increasing the spatial resolution of the detectable signal. The detectable probe may be configured to provide spatial resolution of the detectable signal in several ways, including: 1) Concentrating the label in a small area of the retained component to produce an optically dense signal source; 2) Distributing the labels over a large area of the retained component to produce an optically uniform signal source; or 3) concentrating the label on all or most of the retained components to produce an optically dense and uniform signal source. Fig. 12A-12B depict examples of spatial resolution of luminescent signals (e.g., fluorescent signals) using detectable probes. Fig. 12A depicts fluorescence count data, including background fluorescence counts, over a region (e.g., a surface of a material) when a single binding component comprising multiple fluorophores binds to a binding partner in the center of the region. The region may be uniformly divided into nine sub-regions, where each sub-region may be individually quantified for fluorescence (e.g., each sub-region may correspond to a sensor pixel). The number of fluorophores attached to an affinity reagent may be limited due to the structure and affinity of the affinity reagent. The fluorescence counts in fig. 12A show a modest increase in fluorescence counts for the central subregion, possibly due to binding interactions between the affinity reagent and the binding partner, but the signal intensity may not be sufficiently higher than the background counts for the eight subregions surrounding to conclude that binding interactions occur. FIG. 12B depicts a similar situation to FIG. 12A, but the detectable probe includes a significantly greater number of fluorophores than the affinity reagent depicted in FIG. 12A. The significantly increased fluorescent signal observed in the central subregion, as well as the increase in signal from the surrounding subregions (possibly due to signal crosstalk from the central subregion), increases the confidence that the observed signal is due to the binding interaction of the detectable probe with the binding partner.

The size of the detectable probes may be adjusted to provide a detectable signal on the solid support, surface, or region of field of view. The size of the region on which the detectable probe provides a signal may be determined by the size of the features within the region. For example, the size of the detectable probe may be adjusted to provide a signal over a region that is greater than the region occupied by the binding partner (e.g., polypeptide). The detectable probe may be designed to be larger than the binding partner to reduce background signal, for example by reducing autofluorescence from the binding partner or material adjacent to the binding partner. Alternatively, the detectable probe may be designed to be smaller than the binding partner to increase the spatial resolution of the detectable signal from the detectable probe.

In some configurations, the detectable probe composition may be designed to produce a detectable signal on a solid support, surface, or area of field of view that is larger than the binding partner to which the detectable probe may bind or have affinity. The observability of the probe over a specific area can be improved by increasing the distribution and/or concentration of the labeling component on the surface of the detectable probe. By including additional detection molecules that enhance the detectable signal provided by the detectable probe, the probe can generate a detectable signal over a relatively large area. Figure 13 shows a detectable probe composition for producing a detectable signal over a larger area relative to the area occupied by its recognized binding partner. The detectable probes 1310 bind to binding partners 1330 (e.g., polypeptides) that are optionally anchored to a surface or solid support 1370 by an anchor group 1340 (e.g., a nucleic acid, such as a structured nucleic acid particle, or a functional group linkage). The region of the detectable signal generated by the detectable probe 1310 is increased by linking the detectable probe 1310 to additional detection molecules 1320. The detection molecule 1320 may include a retention component and a plurality of label components (e.g., fluorophores). The retained component of the detection molecule 1320 need not be attached to the binding component. However, the remaining components of the detection molecules 1320 may be attached to binding components other than those present in the detectable probes 1310. The detection molecule 1320 is coupled to the detectable probe 1310 via a linker 1325. The linker 1325 may be a permanent linker (e.g., heterobifunctional linker, click reaction product, streptavidin-biotin linkage), or may be a non-permanent linker (e.g., hybridized nucleic acid). The linker may be flexible, e.g., allowing for direct interaction between the detection molecule 1320 and the detectable probe 1310; or the linker may be rigid, e.g., limiting the detection molecules 1320 to not interact with the detectable probes 1310. The complex formed by detectable probe 1310 and detection molecule 1320 may be formed prior to binding of detectable probe 1310 to binding partner 1330, or may be formed by binding the detectable probe followed by contacting detection molecule 1320 with detectable probe 1310.

The size of the detectable probe configured to bind to the binding partner may be smaller or larger relative to the size of the polypeptide. The size may be measured as volume, molecular weight, longest dimension, effective diameter, radius of gyration, hydrodynamic radius, projection (e.g., footprint), and the like. Alternatively, the size of the detectable probe configured to bind to the binding partner at a site (e.g., a site in a polypeptide array) may be smaller or larger relative to the size of the site. The size of the detectable probe or complex comprising the detectable probe may be adjusted to at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more compared to the size of the binding partner or site. Alternatively or additionally, the size of the detectable probe or complex comprising the detectable probe may be adjusted to be no more than about 1000%, 900%, 800%, 700%, 600%, 500%, 400%, 300%, 250%, 200%, 175%, 150%, 140%, 130%, 120%, 110%, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less compared to the size of the binding partner or site.

By including more than one type or kind of labeling component on the detectable probe, the labeling component of the detectable probe can be configured for multiplexed detection. The ability to attach multiple binding components to a detectable probe increases the dynamic flexibility of the detectable probe for multiple uses. The detectable probes can have a unique combination of labeling components that constitute a unique fingerprint or signature of the detectable probe. The binding cell may be produced from a mixture of detectable probes having different binding specificities, indicated by specific probe markers or fingerprints, such that the observed specific binding interactions can be determined by observing the markers or fingerprints. The label or fingerprint may be generated by varying the number of types or kinds of binding components and the ratio of labeling components attached to the detectable probes. The detectable probe can include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different kinds of label components. Alternatively or additionally, the detectable probes for the multiplex use compositions may comprise no more than about 10, 9, 8, 7, 6, 5, 4, 3, or less of different kinds of labeling components. The ratio of the first species of marker component to the second species of marker component may be at least about 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or greater. Alternatively or additionally, the ratio of the first species of marker component to the second species of marker component may be no greater than about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.1:1 or less.

The multiplex probe composition may comprise a plurality of detectable probes having different binding affinities, wherein the binding affinity of any given detectable probe may be determined by a unique fingerprint or signature of a labeling component on the detectable probe. The number of unique types of detectable probes in a multiplex probe composition can be determined by the type of detection system used to observe the binding interactions and the sensitivity of the detection system to distinguish between differences in probe markers or fingerprints. For fluorescent labeling, a fluorescent detection system may have a limited number of detection channels based on the fluorescence wavelength of the fluorophore on the detectable probe. The detection range within each detection channel may also limit the amount of each fluorophore added to the detectable probe.

The multiplex probe composition can include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more unique types of detectable probes. Alternatively or additionally, the multiplex probe composition can include no greater than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or fewer unique types of detectable probes. The uniqueness may be manifested as a number or diversity of components present in the detectable probe, such as a number or diversity of binding components present in the probe, a number or diversity of labeling components present in the probe, a number or diversity of retention components present in the probe, and/or a number or diversity of labels or barcodes present in the probe.

The detectable probe may be configured to detect by Forster Resonance Energy Transfer (FRET). In some configurations, the detectable probe may contain an attached fluorophore pair configured to be detected by a FRET mechanism. Without wishing to be bound by theory, efficient FRET detection may utilize an absorbing fluorophore and an emitting fluorophore located within a sufficient distance to allow efficient transfer of energy between fluorophores. In some configurations, FRET may provide the advantage of increasing the number of probes distinguishable from each other using a given excitation source. For example, the first probe may include a fluorophore component D that emits at a first wavelength when excited by an excitation source. The second probe may comprise a fluorophore component D and a fluorophore component a, wherein excitation of the fluorophore component D by the excitation source results in energy transfer to the fluorophore component a, which in turn emits at a second wavelength. Standard optics can be used to resolve the two emission wavelengths to distinguish the two probes from each other.

FIGS. 14A-14B depict various configurations of detectable probes that can be detected by a FRET mechanism. FIG. 14A depicts a method for using the helical structure of a nucleic acid to pair a donor fluorophore and an acceptor fluorophore by a critical pairing distance Δs _FRET Positioning to create a configuration of FRET fluorophore pairs. The retention component may include a continuous nucleic acid strand 1410. Oligonucleotide 1420 may hybridize to continuous nucleic acid strand 1410, creating regions of double-stranded nucleic acid and single-stranded nucleic acid 1425. The oligonucleotides may include donor fluorophores 1430 or acceptor fluorophores 1432 in an alternating pattern to provide a suitable spacing Δs _FRET A FRET fluorophore pair is generated. FIG. 14B depicts an alternative method of generating a FRET fluorophore pair using a binding molecule configured to hybridize to a detectable probe. The detectable probe 1440 can include a plurality of donor fluorophores 1430 along a portion of the detectable probe 1440 configured to bind to the binding molecule 1445. Binding molecules 1445 include a plurality of acceptor fluorophores 1432 arranged along the edges of the binding molecules 1445 that bind to the detectable probes 1440. Contact of the detectable probes 1440 with the binding molecules 1445 can result in the formation of a binding region 1450, aligning the donor and acceptor fluorophores 1430, 1432 to form a probe region at the appropriate FRET spacing Δs _FRET A FRET fluorophore pair within. In some configurations, binding molecule 1445 may be coupled to a binding partner or the location where the binding partner is located, allowing FRET interaction to occur when detectable probe 1440 binds to the binding partner and binding molecule 1445 then binds to detectable probe 1440. In some configurations, binding molecule 1445 may be SNAP or other substance that is attached to a binding partner of one or more binding components attached to detectable probe 1440. Thus, binding of the probe to the binding partner can be determined based on observation of FRET between donor fluorophore 1430 and acceptor fluorophore 1432.

The detectable probe composition can include a coupled donor and acceptor luminophore pair. Acceptable donor/acceptor pairs may include Cy2/Cy3, cy3/Cy5, FITC/TRITC, PE/APC, alexa-488/Alexa-/>514、Alexa-/>488/Alexa-/>532、Alexa-/>488/Alexa-/>546、Alexa-488/Alexa-/>610、Alexa-/>647/Alexa-/>680、Alexa-/>647/Alexa-/>700、Alexa-/>647/Alexa-/>750. Cyan FP/YFP, cerulean FP/YFP, GFP/mRFP or combinations thereof. />

The FRET dye pair may be coupled to a nucleic acid, such as an oligonucleotide or a scaffold strand forming part of a structured nucleic acid particle. Nucleic acids containing FRET dye pairs may have modified nucleotides to which the dye is attached at sufficient spacing to allow FRET interactions to occur. The appropriate spacing may be determined by the naturally occurring helical structure that occurs when the oligonucleotide hybridizes to the scaffold strand. Two adjacent dyes in a FRET pair may be separated by at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. Alternatively or additionally, two adjacent dyes in a FRET pair may be separated by no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or fewer nucleotides apart.

Binding interactions involving detectable probes can be observed using a label component other than a fluorophore. The detectable probe can include a barcode label component (e.g., a nucleic acid barcode). The barcode label component may include a unique sequence (e.g., DNA, RNA, amino acids) that, when decoded, provides information identifying the type of detectable probe. For example, a plurality of identical detectable probes may each be labeled with the same kind of barcode (i.e., barcodes having the same sequence) to provide a uniform and single binding interaction signal for that particular detectable probe. The barcode signal can be detected and decoded by methods such as next generation sequencing to obtain an observation of the interaction between the detectable probe and the binding partner, epitope or target moiety.

FIGS. 15A-15D depict a system for recording the binding interactions between a detectable probe and a binding partner, epitope or target moiety using a nucleic acid barcode. Fig. 15A depicts a system containing a detectable probe 1510 that includes a nucleic acid barcode sequence linked to the detectable probe 1510 through a linker 1520. The nucleic acid barcode sequence includes two priming sequences 1522 and a recognition sequence 1525 between the two priming sequences 1522. The detectable probes 1510 bind to a binding partner, epitope or target moiety 1530 that is optionally bound to a solid support 1570. The binding partner, epitope, or target moiety 1530 may be bound to solid support 1570 by an anchor group 1540 (e.g., SNAP or chemical linker). The binding partner, epitope, or target moiety 1530 may have an associated linker 1550 (e.g., attached to solid support 1570, anchor group 1540, or binding partner, epitope, or target moiety 1530). The associated linker 1550 can terminate with a complementary priming sequence 1552 that forms a hybrid bond with the priming sequence 1522 of the nucleic acid barcode of the detectable probe 1510. The complex formed by the detectable probe 1510 and the binding partner, epitope, or target moiety 1530 is contacted with a polymerase 1560 configured to bind nucleic acid. Fig. 15B shows that polymerase 1560 binds to a hybridized nucleic acid sequence formed by ligation of priming sequence 1522 and complementary priming sequence 1552 to initiate an extension reaction. Fig. 15C depicts the final step of the extension reaction. Recognition sequence 1525 and priming sequence 1522 have been added to the terminal sequence of association linker 1550 by extension to add complementary recognition sequence 1555 and additional complementary priming sequence 1552. Fig. 15D depicts the binding partner, epitope or target portion 1530 after multiple cycles of detectable probe 1510 binding and primer extension. Each probe adds a unique recognition sequence (e.g., 1555, 1556, 1557) to the sequence at the end of linker 1550. The addition of complementary priming sequences 1552 at the end of each validation sequence (1555, 1556, 1557) allows subsequent binding of the linker 1550 to the detectable probe. The detectable probes 1510 are shown with an optional plurality of marker components (shown as 6 pointed stars). It will be appreciated that the detectable probe need not include a label, for example in a configuration in which the confirmation sequence is decoded to determine the binding history of the target moiety 1530. Thus, the affinity reagent may be configured or used as illustrated in FIGS. 15A-15D.

Other examples of tags that can be attached to the affinity reagents of the present disclosure, and methods of using and detecting such tags, for example in assays to detect, sequence or quantify polypeptides, are set forth in U.S. patent application publications 2020/03481308 A1, 2020/0348307A1 or 2019/0145982A1, each of which is incorporated herein by reference.

Method for producing detectable probes and affinity reagents

The detectable probes or affinity reagents as described in the present disclosure can be made by suitable methods. The preparation of the detectable probe or affinity reagent may comprise one or more of the following steps: 1) Generating a retention component configured to attach a plurality of binding components and/or a plurality of labeling components; 2) Attaching one or more binding components to the retention component; 3) Attaching one or more marker components to the retention component; and 4) attaching an additional component to the retained component.

The retained components may be obtained by the manufacturing process. Non-nucleic acid retaining components (e.g., polymers, metals, ceramics, carbon, or semiconductor nanoparticles) can be fabricated by batch fabrication and/or purification processes. After the non-nucleic acid retaining component is produced, one or more processing steps may be performed to add one or more surface functional groups to the retaining component. The functional groups may be added for the purpose of improving the solvent solubility characteristics of the retention component or to provide attachment sites for the binding component and/or the labeling component. The surface functional groups may include functional groups configured to attach the binding component and/or the labeling component (e.g., functional groups configured to undergo a click reaction), or nucleic acids configured to hybridize to complementary nucleic acids containing the attached binding component and/or labeling component. For example, the retention component comprising silicon or silica nanoparticles may be functionalized with a silylated compound to covalently add multiple functional groups to the silicon-containing surface of the particles. Following functionalization of the non-nucleic acid retaining component, the affinity group can be attached to the retaining component by any suitable technique, such as a click reaction or nucleic acid hybridization.

The preparation of the nucleic acid retaining component (e.g., nucleic acid folded paper, nucleic acid nanospheres) can be formed by conventional techniques. Nucleic acid nanospheres can be made by methods such as rolling circle amplification to create scaffold chains that can be further modified to attach multiple binding and/or labeling components. An exemplary method of preparing nucleic acid nanospheres is described, for example, in U.S. patent No. 8,445,194, which is incorporated herein by reference. Nucleic acid retaining components, including nucleic acid paper folding, can be made, for example, using the techniques described in Rothennd, nature440:297-302 (2006), and U.S. Pat. Nos. 8,501,923 and 9,340,416, each of which is incorporated herein by reference.

FIG. 21A shows a first approach to forming a detectable probe with a nucleic acid retaining component. The oligonucleotides with attached binding component 2120 and the oligonucleotides with attached labeling component 2130 are prepared prior to assembly of the retention component. The oligonucleotide with attached binding component 2120 and the oligonucleotide with attached labeling component 2130 are contacted with single stranded scaffold 2110 (e.g., M13 phage DNA, plasmid DNA) and additional structural nucleic acid 2140. The nucleic acids are contacted in a suitable DNA buffer at an elevated temperature (e.g., at least about 50 ℃, 60 ℃, 70 ℃, 80 ℃, or 90 ℃) and then cooled. The oligonucleotides will hybridize to the support strand 2110 at the appropriate sequence-dependent positions to form detectable probes 2150.

FIG. 21B shows an alternative approach to forming a detectable probe with a nucleic acid retaining component. An oligonucleotide having a handle configured to attach the binding component 2125 and an oligonucleotide having a handle configured to attach the labeling component 2135 are prepared prior to assembly of the retention component. An oligonucleotide having a handle configured to attach a binding component 2125 and an oligonucleotide having a handle configured to attach a labeling component 2135 are contacted with single stranded scaffold 2110 (e.g., M13 phage DNA or plasmid DNA) and additional structural nucleic acid 2140. The nucleic acids are contacted in a suitable buffer at an elevated temperature (e.g., at least about 50 ℃, 60 ℃, 70 ℃, 80 ℃, or 90 ℃) and then cooled. Upon cooling, a retention component 2155 is formed that is configured to bind the plurality of binding components and/or labeling components. The retention component 2155 is contacted with a plurality of binding components 2128 and/or labeling components 2138 having a stem complementary to the stem on the retention component 2155 in a suitable attachment buffer. After attachment of the plurality of binding components 2128 and/or the plurality of labeling components 2138, detectable probes 2150 are formed.

In some configurations, the detectable probe or affinity reagent may be formed by attaching the binding component and/or the labeling component by the following reaction: reactions of functional groups configured to form a bond with another molecule or group, such as bio-orthogonal reactions or click chemistry (see, e.g., U.S. patent nos. 6,737,236 and 7,427,678, each of which is incorporated herein by reference in its entirety); azide alkyne Huisgen cycloaddition reactions using copper catalysts (see, e.g., U.S. patent nos. 7,375,234 and 7,763,736, each of which is incorporated herein by reference in its entirety); the following portions of the copper-free Huisgen reaction ("no metal click") were used: strained alkyne or triazine-hydrazine moieties, which can be linked to aldehyde moieties (see, e.g., U.S. patent No. 7,259,258, which is incorporated by reference); a triazine chloride moiety, which may be linked to an amine moiety; a carboxylic acid moiety that can be attached to the amine moiety using a coupling reagent such as EDC; a thiol moiety, which may be attached to the thiol moiety; an olefinic moiety, which can be linked to a diene moiety coupled by a diels-alder reaction; and an acetyl bromide moiety, which may be linked to a phosphorothioate moiety (see, e.g., WO 2005/065814, which is incorporated by reference). The functional group may be configured to be via a click reaction (e.g., metal-catalyzed azide-alkyne cycloaddition, strain-promoted azide-nitrone cycloaddition, strain-alkene reaction, thiol-alkene reaction, diels-alder reaction, reverse electron demand diels-alder reaction, [3+2] cycloaddition, [4+1] cycloaddition, nucleophilic substitution, dihydroxylation, thiol-alkyne reaction, photocclick, nitrone dipolar cycloaddition, norbornene cycloaddition, oxanorbornadiene cycloaddition, tetrazine ligation, tetrazole click reaction). Exemplary silane derivative CLICK reactants may include alkenes, alkynes, azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines (e.g., dibenzocyclooctyne-azide, methyltetrazine-trans-cyclooctene, epoxide-thiols, etc.). Click reactions can provide an advantageous method of rapid bonding under mild conditions (e.g., room temperature, aqueous solvents).

In some configurations, the retention component or other component of the detectable probe or affinity reagent may include different types of functional groups. The use of different functional groups may provide a level of control over the number and location of different components to be attached to the detectable probe or affinity reagent. In a particular configuration, the different functional groups exhibit orthogonal reactivity whereby the first component has a portion that is reactive with the first functional group on the probe but substantially non-reactive with the second functional group on the probe, and whereby the second component has a portion that is reactive with the second functional group but non-reactive with the first functional group. Thus, the number and location of the different binding components may be adjusted by appropriate use of orthogonal functional groups on the detectable probes or affinity reagents, or the number and location of the different labeling components may be adjusted by appropriate use of orthogonal functional groups on the detectable probes or affinity reagents. Furthermore, by appropriately using orthogonal functional groups on the detectable probe or the affinity reagent, respectively, the binding component can be localized differently to the labeling component on the detectable probe or the affinity reagent.

The retention component comprising the non-nucleic acid may be formed by a suitable method. Of particular interest are methods that allow a degree of spatial control in attaching the binding, labeling or other components to the retention components during probe assembly. Spatial control may include the ability to separate or isolate probe components or to change the orientation of the probe components. For example, spatial control may include separating the binding and/or labeling components from adjacent or neighboring binding and/or labeling components. In another example, spatial control may include isolating regions of the attached binding component from regions of the attached labeling component. In yet another example, spatial control may include controlling the relative dispersion or distribution of the binding and/or labeling components, e.g., providing the binding and labeling components in a 10:1 ratio in the region where the components are retained.

FIGS. 32A-32B depict schemes for controlling the position of probe components during assembly of a detectable probe or affinity reagent that includes non-nucleic acid retaining components. Fig. 32A depicts a plurality of particles 3210 (e.g., nanoparticles, nanobeads, nanospheres, etc.) configured to associate with an interface 3220, such as a multiphase boundary (e.g., air/liquid interface, oil/water interface). An interface 3220 is formed between first fluid medium 3222 and second fluid medium 3224. Portions of each particle of the plurality of particles 3210 are exposed to a different modifying chemical depending on whether the first liquid medium 3222 or the second liquid medium 3224 is exposed. The portion of each particle of the plurality of particles 3210 exposed to the first liquid medium 3222 forms a first plurality of functional groups 3235 on a surface of the particle. The portion of each particle of the plurality of particles 3210 that is exposed to the second liquid medium 3224 forms a second plurality of functional groups 3230 on the surface of the particle. The first plurality of functional groups 3235 can provide attachment sites for a first type of probe component (e.g., a binding component), and the second plurality of functional groups 3230 can provide attachment sites for a second type of probe component (e.g., a labeling component).

FIGS. 32C-32E depict schemes for controlling the position of probe components during assembly of a detectable probe or affinity reagent that includes non-nucleic acid retaining components. Fig. 32C depicts a plurality of particles 3210 (e.g., nanoparticles, nanobeads, nanospheres, etc.) partially embedded or immobilized within a medium 3226. A coating or layer 3240 (e.g., a metal, metal oxide, polymer, or hydrogel) is applied to the exposed portion of each of the plurality of particles 3210. As shown in fig. 32D, after the coating or layer 3240 is applied to the plurality of particles 3210, the medium 3226 may be removed, thereby providing the plurality of particles 3210 with a partial coating or layer 3240. As shown in fig. 32E, different surface chemistries of the uncoated and coated portions of the plurality of particles 3210 may be used to differentially functionalize the plurality of particles 3210 with the partial coating or layer 3240. The coating or layer 3240 can be provided with a first plurality of functional groups 3235, which can provide attachment sites for a first type of probe component (e.g., binding component). The uncoated portion of each particle can be provided with a second plurality of functional groups 3230, which can provide attachment sites for a second type of probe component (e.g., binding component).

The location or positioning of the binding component and/or the labeling component on the non-nucleic acid retaining component can also be controlled by controlling the spatial or surface density of the attachment sites on the surface of the non-nucleic acid retaining component. Fig. 33 depicts the preparation of particle or nanoparticle retention groups. The particle or nanoparticle 3310 is combined with a mixture of moieties (e.g., binding component attachment sites 3320, labeling component attachment sites 3330, or modifying groups 3340) to be attached to the surface of the particle or nanoparticle 3310. The ratio of the components within the mixture of the portions is balanced to ensure that the particle or nanoparticle 3310 surface is proportionally modified. The end result is a retention of components, each component being uniformly or nearly uniformly distributed over the surface of the particle or nanoparticle 3310 based on the concentration of the component in the portion of the mixture. Alternatively, the components may be added sequentially by initially adding a two-component mixture consisting of the first type of attachment site and blocking group. After surface functionalization, the blocking groups can be removed, either completely or partially, to provide surface sites for attachment of other types of attachment sites.

The plurality of affinity reagents, the plurality of detectable probes, or the combination of at least one affinity reagent and at least one detectable probe may be conjugated to form a multi-probe complex. Similarly, a detectable or affinity reagent may be configured with one or more coupling groups that allow the reagent or probe to attach to one or more other reagents or probes. The coupling groups may be configured to form covalent interactions, non-covalent interactions, electrostatic interactions, magnetic interactions, or any other interactions that form an association between the detectable probe affinity reagent or both. The association between two or more probes in a multiprobe complex may be weak, temporary, or reversible. The association between two or more probes in a multiprobe complex may be strong, permanent or irreversible. In some cases, the detectable probes or affinity reagents may be coupled by hybridization of complementary nucleic acid strands or streptavidin-biotin coupling groups. In other cases, the detectable probes or affinity reagents may be covalently coupled by, for example, a click reaction or cross-linking (e.g., chemical or photoinitiated cross-linking).

The multi-probe complex may be formed as part of the probe synthesis process. The formation of probes or retention components can facilitate the positioning, orientation, and attachment of probe components, such as binding components and/or labeling components. FIGS. 37A-37C depict a process for using a complex to adjust the position of a binding component on a non-nucleic acid retaining component. Fig. 37A depicts two particle retention components 3710 (e.g., nanoparticles) associated with a phase boundary 3720 formed between a first medium 3722 and a second medium 3724. The retention component 3710 is configured to form a complex through attractive interactions (e.g., electrostatic or magnetic attraction, miscible surface functionalities, etc.) between the particles. The retention component 3710 is contacted with a plurality of binding components 3730 within the second medium 3724, the plurality of binding components configured to attach to attachment sites on the particles 3710. Fig. 37B depicts composite particle 3710 after a plurality of binding components 3730 have been attached to the particle surfaces within second medium 3724. Because the surface area within first medium 3722 is excluded and the surfaces near the inter-particle association regions are excluded, binding component 3730 is limited to certain regions of the surface of particles 3710. Fig. 37C shows an optional process in which interactions between particles are disrupted, releasing individual detectable probes 3740. In other configurations, the particles may be retained as multi-probe complexes.

The nucleic acid-based retention component (e.g., a scaffold as set forth in U.S. provisional application No. 63/112,607) can be synthesized by standard nucleic acid synthesis chemistry and provided with specific labeling groups, e.g., by incorporating pre-labeled nucleotides, or by providing attachment sites for labeling groups to be added later, e.g., providing functional side groups and one or more sites for coupling with binding components. Also, PEG scaffolds may be synthesized and functionalized with functional groups that will allow either or both of the attachment component or the labeling component. In another example, a polypeptide scaffold can be synthesized that includes groups that will allow attachment of either or both of the probe component or the label component. In some cases, the retention component may be much larger than the tag to which it is attached. In other cases, the retention component may be similar in size to the marker component to which it is attached. In other cases, the tag component may be larger than the retention component to which it is attached.

In some cases, the retention component, such as a nucleic acid scaffold, may be synthesized directly on top of the binding component. Reagents for synthesizing the retention component may include a binding component. The retention component, such as a nucleic acid scaffold, may be synthesized directly on the linker molecule to be attached to the binding component. For example, reagents for synthesizing the retention component may include linkers. In some embodiments, the retention component may be produced by a template-mediated polymerase extension reaction using a mixture of nucleotides in which some or all of the nucleotides to be incorporated have a functional moiety for attachment of the labeling component. For example, the three nucleotides used by the polymerase during extension may be unlabeled (and/or may lack a functional group), and the fourth nucleotide may be labeled with a fluorescent moiety or other labeling moiety (or the fourth nucleotide may have a functional group). In this example, the complementary base of the fourth nucleotide may appear in the template in a predetermined pattern. For example, the complementary base of the fourth nucleotide may occur at the nucleotide spacing set forth elsewhere herein.

As shown in fig. 49A, the binding component, shown here as a nucleic acid aptamer, may include a primer sequence at one end (e.g., the 3' end). The template nucleic acid comprising a sequence segment complementary to the primer sequence as described above may then be hybridized to the primer segment of the binding component, for example (see fig. 49B, step 1). As described above, for example, in the presence of unlabeled and labeled nucleotides, polymerase-mediated extension of a template-based primer sequence over a binding component then results in an affinity probe that includes the binding component (e.g., aptamer) and a labeling component that includes a nucleic acid with a labeled retention component incorporated (extension product of a polymerase reaction with labeled nucleotides (see FIG. 49B, step 2)), in some cases, the template may include a particular nucleotide species, e.g., guanosine nucleotides at a desired interval or at a desired position, while the extension reaction is performed in the presence of unlabeled adenosine triphosphate, thymidine and guanosine and labeled cytosine triphosphate, this results in the labeled cytosine nucleotides being periodically incorporated into the extension product scaffold, for example, by including a template with guanosine nucleotides at every 3 rd, 4 th, 5 th position in the template, it may allow incorporation of labeled cytosine at every 3 rd, 4 th, 5 th position of the retention component, it may be that a particular label may be at a desired interval or at a desired position, and the label may be selectively incorporated at a particular site in the template, or may be later in the context of a particular label, as described herein, for example, the nucleotide may be provided for the label to be selectively incorporated in the label, or in situ.

In some cases, templates for scaffolds may include circular nucleic acids, where extension of the primer sequence produces an extended concatemer with a repeat sequence segment, e.g., by rolling circle amplification. In an optional form, a template sequence coupled to the binding component may be provided. The primer can be hybridized to the template and extended by polymerization in the presence of labeled and unlabeled nucleotides to produce a second association strand comprising a labeling group as described above.

In some cases, the affinity reagents or detectable probes may be constructed in a modular format that allows for more targeted adjustment of the end product labeled probes. For example, in some cases, binding components such as aptamers or antibodies can be generated and maintained as unlabeled libraries. In such cases, these binding components may be maintained with an attachment region for coupling the labeling component to the binding component, depending on the desired labeling protocol for a given experiment. Such attachment regions may include chemical coupling moieties such as NHS esters, "Click" Chemistry components (see, e.g., H.C.Kolb; M.G.Finn; K.B.Sharpless (2001), "Click Chemistry: diverse Chemical Function from a Few Good Reactions" Angewandte Chemie International edition.40 (11): 2004-2021), as well as other conventional chemical coupling methods, wherein the labeling components include any necessary complementary coupling moieties. The advantage of such modularity is that a plurality of different detectable probes or affinity reagents can be readily formed using the same or similar retention components. Thus, the plurality of different detectable probes or affinity reagents may differ in one or more of the number of label components, the type of label component, the number of binding components, and the type of binding component, while having a retention component that shares a common structural feature. The common structural features may be, for example, the size of the retention component, the shape of the retention component, the chemical composition of the retention component, the nucleic acid sequence of the retention component, the three-dimensional structure of the fold in the retention component, the three-dimensional structure of the scaffold in the fold in the retention component, and the like. In some configurations, the plurality of different detectable probes may differ in the number of annealed staple oligonucleotides in the paper folding structure, the location on the scaffold chain to which one or more staple oligonucleotides are annealed in the paper folding structure, the length of one or more staple oligonucleotides on the scaffold chain to which they are annealed in the paper folding structure, the sequence of one or more staple oligonucleotides on the scaffold chain to which they are annealed in the paper folding structure, or the number, type, or location of functional groups in the paper folding structure.

In some cases, the binding component may comprise one member of a binding or coupling pair (e.g., a receptor), while the labeling component comprises a complementary member of the binding or coupling pair (e.g., a ligand for the receptor). For example, in some cases, the binding component may be coupled to a single stranded nucleic acid sequence, while the labeling component is coupled to a second strand having a sequence complementary to the single strand. In such a case, the coupling may be performed by hybridization of the label-bound nucleic acid strand with the nucleic acid strand bound by the binding component. It will be appreciated that in the case of an aptamer binding component, the production of the aptamer and the tag-coupling component may be produced in a manner employing, for example, PCR amplification of the single-stranded probe and the tag-coupling component, followed by removal of the complementary strand prior to hybridization of the tag component.

In any of the above contexts, the labeling component and the retention component may be synthesized separately from the binding component, either with the labeling group attached, or as a functionalized retention component, to which the labeling component may be subsequently attached either before or after coupling with the binding component. In the case of nucleic acid-based retained components, such structures can generally be synthesized by well-known solid phase nucleic acid synthesis techniques, wherein known nucleotides are added consecutively to construct the polynucleotide structure, or amplified by polymerase chain reaction of the scaffold sequence. As noted, such processes may employ periodic introduction of labeled nucleotides during synthesis in order to construct multi-labeled probes in a desired form. Alternatively, modified nucleotides may be incorporated during synthesis, which allows for easy addition of the labeling component after synthesis.

Covalent attachment of the separately synthesized retention component to the other component (e.g., the binding component, the labeling component, or the other retention component) may be accomplished via chemical or biochemical means, such as chemical coupling of the nucleic acid scaffold to the binding component, e.g., using known chemical coupling techniques, such as click chemistry, or by biochemical means, such as attachment of the nucleic acid scaffold to the nucleic acid component of the binding component. Alternatively, the retention component may be non-covalently attached to the binding component by hybridization to a complementary nucleic acid component that has been coupled to the binding component. An example of such attachment is schematically shown in fig. 51. As shown, the binding component 400 includes a nucleic acid probe sequence 400b attached thereto. A separately synthesized labeled nucleic acid retaining component 401 having a sequence complementary to the probe sequence 400b is then hybridized to the binding component to provide a labeled affinity probe.

Any of a variety of covalent or non-covalent chemicals may be used to attach or link the components of the detectable probes or affinity reagents set forth herein. The chemicals and methods set forth herein in the context of attaching a retention component to other components can also be used to attach a detectable probe or affinity reagent to other substances, such as binding partners (e.g., polypeptides), surfaces, solid supports, sites of arrays, or particles. In addition, the chemicals and methods may be used to synthesize retention, binding or labeling components, or to add functional groups or linkers to such components.

In some configurations employing polypeptide-based binding components, such as antibodies or antibody fragments, a coupling method may be used to couple only a single labeling component to a single binding component by incorporating a single coupling group into a given polypeptide. In particular, a binding component such as an antibody or antibody fragment may be provided with a first coupling handle or functional group, while a separate labeling component (or component that may be readily labeled) is provided with a second coupling handle or functional group that reacts or binds with the first coupling moiety to effect attachment between the binding component and the labeling component.

An example of such a method is the SpyTag/SpyCatcher labeling method (see, e.g., zaker B, fierer JO, celik E, chittock EC, schwarz-Linek U, moy VT, howarth M (3. 2012.) "Peptide tag forming a rapid covalent bond to aprotein, through engineering a bacterial adhesin". Proceedings of the National Academy of Sciences of the United States of America.109 (12): E690-7). In this method, a 13 amino acid Tag polypeptide (SpyCatcher) forms a first coupling handle, and a 12.3kDa protein (Spy Tag) forms another coupling handle. For example, spy catchers can be integrated into a first component (e.g., a binding component or a labeling component) as a recombinant fusion protein. The Spy Catcher component irreversibly binds to Spy Tag through an isopeptide bond, which can be fluorescently labeled or otherwise labeled for detection. It will be appreciated that Tag or latch may be integrated with the first component.

In some cases, the different components may be attached to each other using click chemistry. In the case of components having a nucleic acid portion, these may be attached using ligation and/or hybridization. In some cases, a joint is used for attachment. Examples of linkers include double stranded DNA, single stranded DNA, or polyethylene glycols of different molecular weights. The linker may also include functional groups that allow bioconjugation.

In some cases, the attachment may employ chemical conjugation, bioconjugate, enzymatic conjugation, photo-conjugation, thermal conjugation, or a combination thereof. (Spicer, C.D., pashuck, E.T.,&stevens, M.M., achieving Controlled Biomolecule-biological Condition.chemical reviews, 2018,118, pages 7702-7743, and Greg T.Herman, "Bioconjugate Techniques", academic Press; version 3, 2013, the disclosure for this disclosure is incorporated herein by reference). For example, bioconjugates can be used to form covalent bonds between two molecules, at least one of which is a biomolecule. In some cases, two components of the detectable probe or affinity reagent attached to each other may be functionalized. Functionalization of the two partners can increase the efficiency or speed of the attachment (e.g., conjugation) reaction. For example, a thiol (-SH) or amine (-NH) group of the chemically active site of an aptamer, biological or chemical entity ₂ ) May be functionalized to allow greater reactivity or efficiency of the attachment reaction. Any of a variety of thiol-reactive (or thiol-reactive) or amine-conjugated chemistries may be used to couple the chemical moiety to the thiol or amine group. Real worldExamples include, but are not limited to, the use of haloacetyl, maleimide, aziridine, acryl, arylating agents, vinyl sulfone, pyridyl disulfide, TNB-thiol, and/or other thiol-reactive/amine-reactive/thiol-reactive reagents. Many of these groups are attached to the sulfhydryl group by alkylation (e.g., by formation of a thioether or amine bond) or disulfide exchange (e.g., by formation of a disulfide bond). Further strategies and details regarding the reaction of bioconjugation are described below and can be extended to other suitable molecules.

Attachment may be accomplished in part by chemical reaction of a chemical moiety or linker molecule with a chemically active site on a biological molecule or other substance. Chemical conjugation may be via an amide formation reaction, a reductive amination reaction, an N-terminal modification, a thiol michael addition reaction, a disulfide formation reaction, a copper (I) -catalyzed alkyne-azide cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition reaction (sparc), a strain-promoted alkyne-nitrone cycloaddition (sparc), a retro-electron demand diels-alder (IEDDA) reaction, an oxime/hydrazone formation reaction, a free radical polymerization reaction, or a combination thereof. The enzyme-mediated conjugation may be via transglutaminase, peroxidase, sortase, spyTag-spycatcheter, or a combination thereof. The photo-conjugation and activation may be via a photo-acrylate cross-linking reaction, a photo-thiol-ene reaction, a photo-thiol-alkyne reaction, or a combination thereof. In some cases, the attachment or conjugation may be via non-covalent interactions, which may be by self-assembling peptides, binding sequences, host-guest chemistry, nucleic acids, or combinations thereof.

In some cases, site-selective methods can be used to modify the reactive moiety of a detectable probe, affinity reagent, or component thereof to increase attachment efficiency, ease of use, and/or reproducibility. Three common strategies are available for site-selective attachment, (i) a single motif may be selected among a multitude of motifs rather than a modification strategy that targets a general functional group or moiety. This can be determined by the surrounding sequence, local environment or fine differences in reactivity. The ability of enzymes to modify specific amino acids or glycans at individual positions within a protein sequence is particularly pronounced. Reactions exhibiting fine chemoselectivity also fall into this category, such as those that are uniquely reactive towards the N-terminus of the protein or the anomeric position of the glycan. (ii) Site-specific incorporation of non-native functional groups by hijacking the native biosynthetic pathway can be utilized. (iii) The unique reactivity of the installation via chemical synthesis can be exploited. Complete or partial synthesis of polypeptides and oligonucleotides is common, particularly by solid phase methods. These techniques allow sequences of up to 100 amino acids or 200 nucleotides and allow the installation of a wide variety of functionalized monomers with precise positional control.

In some cases, chemical conjugation techniques may be applied to create attached substances, such as biomaterial-biomolecule conjugates. The functional groups for attachment may be natural to the substance to be modified (e.g., the biomolecule to be modified) or may be synthetically incorporated. In the following illustrations, R and R' may be biomolecules (e.g., without limitation, SNAP, proteins, nucleic acids such as nucleic acid paper folding or nucleic acid nanospheres, carbohydrates, lipids, metabolites, small molecules, monomers, oligomers, polymers), affinity reagents, detectable probes, binding components, labeling components, retention components, and/or solid supports.

In some cases, reductive amination may be used for attachment, such as via bioconjugation. The amine can react reversibly with the aldehyde to form a transient imine moiety with elimination of water. This reaction occurs at a rapid equilibrium, and due to the high concentration of water, unconjugated starting materials are very advantageous under aqueous conditions. However, in the second step, the labile imine may be irreversibly reduced to the corresponding amine via treatment with sodium cyanoborohydride. Such mild reducing agents can selectively reduce imines even in the presence of unreacted aldehyde. As a result, the biomolecule or other substance may gradually undergo irreversible conjugation with a second substance, such as a biological material of interest. In contrast, stronger reducing agents such as sodium borohydride are also capable of reducing aldehydes. This two-step reductive amination process can also be used for ketone modification. For example, reductive amination is thus mainly used to modify sodium periodate treated alginate and chitosan scaffolds. The order of reactivity of the attached reducing sugars can also be reversed by using terminal aldehydes/ketones produced in open chain form. This strategy can be used, for example, to mimic the glycosylation and galactosylation patterns of native collagen in ECM via reductive amination of maltose and lactose, respectively.

In some cases, isothiocyanates can be used to attach substances to each other. For example, isothiocyanates of biomolecules or solid supports can be used for bioconjugation. The isothiocyanate moiety can react with nucleophiles such as amines, sulfhydryl groups, phenolic acid ions of tyrosine side chains, or other molecules to form stable linkages between the two molecules.

In some cases, isocyanates may be used to attach two substances. For example, biomolecules or isocyanates of a solid support may be used for bioconjugate. For example, isocyanates may react with amine-containing molecules to form stable isourea linkages.

In some cases, acyl azide may be used to attach two substances. For example, biomolecules or acyl azides of solid supports can be used for bioconjugation. For example, acyl azide is an activated carboxylate group that can react with a primary amine to form an amide bond.

In some cases, amides may be used to attach two substances. For example, amides of biomolecules or solid supports can be used for bioconjugation. For example, the use of reactive N-hydroxysuccinimide (NHS) esters is particularly widespread. Although NHS-esters may be preformed, they are typically instead generated in situ using N- (3- (dimethylamino) propyl) -N' -Ethylcarbodiimide (EDC) coupling chemistry and coupled directly with the substance of interest. While formation of activated NHS-esters is favored under mildly acidic conditions (pH 5), subsequent amide coupling is accelerated at higher pH where the amine coupling partner is not protonated. One-step modification at an intermediate pH of 6.5 is possible. Attachment is typically performed by first forming the active NHS-ester at pH 5, then raising the pH to 8 and adding the amine coupling partner in a two-step procedure. In some cases, the water-soluble derivative sulfo-NHS may be used as a surrogate. In some cases, NHS esters of biomolecules or other substances can react with tyrosine, serine, and threonine-OH groups and couple, as opposed to N-terminal alpha-amines and lysine side chain epsilon-amines.

In some cases, sulfonyl chloride may be used to attach two substances. For example, the sulfonyl chloride of a biomolecule or solid support can be used for bioconjugation. For example, the reaction of a sulfonyl chloride compound with a primary amine-containing molecule loses a chlorine atom and forms a sulfonamide linkage.

In some cases, tosylate may be used to attach two substances. For example, toluene sulfonate of a biomolecule or solid support can be used for bioconjugation. For example, functional groups including tosylate esters may be formed from the reaction of 4-toluenesulfonyl chloride (also known as methylbenzenesulfonyl chloride or TsCl) with hydroxyl groups to produce sulfonyl ester derivatives. The sulfonyl esters can be coupled with nucleophiles to create covalent bonds, and can create secondary amine linkages to primary amines, thioether linkages to sulfhydryl groups, or ether linkages to hydroxyl groups.

In some cases, carbonyl groups may be used to attach two species. For example, carbonyl groups of biomolecules or solid supports can be used for bioconjugation. For example, carbonyl groups such as aldehydes, ketones, and glyoxal can react with amines to form schiff base intermediates in equilibrium with their free forms. In some cases, the addition of sodium borohydride or sodium cyanoborohydride to a reaction medium containing an aldehyde compound and an amine-containing molecule will result in the reduction and covalent bond formation of the schiff base intermediate, thereby creating a secondary amine linkage between the two molecules.

In some cases, an epoxide or ethylene oxide may be used to attach both substances. For example, epoxides or oxiranes of biomolecules or solid supports can be used for bioconjugate. For example, epoxide or oxirane groups can be reacted with nucleophiles during the ring opening process. Can react with primary amine, sulfhydryl or hydroxyl groups to produce secondary amine, thioether or ether linkages, respectively.

In some cases, carbonates may be used to attach both substances. For example, biomolecules or carbonates of solid supports may be used for bioconjugation. For example, carbonates may be reacted with nucleophiles to form urethane linkages, and disuccinimidyl carbonates may be used to activate hydroxyl-containing molecules to form amine-reactive succinimidyl carbonate intermediates. In some cases, this carbonate activation procedure can be used to couple polyethylene glycol (PEG) to proteins and other amine-containing molecules. In some cases, a primary amino group of a nucleophile such as a protein may react with a succinimidyl carbonate functional group to provide a stable urethane (aliphatic urethane) linkage

In some cases, aryl halides may be used to attach two species. For example, aryl halides of biomolecules or solid supports can be used for bioconjugation. For example, aryl halide compounds such as fluorobenzene derivatives can be used to form covalent bonds with amine-containing molecules such as proteins. Other nucleophiles such as thiol, imidazolyl and phenolic acid groups can also react to form stable bonds. In some cases, fluorobenzene-type compounds have been used as functional groups in homobifunctional crosslinkers. For example, their reaction with amines involves nucleophilic displacement of a fluorine atom with an amine derivative, resulting in a substituted arylamine bond.

In some cases, imidoesters may be used to attach two substances. For example, the imidoesters of biomolecules or solid supports can be used for bioconjugation. For example, the alpha-amine and epsilon-amine of a protein can be targeted and crosslinked by reaction with homobifunctional imidoesters. In some cases, after conjugation of the two proteins with the difunctional imidate crosslinker, the excess imidate functionality may be blocked by ethanolamine.

In some cases, carbodiimides may be used to attach two substances. For example, carbodiimides may be used for bioconjugate. In general, carbodiimides are zero length cross-linking agents that can be used to mediate the formation of amide or phosphoramidate linkages between carboxylate groups and amines or between phosphates and amines, respectively. Carbodiimides are zero length reagents because no additional chemical structure is introduced between the conjugated molecules when these bonds are formed. In some cases, the N-substituted carbodiimide can react with carboxylic acids to form highly reactive O-acylisourea derivatives. This active material may then react with a nucleophile such as a primary amine to form an amide bond. In some cases, a thiol group may attack an active species and form a thioester linkage. In some cases, the hydrazide-containing compound may also be coupled to the carboxylate group using a carbodiimide-mediated reaction. Using a difunctional hydrazide reagent, the carboxylate may be modified to have terminal hydrazide groups that can be conjugated with other carbonyl compounds.

In some cases, phosphate esters may be used to attach two substances. For example, phosphorylated amino acids of a 5' phosphate or polypeptide of a biological molecule containing a phosphate group, such as an oligonucleotide, may also be conjugated to an amine-containing molecule by utilizing a carbodiimide-mediated reaction. For example, a carbodiimide of a biomolecule may activate a phosphate to an intermediate phosphate similar to its reaction with a carboxylate.

In the presence of an amine, the esters react to form stable phosphoramidate linkages.

In some cases, anhydrides may be used to attach both materials. For example, biomolecules or anhydrides of solid supports may be used for bioconjugation. Anhydrides are highly reactive towards nucleophiles and are capable of acylating many important functional groups of proteins and other molecules. For example, protein functional groups capable of reacting with anhydride include, but are not limited to, N-terminal alpha-amine, epsilon-amine of lysine side chains, cysteine sulfhydryl, phenolic acid root ion of tyrosine residues, and imidazole ring of histidine. In some cases, the reactive site of the anhydride in the protein molecule is a modification of any attached carbohydrate chain. In some cases, in addition to amino modifications in the polypeptide chain, glycoproteins may also be modified at their polysaccharide hydroxyl groups to form esterified derivatives.

In some cases, fluorophenyl esters may be used to attach both substances. For example, the fluorophenyl esters of biomolecules or solid supports can be used for bioconjugation. The fluorophenyl ester may be another type of carboxylic acid derivative that can react with amines, consisting of esters of fluorophenol compounds, which create groups capable of forming amide bonds with proteins and other molecules. In some cases, the fluorophenyl ester may be: pentafluorophenyl (PFP) ester, tetrafluorophenyl (TFP) ester or sulfo-tetrafluorophenyl (STP) ester. In some cases, the fluorophenyl ester is reacted with an amine-containing molecule at a slightly basic pH to give the same amide linkage as the NHS ester.

In some cases, hydroxymethylphosphine may be used to attach two substances. For example, a biomolecular or hydroxymethylphosphine of a solid support may be used for bioconjugation. Phosphine derivatives with hydroxymethyl substitution can be used as attachment agents for coupling or crosslinking purposes. For example, tris (hydroxymethyl) phosphine (THP) and beta- [ tris (hydroxymethyl) phosphino ] propionic acid (THPP) are small trifunctional compounds that spontaneously react with nucleophiles such as amines to form covalent linkages.

In some cases, thiols may be used to attach two substances. For example, thiol reactivity of biomolecules or solid supports can be used for bioconjugation. For example, the thiol group of cysteine is the most nucleophilic functional group found in the 20 proteinogenic amino acids. By carefully controlling the pH, selective modification of other nucleophilic amino acid residues, such as lysine, can be readily achieved. As another example, thiol modification of oligonucleotides can be used to achieve derivatization, however the ease with which alternative functional groups with enhanced chemical orthogonality can be installed limits the use for biomaterial conjugation. Further, the conjugated addition of thiols, also known as michael addition, to α, β -unsaturated carbonyl groups can be used to form polypeptide conjugates in the fields of tissue engineering, functional materials, and protein modification. In general, the reaction rate and conjugation efficiency are mainly controlled by three factors, and can be modified as needed: (i) pK of thiols _a The method comprises the steps of carrying out a first treatment on the surface of the (ii) electrophilicity of michael acceptors; (iii) selection of the catalyst. Regarding (i): mercaptide anions are active nucleophiles during the michael addition and the propensity of thiols to undergo deprotonation may determine mercaptide concentration and thus reaction rate. For example, aromatic thiols have a lower pK when compared to their aliphatic counterparts _a Resulting in a higher reaction rate using weak base catalysis. As a result, local structures can significantly alter conjugation efficiency, particularly for polypeptide substrates. The pK of cysteine-containing peptides can be significantly altered by the rational selection of the surrounding amino acids _a And reactivity, the presence of positively charged amino acids such as lysine and arginine serves to reduce the thiol pK _a And thus enhances reactivity. Regarding (ii): the Michael acceptors become more electron deficient, which becomes more active against nucleophilic attackAnd thus the reaction rate increases. Within the receptors most widely used in the field of biological materials, the trend of reactivity can be summarized as maleimide>Vinyl sulfones>Acrylic esters>Acrylamide>And (3) a methacrylate. With respect to (iii), the michael addition can be accelerated by basic or nucleophilic catalysis (although both work by increasing the concentration of active thiolates).

In some cases, the unique nucleophilicity of thiols may be utilized to selectively react with a number of alternative electrophiles, which allows for efficient and selective attachment. For example, one such group includes an α -halocarbonyl group, and iodoacetamide-based reagents are particularly useful. Higher thiol selectivity can be achieved using brominated and even chlorinated derivatives of lower electrophilicity, although the reactivity is also greatly reduced. Recently, methylsulfonyl heteroaromatic derivatives have become promising reagents for thiol-specific conjugation. In other cases, alternative thiol functionalities such as disulfide-bridged pyridazindiones, carbonyl-acrylic reagents, and cyclopropenyl ketones may be used for bioconjugate.

In some cases, sulfhydryl groups may be used to attach two substances. For example, thiol groups of biomolecules or solid supports may be used for bioconjugation. In some cases, three forms of activated halogen derivatives can be used to produce thiol-reactive compounds: haloacetyl, benzyl halide and alkyl halide. In each of these compounds, the halogen group can be easily replaced by an aggressive nucleophile to form an alkylated derivative that loses HX (where X is halogen and hydrogen is from the nucleophile). The haloacetyl compounds and benzyl halides are typically iodine or bromine derivatives, whereas the halomustards are predominantly in the chlorine and bromine forms. Iodoacetyl groups have also been successfully used to couple affinity ligands to chromatographic supports.

In some cases, maleimide may be used to attach two substances. For example, a biomolecule or maleimide of a solid support may be used for bioconjugation. The double bond of maleimide can undergo alkylation reaction with thiol groups to form stable thioether linkages.

In some cases, aziridines may be used to attach two substances. For example, biomolecules or aziridines of a solid support may be used for bioconjugate. The highly hindered nature of this heterocycle makes it highly reactive towards nucleophiles. For example, a sulfhydryl group will react with an aziridine-containing reagent during ring opening to form a thioether linkage. The simplest aziridine compound, ethyleneimine, can be used to convert available sulfhydryl groups into amines. In some cases, substituted aziridines may be used to form homobifunctional and trifunctional crosslinkers.

In some cases, thiol-maleimide reactions are particularly useful for conjugation at low concentrations or when extremely high efficiencies are required due to the value of the biomolecular substrate. The ease with which maleimides are incorporated into a wide range of materials further enhances their use for attachment by modification of the amine with the bifunctional reagent succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (commonly referred to simply as SMCC). For example, this reagent has been widely used to introduce maleimide functionality onto selected biological materials, and then enable attachment of both peptides and growth factors to create bioactive scaffolds.

In some cases, an acryl group may be used to attach both substances. For example, the acryloyl groups of biomolecules or solid supports can be used for bioconjugation. The reactive double bond is able to undergo further reactions with sulfhydryl groups. In some cases, the acryloyl compound reacts with the thiol group to produce a stable thioether bond. In some cases, acryl can be used to design a thiol-reactive fluorescent probe, 6-acryl-2-dimethylaminonaphthalene.

In some cases, aryl groups may be used to attach two species. For example, aryl groups of biomolecules or solid supports may be used for bioconjugate with sulfhydryl groups. While aryl halides are commonly used to modify amine-containing molecules to form aryl amine derivatives, they can also react quite easily with sulfhydryl groups. For example, fluorobenzene-type compounds have been used as functional groups in homobifunctional crosslinkers. Their reaction with nucleophiles involves a bimolecular nucleophilic substitution, resulting in the replacement of the fluorine atom with a sulfhydryl derivative and the creation of a substituted aryl bond. Conjugates formed with thiol groups are reversible by cleavage with excess thiol (e.g., DTT).

In some cases, disulfides may be used to attach two substances. For example, disulfide groups of biomolecules or solid supports can be used for bioconjugation. In some cases, a compound containing a disulfide group is capable of participating in a disulfide exchange reaction with another thiol. The disulfide exchange (also known as interchange) process involves attack of thiols at the disulfide, breaking the-S-bond, followed by the formation of a new mixed disulfide that includes a portion of the original disulfide compounds. The reduction of disulfide groups in proteins to thiol groups using thiol-containing reducing agents is performed by intermediate formation of mixed disulfides. In some cases, the crosslinking or modification reaction may utilize a disulfide exchange process to form disulfide linkages with thiol-containing molecules.

In some cases, disulfide bonds may be used for attachment, such as bioconjugation. For example, the use of disulfide exchange reactions may be advantageous for modifying a polypeptide of interest. The most commonly used reagents in tissue engineering are based on reactive pyridylthio-disulfides, which undergo rapid thiol-exchange to release 2-mercaptopyridine, which is poorly nucleophilic and spectrally active. In addition, due to the reversibility of disulfide bond formation, cleavage can be controlled with time accuracy by adding a reducing agent such as Dithiothreitol (DTT) or glutathione.

In some cases, a pyridyldithiol may be used to attach two substances. For example, the pyridyldithiol functionality can be used to construct cross-linking agents or modifying agents for bioconjugation. Pyridyl disulfides can be produced from available primary amines on the molecule by the reaction of 2-iminothiolane in tandem with 4,4' -bipyridine disulfide. For example, the simultaneous reaction between a protein or other molecule, 2-iminothiolane, and 4,4' -bipyridine disulfide produces a modification containing reactive pyridyl disulfide groups in a single step. The pyridyl disulfide will readily undergo an exchange reaction with the free sulfhydryl group, yielding a single mixed disulfide product.

In some cases, thiol activated with a leaving group of 5-thio-2-nitrobenzoic acid can be used to couple free thiols by disulfide exchange similar to pyridyl disulfide as described herein. The disulfide of the elman reagent readily undergoes disulfide exchange with the free thiol group to form a mixed disulfide, while releasing one molecule of chromogenic substance 5-thioxo-2-nitrobenzoate, also known as 5-thio-2-nitrobenzoic acid (TNB). The TNB-thiol groups can again undergo exchange with thiol-containing target molecules, resulting in disulfide cross-links. After coupling with the thiol compound, the TNB groups are released.

In some cases, disulfide reduction may be performed using thiol-containing compounds, such as TCEP, DTT, 2-mercaptoethanol, or 2-mercaptoethylamine.

In some cases, vinyl sulfones may be used to attach two substances. For example, vinyl sulfone groups of biomolecules or solid supports can be used for bioconjugation. For example, michael addition of thiols to activated vinyl sulfones to form biomolecule-material conjugates has been used to demonstrate that cysteine-capped peptides can crosslink vinyl-sulfone functionalized multi-arm PEG to form protease-reactive hydrogels enabling cell invasion during tissue growth. In some cases, in addition to thiols, vinyl sulfone groups can also react with amines and hydroxyl groups at higher pH conditions. The product of the reaction of a thiol with a vinyl sulfone gives a single stereoisomer structure. In addition, vinyl sulfone-containing crosslinkers and modifying agents can be used to activate surfaces or molecules to contain thiol-reactive groups.

In some cases, thiol-containing molecules can interact with metal ions and metal surfaces to form dative bonds for bioconjugation. In some cases, oxygen-and nitrogen-containing organic or biological molecules can be used to chelate metal ions, such as in various lanthanide chelates, difunctional metal chelate compounds, and FeBABE. Furthermore, amino acid side chains and pseudogroups in proteins often form bioinorganic motifs by coordinating metal ions as part of the active center.

In some cases, thiol organic compounds may be conventionally used to coat metal surfaces or particles to form biocompatible layers or to create functional groups for further conjugation of substances such as biomolecules. For example, thiol-containing aliphatic/PEG linkers have been used to form self-assembled monolayers (SAM) on planar gold surfaces and particles.

In some cases, many alternative coupling systems may be used for attachment between substances or biomolecular functionalization. These include the use of O-nitrophenyl esters (which have reduced stability under aqueous conditions) or 1,1' -Carbonyldiimidazole (CDI) to form amine bridged urethane linkages instead of amides. Hydrazine may also be used in place of amine during EDC/NHS mediated coupling. The hydrazine functionalized peptide can be coupled to the biological material in one step at a pH of 5-6. This allows a degree of site-selectivity to the lysine residues present.

In some cases, N-terminal modification of biomolecules may be used for bioconjugation. For example, 2-pyridylaldehyde modified acrylamide hydrogels can specifically react with the N-terminus of ECM proteins, form cyclic imidazolidone products with adjacent amide linkages, and enable directed display of these key bioinformatic motifs.

In some cases, acrylates, acrylamides, and methacrylates of substances such as biomolecules or solid supports may be used for attachment. In some cases, a thiol of a substance such as a biomolecule or solid support may be used for bioconjugation.

In some cases, thiol-reactive conjugation such as Native Chemical Ligation (NCL) can be used to form attachment substances via peptide bonds, e.g., attach peptides and proteins to biomaterial scaffolds. For example, a peptide having a C-terminal thioester reacts with an N-terminal cysteine residue in another peptide to undergo a trans-thioesterification reaction, which results in the formation of an intermediate thioester with a cysteine thiol.

In some cases, strong binding of (streptavidin) to small molecule biotin can be used for attachment. In some cases, (streptavidin) may be attached to a first substance and biotin may be attached to a second substance, such that the substances may be attached via binding of (streptavidin) to biotin. In some cases, the modifying reagent may add functional biotin groups to proteins, nucleic acids, and other molecules. In some cases, depending on the functional groups present on the biotinylated compound, specific functional groups on the antibody or other protein may be modified to create (streptavidin) binding sites. By appropriate selection of biotin derivatives, amine, carboxylate, sulfhydryl and carbohydrate groups can be specifically targeted to biotinylation. In some cases, photoreactive biotinylating reagents are used to non-selectively add biotin groups to molecules that do not contain convenient functional groups for modification. In some cases, biotin-binding proteins can be immobilized on surfaces, chromatographic carriers, microparticles and nanoparticles for coupling biotinylated molecules. In some cases, a series of (streptavidin-biotin interactions can be established with each other to take advantage of the multivalent nature of each tetrameric (streptavidin) molecule and enhance the detection ability of the target. In some cases, amine-reactive biotinylation reagents, which may contain functional groups that are pendant from the valeric acid side chains of biotin, are capable of forming covalent bonds with primary amines in proteins and other molecules. In some cases, NHS esters spontaneously react with amines to form amide linkages, while carboxylate-containing biotin compounds can be coupled with amines via carbodiimide-mediated reactions using EDC. In some cases, NHS-iminobiotin can be used to label amine-containing molecules with iminobiotin tags, thereby providing reversible binding potential to avidin or streptavidin. In some cases, sulfo-NHS-SS-biotin (also known as NHS-SS-biotin) is sulfosuccinimidyl-2- (biotinylamino) ethyl-1, 3-dithiopropionate, a long chain cleavable biotinylation agent useful for modifying amine-containing proteins and other molecules. In some cases, 1-biotinylamino-4- [4' - (maleimidomethyl) cyclohexane-carboxamido ] butane is a biotinylating agent containing a maleimide group at the end of the extended spacer, and reacts with thiols in proteins and other molecules to form stable thioether linkages. In some cases, N- [6- (biotinamido) hexyl ] -3'- (2' -pyridyldithio) propanamide, wherein the reagent contains a 1, 6-diaminohexane spacer attached to the valeric acid side chain of biotin, the terminal amino group of the spacer can be further modified via amide linkage with the acid precursor of SPDP to produce a terminal thiol-reactive group. The pyridyl disulfide end of biotin-HPDP can react with free thiol groups in proteins and other molecules to form disulfide bonds while simultaneously losing pyridine-2-thione.

In some cases, carboxylic acid esters may be used to attach two substances. For example, carboxylic acid esters of biomolecules or solid supports can be used for bioconjugation. In some cases diazomethane and other diazoalkyl derivatives may be used to label carboxylate groups. In some cases, N' -Carbonyldiimidazole (CDI) can be used to react with carboxylic acids under non-aqueous conditions to form highly reactive N-acyl imidazoles. The activated carboxylic acid ester may then be reacted with an amine to form an amide linkage, or with a hydroxyl to form an ester linkage. In addition, activation of the styrene/4-vinylbenzoic acid copolymer with CDI can be used to immobilize the enzyme lysozyme or other molecules to the carboxyl groups on the substrate through the amino groups available for them.

In some cases, the carbodiimide acts as a zero length cross-linker that is capable of activating carboxylate groups for coupling with amine-containing compounds for attachment. In some cases, carbodiimides are used to mediate the formation of amide or phosphoramidate linkages between carboxylic acid esters and amines or between phosphoric acid esters and amines.

In some cases, N' -disuccinimidyl carbonate or N-hydroxysuccinimidyl chloroformate may be used, for example, via bioconjugation of attachment substances. N, N' -disuccinimidyl carbonate (DSC) consists of a carbonyl group containing essentially two NHS esters. The compounds are highly reactive towards nucleophiles. In aqueous solution, DSC will hydrolyze to form two molecules of N-hydroxysuccinimide (NHS) while releasing one molecule of CO2. In a non-aqueous environment, the reagent can be used to activate hydroxyl groups into succinimidyl carbonate derivatives. DSC-activated hydroxyl compounds are useful for conjugation with amine-containing molecules to form stable crosslinked products.

In some cases, sodium periodate can be used to oxidize hydroxyl groups on adjacent carbon atoms to form reactive aldehyde moieties suitable for coupling with amine-or hydrazide-containing molecules for attachment, e.g., via bioconjugation. For example, these reactions can be used to create cross-linking sites in carbohydrates or glycoproteins for subsequent conjugation of amine-containing molecules by reductive amination.

In some cases, enzymes can be used to oxidize hydroxyl-containing carbohydrates to produce aldehyde groups for bioconjugation. For example, galactose oxidase on the terminal galactose or N-acetyl-d-galactose moiety reacts to form C-6 aldehyde groups on the polysaccharide chain. These groups can then be used for conjugation reactions with amine-or hydrazide-containing molecules.

In some cases, reactive alkyl halide compounds may be used to specifically modify hydroxyl groups in carbohydrates, polymers, and other materials for attachment.

In some cases, aldehydes or ketones may be used to attach both substances. For example, aldehydes or ketones of biomolecules or solid supports may be used for bioconjugation. For example, hydrazine derivatives, particularly hydrazide compounds formed from carboxylate groups, can react specifically with aldehyde or ketone functionalities in the target molecule. To further stabilize the bond between the hydrazide and the aldehyde, the hydrazone may be reacted with sodium cyanoborohydride to reduce the double bond and form a stable covalent bond.

In some cases, an aminooxy group can be used to attach two species. For example, aminooxy groups of biomolecules or solid supports can be used for bioconjugation. For example, chemoselective ligation reactions occurring between aldehyde groups and aminooxy groups produce oxime linkages (aldoxime), which have been used in many bioconjugation reactions, as well as the coupling of ligands to insoluble supports including surfaces. This reaction is also quite efficient for ketones, forming an oxime called ketoxime.

In some cases, cycloaddition reactions may be used for attachment, such as via bioconjugation. In cycloaddition reactions, two or more unsaturated molecules are brought together to form a cyclic product of reduced unsaturation, these reaction partners are not normally present in the natural system, and conjugation using cycloaddition thus makes use of the introduction of non-natural functionalities within the coupling partner.

In some cases, copper-catalyzed azide-alkyne cycloaddition (CuAAC) can be used to attach two substances. For example, cuAAC can be used for bioconjugation. In some cases the (3+2) cycloaddition between azide and alkyne occurs spontaneously at high temperature (> 90 ℃) to produce a mixture of the two triazole isomers. In some cases, this reaction is carried out at room temperature, ambient, oxygenated, and/or aqueous environment. In some cases, the peptide-substance conjugate is formed by CuAAC, for example, using alkyne-terminated peptides, thereby forming a hydrogel with azide-functionalized PEG. In some cases, to achieve conjugation via CuAAC, the copper (I) catalyst may be added directly, or generated in situ by reduction of the original copper (II) complex, most typically using ascorbic acid. The addition of the reducing agent further reduces the sensitivity of the CuAAC linkage to oxygen. Although triazole formation does not require additional ligands, the addition of tertiary amine-based ligands may be employed.

In some cases, strain-promoted azide-alkyne cycloaddition (sparc) can be used to attach two substances. Sparc can be used for bioconjugation. In some cases, highly strained cyclooctyne readily reacts with azide under physiological conditions to form triazole without the need for any catalyst addition. In some cases, in addition to peptide conjugation using sparc, many important reports also use sparc to attach protein substrates to cyclooctyne-functionalized biomaterials via the introduction of non-native azide motifs into the protein coupling partners. In some cases, this is achieved, for example, via enzyme-mediated N-terminal modification of IFN-gamma, or via codon reassignment with the unnatural amino acid 4-azidophenylalanine in many protein substrates, by maleimide functionalization including the natural cysteines present in bone morphogenic protein-2 (BMP-2). In some cases, supramolecular host-guest interactions may also be used to promote azide-alkyne cycloaddition. For example, by bringing the two reactive partners into close proximity within the lumen of the cucurbituril [6] uril host, an efficient cycloaddition can be achieved on the surface of the protein, and this strategy can be extended to other suitable molecules.

In some cases, inverse electron demand Diels-Alder reactions (IEDDA) may be used for attachment, such as via bioconjugation. For example, IEDDA reactions between 1,2,4, 5-tetrazine and strained alkenes or alkynes may be employed. A wide range of suitable derivatives have been reported for use in performing molecular conjugation, for example, trans-cyclooctene can be utilized with a range of increasingly strained (and thus reactive) trans-cyclooctenes. In some cases, functionalized norbornene derivatives can be used to carry out the IEDDA reaction. In some cases, triazines may be utilized. In some cases, spirohexene may be utilized. These strategies can be extended to other suitable molecules. In some cases, a heterodiels-alder cycloaddition of maleimide and furan may be used for attachment. For example, coupling of furan functionalized RGDS peptides to maleimide functionalized PEG-hydrogels can be utilized and this strategy can be extended to other suitable molecules. In some cases, furan functionalized hyaluronic acid hydrogels can be crosslinked with bismaleimide functionalized peptides via diels-alder cycloadditions.

In some cases, oximes and hydrazones can be used to attach both materials. For example, oxime and hydrazone formation may be used for bioconjugation. In some cases, stable attachment of peptides and DNA to biological materials via hydrazone formation can be achieved via bifunctional crosslinking, and this strategy can be extended to other suitable molecules. For example, protein crosslinked hydrogels can be produced by oxime modification of the N-and C-termini of proteins.

In some cases, the diels-alder reaction consists of covalent coupling of a diene with an olefin to form a six-membered ring complex for attachment.

In some cases, transition metal complexes may be used for attachment, such as via bioconjugation. The nature of the late transition metal may make the transition metal complex well suited for handling unsaturated and polarizable functional groups (alkene, alkyne, aryl iodide, aryl boronic acid, etc.). For example, pd (0) -functionalized solid supports can mediate allyl carbamate deprotection and suzuki-miyaura cross-coupling in the cytoplasm. In other examples, ruthenium catalysts can be used to mediate allyl carbamate deprotection of caged fluorophores inside living cells. In some cases, the use of palladium-based applications in cell culture include copper-free Sonagashira coupling, extracellular suzuki coupling on the surface of e.coli cells, and conjugation of thiol groups to allyl selenosulfate. In some cases, olefin metathesis can be used for bioconjugation. For example, with ruthenium complexes, S-allyl cysteine can be readily incorporated into proteins by a variety of methods, including conjugated addition of allyl mercaptan to dehydroalanine, direct allylation of cysteine, desulfurization of allyl disulfide, or metabolic incorporation in methionine auxotrophic E.coli as a substitute for methionine.

In some cases, forming complexes with boric acid derivatives can be used to attach substances, for example via bioconjugation. For example, boric acid derivatives can form a ring structure with other molecules having adjacent functional groups consisting of 1, 2-or 1, 3-diol, 1, 2-or 1, 3-hydroxy acid, 1, 2-or 1, 3-hydroxylamine, 1-2-or 1, 3-hydroxyamide, 1, 2-or 1, 3-hydroxyoxime, and various sugars or biomolecules containing these substances.

In some cases, enzyme-mediated conjugation may be used to attach the substance. For example, the transglutaminase family catalyzes the formation of isopeptidic linkages between primary amines of lysine side chains and amide linkages of complementary glutamine residues, and this strategy can be extended to other suitable molecules. In other cases, peroxidase-mediated conjugation may be used for conjugation. For example, horseradish peroxidase (HRP) can be used to oxidize the phenolic groups of a wide range of organic substrates such as tyrosine to produce highly reactive free radicals or quinone intermediates that undergo spontaneous dimerization, resulting in the formation of ortho carbon-carbon bonds between two tyrosine residues, which strategy can be extended to other suitable molecules. In some cases, short peptide tags may be used for bioconjugation. These peptide tags can be as short as 5 amino acids in length and can be attached to polypeptides that allow for subsequent modification thereof.

In some cases, polymerization of low molecular weight monomers may be used to attach the substance, for example via bioconjugation. Aggregation can be categorized as occurring via one of two mechanisms, namely chain growth or step growth. During chain growth polymerization, monomers are added at the "living" end of the growing polymer chain, resulting in the formation of high molecular weight species even at low conversions. During step-growth polymerization, short oligomer chains are coupled to form polymeric species, requiring high conversion in order to reach high molecular weights. Both techniques can be used to form conjugates, such as biomolecule-polymer conjugates. Polymerization of acrylate and methacrylate monomers has proven particularly effective. For example, acrylate and methacrylate modified proteins can be readily polymerized. Similarly, free radical polymerization remains one of the most common methods by which DNA and RNA functional materials are formed due to the availability of the synthetic oligonucleotide phosphoramidite building block "Acrydite". By conducting the polymerization in the presence of a comonomer, the density at which the molecule assumes can be easily adjusted, allowing the potential difficulties presented by steric hindrance to be overcome. Initiation of polymerization can be triggered by a variety of means including heat, UV and visible light, redox reactions, and electrochemistry. The acrylate-modified proteins may also undergo polymerization to produce functional species while retaining biological activity. In some cases, living Radical Polymerization (LRP) may be used for bioconjugation. For example, LRPs most commonly used to form bioconjugates include Atom Transfer Radical Polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and Nitroxide Mediated Polymerization (NMP).

In some cases, photo-conjugation may be used to attach a substance, for example via bioconjugation. In some cases, polymerization is initiated by the generation of free radical species, which then propagate through bond formation to produce living polymer chains. The initiation step may be induced by a variety of stimuli, with thermal decomposition, redox activation and electrochemical ionization of the initiating species being the most common. Alternatively, many initiators may be activated via photo-induced photolytic bond cleavage (type I) or extraction of protons (type II) photo-activated from co-initiators. Photoinitiation provides the benefits of being suitable for a wide temperature range, using a narrow and tunable activation wavelength depending on the initiator used, fast generation of free radicals, and being able to control polymerization by removing the light source. Importantly, the resistance of the polymerization to oxygen is greatly enhanced, enabling the polymerization to be carried out in the presence of cells and tissues. Incorporation of acrylate functionalized peptides and proteins during photopolymerization can be used as a method to produce biomaterial conjugates. Alternatively, photoinitiated polypeptides have also been widely reported attached to a pendant vinyl group on a preformed substance, and recently used for 3D patterning via two-photon excitation. A wide variety of photoinitiators are available for photoconjugation conjugation. For example, but not limited to, eosin Y, 2-dimethoxy-2-phenyl-acetophenone, igracure D2959, phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate, and riboflavin may be used as photoinitiators. Photoinitiators typically absorb light to initiate the photoreaction process. In some cases, the photo-conjugation may utilize a photo-thiol-ene reaction. Mercaptans can also react with olefins via free radical mechanisms. The thiol radicals first react with the olefin, producing carbon-centered radicals, which can then extract protons from another thiol, thereby propagating the reaction. The electron rich olefins can accelerate the photo thiol-ene reaction, which produces an unstable carbon-radical intermediate that can rapidly extract thiol-hydrogen. An exception to this rule is norbornene derivatives, where the reactivity is instead driven by the release of the ring tension after addition of the thiol. This results in a general trend in reactivity of norbornene > vinyl ether > propenyl > allyl ether > acrylate > maleimide. Norbornene and allyloxycarbonyl (alloc groups) have been particularly widely used for peptide/protein-biomaterial functionalization, respectively, due to the almost negligible contribution of chain transfer and their ease of introduction during peptide synthesis. For example, the alloc group, which is typically used as an orthogonal lysine protecting group during solid phase peptide synthesis, is a potent photo thiol-ene function. In other examples, norbornene photo thiol-ene reactions can be used for the tethering and spatial patterning of bioactive peptides and growth factor proteins. In addition to the most commonly used alloc and norbornene functionalities, other olefins have also been used for biomaterial functionalization. For example, codon reassignment has been used to site-specifically incorporate allyl-cysteine residues into proteins, which can then undergo conjugation by utilizing a photo thiol-ene reaction. Alternatively, acrylates may undergo mixed mode photopolymerization in the presence of cysteine-terminated peptides, while the allylic disulfide structure has recently been demonstrated to undergo reversible and controlled exchange of conjugated thiols.

In some cases, aryl azide or halogenated aryl azide may be used to attach the substance.

In some cases, photoreactive groups such as benzophenone may be used for the attachment substance.

In some cases, the photoreactive group anthraquinone can be used for attachment of substances, such as via bioconjugation. In some cases, the photo thiol-alkyne reaction can be used to attach a substance, such as via bioconjugation. Most examples of photo thiol-alkyne reactions utilize simple propargyl-ether or-amine functionalities.

In some cases, photocaging and activation of reactive functional groups can be used to attach a substance, such as attachment via bioconjugation. Generally, transient reactive species are formed, whether they are acrylate or thiol derived free radicals. In some cases, photo-trapping can be used to mask or protect the functional group until it is desired to expose it. In some cases, the most widely used cages are based on ortho-nitrobenzyl and coumarin chromophores. For example, nitrobenzyl-terminated cysteine residues can be caged by irradiation with 325nm UV light, and the liberated thiol can then be reacted via michael addition with maleimide-functionalized peptides to produce a patterned hydrogel capable of directing cell migration. In some cases, 6-bromo-hydroxycoumarin may be used for thiol-caging. In some cases, photoaffinity probes can be used for bioconjugation, wherein highly reactive intermediates are irradiated and then rapidly reacted with recently accessible functional groups with high spatial precision. In some cases, phenyl azide, benzophenone, and phenyl-biaziridine are most commonly used. In some cases, a photocaged cycloaddition may be employed. For example, UV irradiation of tetrazoles has been demonstrated to produce reactive nitrile-imine intermediates that can undergo rapid cycloaddition with electron-deficient olefins such as acrylates or acrylamides. In some cases, the side-reactivity of nitrile-imines with thiols can be used for site-specific conjugation of cysteine-containing proteins to tetrazole functionalized surfaces.

In some cases, non-covalent interactions may be used to attach a substance, such as attachment via bioconjugation. In some cases, non-covalent sequences that exhibit binding affinity for the biomolecules of interest allow for simple isolation of post-fabrication modifications or native biomolecules from the environment within the biological sample. Useful binding sequences are short peptides of between 7 and 20 amino acids in length from a variety of sources, including known protein binding domains that are present in vivo or determined by techniques such as phage display. In some cases, the aptamer may also be used to bind a variety of protein substrates, including the cytokines Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF) as well as cell surface proteins such as Epidermal Growth Factor Receptor (EGFR). In some cases, the binding sequence may also be incorporated into biological materials that have affinity for natural biopolymers such as heparin. In some cases, adsorption of the biomaterial scaffold by the added or endogenous growth factors or signaling proteins can then be controlled by first inducing biopolymer binding. In some cases, binding affinity at the amino acid level may also be utilized to enable peptides and proteins to be conjugated to certain biomaterial substrates. For example, the binding of unnatural catechol-based amino acids can be used to induce binding to bioglass and metal implants containing metal oxides, enabling enhancement of the biological activity of these important technologies.

In some cases, self-assembling peptides can be used to attach substances, such as via bioconjugation. For example, natural peptides and proteins employ a range of secondary structures, including β -sheet and α -helix, which can both stabilize individual sequences and control inter-protein aggregation. In some cases, self-assembling peptides have been widely used to assemble hydrogels and fibrous materials. In many of these structures, a biological epitope or functional group may be attached to some or all of the peptide building blocks during peptide synthesis to add a desired biological activity to the system. Peptide-ligands and growth factor mimics, ranging from simple adhesion motifs to laminin-derived epitopes, have been shown on the surface of self-assembled fibrils. Alternatively, glycopeptides may be assembled in order to recruit extracellular signaling proteins and growth factors, mimic glycosylation patterns within hyaluronic acid, or study optimal sulfonation ratios in glycosaminoglycan scaffolds. In some cases, self-assembled domains can also be added to the full-length protein, resulting in incorporation of pendent functional groups during hydrogel formation. In some cases, the propensity of peptides to form secondary structures has also been exploited within non-self-assembled scaffolds. This can be achieved by mixing the self-assembled peptide into a covalent hydrogel consisting of, for example, an interpenetrating network of a non-interacting polymer such as PEG between the positively charged peptide and the negatively charged alginate gel or a system in which additional charge interactions further stabilize the final construct. Alternatively, the pendent helical groups may be attached to a covalent species and used to drive non-covalent attachment of a biologically active group such as a growth factor by self-assembly into a coiled coil triple helix.

In some cases, host-guest chemistry may be used to attach the substance, such as via bioconjugation. For example, the adhesion properties of beta-cyclodextrin modified alginate scaffolds can be controlled in situ by adding a guest naphthyl-functionalized RGDS peptide and by subsequently introducing a non-cell-adhering adamantane-RGES peptide with a higher host binding constant, enabling dynamic modulation of fibroblast attachment. Host-guest interactions between cyclodextrins and naphthyl or adamantane functionalized peptides allow alginate functionalization, which can be applied to other suitable molecules.

In some cases, the nucleic acid may be used for attachment of a substance, such as via bioconjugation. In some cases, in a similar manner to self-assembled peptides, the nucleic acids themselves may also form assembled materials to create an adjustable platform for molecular display. In some cases, the DNA-tagged polypeptide may be conjugated to a suitably functionalized substance.

Generally, incorporation of functional groups can be used to attach substances. For example, the introduction of unique reactive motifs into the molecule provides a chemical "tag" that allows for single site selectivity or specificity to be achieved. In some cases, the polypeptide or nucleic acid may be produced via Solid Phase Synthesis (SPS). The versatility of organic synthesis allows overcoming the functionality Difficulties in group incorporation can be addressed with a wide range of appropriately functionalized amino acids and nucleotides as described herein. In some cases, an alternative approach is to introduce Unnatural Amino Acids (UAA) with the desired functionality. This can be achieved by modifying the lysine residue with an amine reactive derivative. In some cases, auxotrophic bacterial strains are used that are not able to biosynthesize a particular amino acid, and therefore require uptake from the growth medium. Bacterial cells can be induced to incorporate UAA during translation by making the bacteria deficient in natural amino acids and complementing structurally related non-natural analogs. This technique can be used to mount azide and alkyne-based mimics of methionine, resulting in the introduction of functional groups for performing CuAAC and sparc reactions. In some cases, the use of codon reassignment uses orthogonal tRNA and tRNA synthetase pairs that selectively recognize and charge UAA during translation. In some cases, this can be accomplished by tRNA from the surrogate world _CUA The tRNA synthetase pair is incorporated into the host cell to reassign the amber stop codon UAG. This pair may be able to install the required UAA, but is virtually invisible to the endogenous cellular machinery. As a result, site-directed mutagenesis can be used to introduce a single TAG codon at a desired position in the encoding DNA, resulting in a single introduction of UAA with high specificity and selectivity.

Compositions and kits

Compositions or kits may be formed comprising detectable probes or affinity reagents as described in the present disclosure. Several configurations are set forth below in the context of the composition. It will be appreciated that the kit may be similarly configured. The composition may include one or more detectable probes or affinity reagents along with solutions or solvents and other constituent components. The specific formulation of the detectable probe or affinity reagent composition may depend on the intended use of the detectable probe or affinity reagent and the specific composition of the detectable probe or affinity reagent in the composition. The composition may be formulated based on several factors, including: 1) Stability and storage requirements of the detectable probes or affinity reagents; 2) Characteristics and/or features of the detectable probe or affinity reagent; and 3) the mode of use of the detectable probe or affinity reagent. Stability and/or storage considerations may include conditions that maintain stability of the retention component, binding component, and/or labeling component. The characteristics or feature considerations may include affinity and/or avidity features, probe dissociation, and labeling component features. The mode of use considerations may include detection methods, multiplexing, and secondary binding interactions.

In general, the composition may include one or more detectable probes or affinity reagents in a liquid medium. In some configurations, the liquid medium may be aqueous or otherwise include water. In some configurations, the liquid medium may include a non-aqueous solvent, such as a polar solvent or a non-polar solvent. The liquid medium may comprise a pH buffered solution. The pH buffer solution may be formulated to maintain the solution pH within a desired range, for example to maintain stability of the retention component or binding component or to adjust the strength of the binding interaction of the binding component. The liquid medium may further comprise one or more salts. Salts in the liquid medium may be added to alter the ionic strength of the liquid medium. The ionic strength may be adjusted, for example, to maintain stability of the retention component or binding component or to adjust the strength of the binding interaction of the binding component. The probe composition may comprise an emulsion or colloidal suspension, such as a water-in-oil emulsion or an oil-in-water emulsion.

Detectable probes or affinity reagents comprising nucleic acids (e.g., DNA fold, DNA nanospheres, aptamers) may be provided in compositions specifically formulated to maintain nucleic acid stability. In some configurations, the detectable probe or affinity reagent comprising the nucleic acid may be a probe comprising a magnesium salt (e.g., mgCl ₂ ) Is provided in a liquid medium of (a). Magnesium salts may be provided in sufficient concentration to maintain nucleic acid stability (e.g., base pair binding, helical structure, etc.). The magnesium salt can have a concentration of at least about 10mM, 50mM, 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, 220mM, 240mM, 260mM, 280mM, 300mM, 350mM, 400mM, 500mM, or more. Alternatively or additionally, the magnesium salt may have a concentration of no greater than about 500mM, 400mM, 350mM, 300mM, 280mM, 260mM, 240mM, 220mM, 200mM, 180mM, 160mM, 140mM, 120mM, 100mM, 50mM, 10mM, or less.

The liquid medium comprising the detectable probe or affinity reagent may further comprise a scavenger material. The scavenger may include any chemical species that is intended to remove chemically unfavorable or harmful species, such as radical scavengers, oxygen scavengers, and metal chelators. Possible scavengers in the detectable probe or affinity reagent composition may include substances such as hydrazine, ascorbic acid, tocopherol, naringenin, glutathione, stannene and EDTA. The scavenger may be included in a liquid medium that is intended for storing the detectable probe or affinity reagent composition prior to the assay or other means of use.

In some configurations, the detectable probe or affinity reagent composition comprising a liquid medium may be formulated as a multi-phase liquid, such as a water-in-oil emulsion or an oil-in-water emulsion. The multi-phase liquid medium may allow for localization and/or confinement of the detectable probe or affinity reagent prior to or during the mode of use. For example, the release of the detectable probe or affinity agent may be controlled by breaking the emulsion, thereby releasing the probe confined within the emulsion. In addition, the multiphasic formulation may increase the stability of the detectable probe or affinity reagent having mixed chemical characteristics (e.g., hydrophobic and hydrophilic components) or the sensitivity to aqueous chemicals (e.g., sensitivity to hydrolysis).

In some configurations, the detectable probe or affinity reagent composition may be formulated to include a competitor. The competitor may comprise an affinity reagent or other molecule configured to bind to a binding partner, epitope or target moiety. The competitor may be characterized by a low affinity for the binding partner, epitope or target moiety. In some configurations, the competitor may be characterized by a low affinity for the same binding partner, epitope or target moiety as the binding partner, epitope or target moiety of the detectable probe or affinity reagent. In other configurations, the competitor may be characterized by a low affinity for multiple binding partners, epitopes, or target moieties (e.g., reduced binding specificity). Without wishing to be bound by theory, the competing materials may include binding materials driven by an increase in the free energy of gibbs bound from displacement of the binding partner, epitope or target moiety. For example, the competitor may have a greater enthalpy of binding to the binding partner than the detectable probe or affinity reagent, thereby energetically favoring displacement of the competitor and binding of the probe. Likewise, a competitor may increase the entropy of the system by dissociating from the binding partner to facilitate a detectable probe or affinity reagent. The competitor may be advantageous in creating a tunable affinity in the detectable probe or affinity reagent composition because: 1) The competitor may thermodynamically promote binding of the detectable probe or affinity reagent to the binding partner, epitope or target moiety, and 2) the competitor may affect the kinetics of probe binding by providing concentration-dependent competition for the binding partner, epitope or target moiety. FIGS. 20A-20B depict the use of detectable probes or affinity reagents comprising binding competitors to compete to promote binding. Fig. 20A shows the contact of a binding partner 2010, including an epitope or target moiety 2020, with a detectable probe 2040. The surface of binding partner 2010 may bind to a plurality of competing affinity reagents 2030 that bind non-specifically to the surface. Fig. 20B shows free competitor affinity reagent 2035 displaced by binding of detectable probe 2040 to epitope or target moiety 2020. Displacement of the competing affinity reagent may energetically or entropy promote binding of the detectable probe to the binding partner 2010.

The competitor may include an affinity agent, such as an aptamer, a peptide aptamer, a designed ankyrin repeat protein (DARPin), an antibody or an antibody fragment. The competitor may be provided as a component of the detectable probe or affinity reagent or as a separate substance from any detectable probe or affinity reagent. FIGS. 16A-16B illustrate a detectable probe composition comprising a competitor. FIG. 16A shows a detectable probe composition including a competitor in a free solution. The composition includes a detectable probe 1610 containing a plurality of attached binding components 1620 (e.g., antibodies or antibody fragments). The composition further includes a competing affinity reagent 1630 (e.g., an aptamer) that is free in solution due to separation from the probe 1610. The competitor 1630 is free to associate with the binding partner, epitope or target moiety, but the competitor is compartmentalizable and can dissociate from the binding partner, epitope or target moiety. Fig. 16B shows a detectable probe composition comprising competing components attached to a detectable probe. The detectable probes 1610 include a plurality of attached binding members 1620 (e.g., antibodies or antibody fragments) and a plurality of attached competing members 1630 (e.g., aptamers). A detectable probe or affinity reagent composition comprising an attached competing component may be advantageous for facilitating dissociation of the bound affinity reagent at a slow dissociation rate, as the probability of the strongest binding species binding to the target at any given time is reduced.

Alternatively, the competitor may comprise a substance that competes with the binding partner, epitope or target moiety for binding to the detectable probe or affinity agent. For example, the competitor may be a short peptide sequence (e.g., 2-15 amino acid residues) containing the target sequence free in solution. The free peptide may be capable of competitively binding with the detectable probe or affinity reagent, thereby altering the apparent affinity of the binding interaction of the detectable probe or affinity reagent with the binding partner, epitope or target moiety in a manner analogous to a competitor.

A competitor may be present when the detectable probe or affinity agent is contacted with the binding partner, epitope or target moiety. The competitor may be introduced either before or after the detectable probe or affinity agent is contacted with the binding partner, epitope or target moiety. The ratio of detectable probes or affinity reagents to competing materials in the composition can be adjusted to adjust the affinity characteristics of the composition. The detectable probe or affinity reagent composition can be adjusted to adjust binding characteristics, such as probe binding or dissociation rates, to achieve a desired level of affinity. The competitor may be present at a mass or molar ratio of at least about 1:1000000, 1:100000, 1:10000, 1:1000, 1:100, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 100:1, 1000:1, 10000:1, 100000:1, 1000000:1 or more relative to the detectable probe or affinity reagent. Alternatively or additionally, the competitor may be present at a mass or molar ratio of no greater than about 1000000:1, 100000:1, 10000:1, 1000:1, 100:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000 or less relative to the detectable probe or affinity reagent.

The presence of a competitor substance, for example in the form of a competitor component, in the detectable probe or affinity reagent composition may affect the binding or affinity characteristics of the detectable probe or affinity reagent. The presence of a competitor substance, e.g., in the form of a competitor component, in the detectable probe or affinity reagent composition may affect the dissociation rate, binding rate, dissociation constant or affinity of the detectable probe or affinity reagent. The presence of a competitor, e.g., in the form of a competitor component, in the detectable probe or affinity reagent composition may increase the dissociation rate, binding rate, dissociation constant or affinity of the detectable probe or affinity reagent. The presence of a competitor, e.g., in the form of a competitor component, in the detectable probe or affinity reagent composition may decrease the dissociation rate, binding rate, dissociation constant or affinity of the detectable probe or affinity reagent. The presence of a competitor, e.g., in the form of a competitor component, may increase or decrease the dissociation rate, binding rate, dissociation constant, or affinity of the detectable probe or affinity reagent by a factor of at least about 2, 5, 10, 25, 50, 100, 250, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, or more. Alternatively or additionally, the presence of a competitor, e.g., in the form of a competitor component, may increase or decrease the dissociation rate, binding rate, dissociation constant or affinity of the detectable probe or affinity reagent by a factor of no more than about 1000000, 500000, 100000, 50000, 10000, 5000, 1000, 500, 250, 100, 50, 25, 10, 5, 2 or less.

The detectable probe or affinity reagent composition can include one or more components configured to alter the binding characteristics of the detectable probe or affinity reagent. Components may be added to the detectable probes or affinity reagent compositions to increase the binding rate, dissociation constant, and/or affinity of the detectable probes or affinity reagent compositions. Components may be added to the detectable probes or affinity reagent compositions to reduce the binding rate, dissociation constant, and/or affinity of the detectable probes or affinity reagent compositions. The component may affect the affinity or avidity of the detectable probe or affinity agent by chemically altering the binding interaction between the detectable probe or affinity agent and the binding partner, epitope or target moiety (e.g., altering the conformation of the binding component, weakening the electrostatic interaction between the binding component and the binding partner). The component may affect the affinity or avidity of the detectable probe or affinity reagent by creating a weak secondary binding interaction with the detectable probe or affinity reagent.

Components may be added to the detectable probe or affinity reagent composition to alter the binding interaction between the detectable probe or affinity reagent composition and the binding partner, epitope or target moiety. The added component may alter the binding interaction by altering the binding partner, epitope or target moiety, altering the detectable probe or affinity reagent, or by altering the binding interaction between the detectable probe or affinity reagent composition and the binding partner, epitope or target moiety. The added component may cause a conformational change in the detectable probe or affinity reagent, the binding component and/or the binding partner, epitope or target moiety that increases or decreases the likelihood and/or strength of the binding interaction. The added components may alter the binding interactions, for example, by electrostatically screening the binding interactions or competing to form binding interactions.

A denaturing agent, surfactant or chaotropic agent may be added to the detectable probe or affinity reagent composition to alter the binding interaction between the detectable probe or affinity reagent and the binding partner, epitope or target moiety. A denaturing agent, surfactant or chaotrope may be added to the detectable probe or affinity reagent composition to alter the binding characteristics of the detectable probe or affinity reagent, such as binding rate, dissociation constant or affinity. The denaturing agent, surfactant or chaotropic agent may facilitate the removal of the detectable probe or affinity agent from the binding partner, epitope or target moiety. A denaturing agent, surfactant or chaotropic agent may be introduced to the detectable probe or affinity reagent composition after probe binding to facilitate the removal of the detectable probe or affinity reagent from the binding partner, epitope or target moiety. Binding of a detectable probe or affinity agent in the presence of a denaturing agent, a surfactant or a chaotropic agent can reduce the rate of binding of the detectable probe or affinity agent upon binding to a binding partner, epitope or target moiety. The concentration of the denaturing agent, surfactant or chaotrope in the detectable probe or affinity reagent composition may be adjusted to adjust the affinity characteristics of the detectable probe or affinity reagent. The concentration of the denaturing agent, surfactant or chaotrope in the detectable probe or affinity reagent composition may be limited to avoid destabilizing the detectable probe or affinity reagent or components of the detectable probe or affinity reagent (e.g., DNA origami components). The denaturant, surfactant or chaotrope in the detectable probe or affinity reagent composition may be bound to heat to facilitate the removal of the detectable probe or affinity reagent from the binding partner, epitope or target moiety.

Salts, such as metal salts, may be added to the detectable probe or affinity reagent composition to alter the binding interaction between the detectable probe or affinity reagent and the binding partner, epitope or target moiety. Salts or ionic species including salts may form interactions with the detectable probe or affinity reagent or binding partner, epitope or target moiety that alter the chemistry of the binding interaction between the detectable probe or affinity reagent and the binding partner, epitope or target moiety. Salts or ionic species including salts can disrupt the chemistry of the binding interaction between the detectable probe or affinity reagent and the binding partner, epitope or target moiety. Salts or ionic species including salts may facilitate binding interactions between the detectable probe or affinity reagent and the binding partner, epitope or target moiety. In some configurations, salts may be added to the detectable probe or affinity reagent composition to facilitate the removal of the detectable probe or affinity reagent from the binding partner, epitope, or target moiety. Salts in the detectable probe or affinity reagent composition may bind thermally to facilitate the removal of the detectable probe or affinity reagent from the binding partner, epitope, or target moiety. Salts may be introduced to the detectable probe or affinity reagent composition after probe binding to alter the binding interaction of the detectable probe or affinity reagent with the binding partner, epitope, or target moiety (e.g., removal of the detectable probe or affinity reagent).

The detectable probe or affinity reagent composition may further comprise a binding molecule that alters the affinity and/or observability of the detectable probe or affinity reagent. Binding molecules may include molecules configured to form reversible or irreversible binding interactions with a detectable probe or affinity reagent. The binding molecules may form weak binding interactions with binding partners, epitopes, target moieties or detectable probes or affinity reagents to hold the detectable probes or affinity reagents in proximity to the potential targets, thereby increasing the likelihood that binding interactions may occur. The binding molecules may include additional labeling components that increase the signal generated upon binding interactions. Exemplary binding molecules are described elsewhere herein. The binding molecule may be provided to the detectable probe or affinity reagent composition before, during, or after the detectable probe or affinity reagent has been contacted with the binding partner, epitope, or target moiety.

The detectable probes or affinity reagent compositions of the present disclosure may be provided in kit form, including suitable packaging materials, if desired. In one embodiment, for example, the detectable probe or affinity reagent composition may comprise a plurality of detectable probes or affinity reagents, e.g., provided in a solution or suspension. In another embodiment, the detectable probes or affinity reagent compositions may comprise a plurality of detectable probes or affinity reagents, e.g., provided as a solid such as a crystal or lyophilized solid. Thus, any combination of reagents or components useful in the methods of the present disclosure, such as those previously set forth herein with respect to a particular method, may be included in the kits provided by the present disclosure. These reagents or components may include, without limitation, buffers, salts, stabilizers, retention components, binding components, labeling components, and competing affinity reagents. For example, the kit may include a plurality of detectable probes or affinity reagents provided in a storage buffer containing a stabilizing surfactant and an oxygen scavenger.

As used herein, the phrase "packaging material" refers to one or more physical structures for containing the contents of a kit, such as detectable probes, affinity reagents, and the like. The packaging material may be constructed by well known methods, preferably providing a sterile, non-contaminating environment. Packaging materials employed herein may include, for example, those conventionally utilized in affinity reagent systems. Exemplary packaging materials include, but are not limited to, glass, plastic, paper, foil, etc., capable of containing within fixed limits components useful in the methods of the present disclosure, such as detectable probes or affinity reagent compositions.

The packaging material may include a label that indicates that the detectable probe or affinity reagent is available for a particular method. For example, the tag may indicate that the kit may be used to detect a particular binding partner, epitope, or target moiety, thereby providing a characterization during polypeptide assays. In another example, the label may indicate that the kit may be used for therapeutic or diagnostic purposes.

Instructions for use of the packaged reagents or components are also typically included in the kits of the disclosure. "instructions for use" generally include tangible representations describing the concentration of reagents or components or at least one assay parameter, such as the relative amounts of kit components and sample to be mixed, the maintenance period of the reagent/sample mixture, temperature, buffer conditions, and the like.

The kit or composition may comprise a plurality of different detectable probes and/or different affinity reagents that differ in the number or type of binding members attached thereto. Alternatively or additionally, the kit or composition may comprise a plurality of different detectable probes and/or different affinity reagents, each attached to a different labeling component. The detectable probes or affinity reagents, although different in terms of the number or type of binding components or in terms of the number or type of labeling components, may still have substantially the same type of retention components. For example, a plurality of different detectable probes and/or different affinity reagents may contain a nucleic acid-based paper folding retention component, wherein the structure of the paper folding or the structure of the scaffold in the paper folding is the same for all different probes and/or reagents. The plurality of different detectable probes and/or different affinity reagents may comprise at least about 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1000 or more different detectable probes and/or different affinity reagents. Alternatively or additionally, the plurality of different detectable probes and/or different affinity reagents may comprise up to about 1000, 500, 250, 100, 50, 25, 10, 5, 4, 3, 2 or less detectable probes and/or different affinity reagents.

Application method

The detectable probes or affinity reagents of the present disclosure can be used in a wide variety of applications, including binding assays. In general, the use of a detectable probe or affinity reagent composition may involve one or more of the following steps: 1) Contacting a detectable probe or affinity reagent with a solution and/or solid support having a binding partner for the detectable probe or affinity reagent; 2) Allowing the detectable probe or affinity reagent to bind to the binding partner in the solution or on the solid support; 3) Washing unbound probes from the solution and/or solid support; 4) Observing the solution and/or solid support to detect signals from one or more detectable probes or affinity reagents; 5) Removing one or more bound probes or reagents from the binding partner; 6) Optionally repeating one or more of steps 1) -5); and 7) using the presence and/or absence of signals from one or more probes or reagents to predict the presence and/or absence of the binding partner in the solution and/or on the solid support.

The method of observing the binding interaction between the detectable probe or affinity reagent and the binding partner can be used for any of a variety of characterizations. In some cases, detectable probes or affinity reagents may be utilized to facilitate polypeptide characterization assays. The assays may be configured to determine or predict one or more characteristics (e.g., size, trait, isoform, etc.) of one or more polypeptides. Alternatively or additionally, the assay may be an assay configured to determine or predict one or more polypeptides in a sample. The polypeptide characterization assay or quantitative assay may be configured for single molecule detection, wherein one or more polypeptide molecules are individually resolved. For example, in a multiplex format, one or more characteristics and/or amounts of each polypeptide of a plurality of polypeptides may be predicted or determined based on detection of the polypeptides individually. In some configurations, polypeptide assays can include single molecule characterization or quantification of a polypeptide to determine the presence of one or more epitopes (e.g., dimeric, trimeric or tetrameric amino acid sequences; post-translational modifications, etc.) in a plurality of polypeptides. Detectable probes or affinity reagents may be advantageous for polypeptide characterization assays because they have strong affinity for binding to a target and their signal output is strong, giving high confidence binding interaction data.

When bound to an affinity reagent or detectable probe, a polypeptide or other analyte may be attached to a Structured Nucleic Acid Particle (SNAP). Fig. 58A shows polypeptide 5840 attached to SNAP 5850. Polypeptide 5840 is an exemplary analyte and may be replaced with other analytes of interest. NAP 5850 acts as a retention component for polypeptide 5840 and may be replaced or modified with a retention component set forth herein in the context of affinity reagents and detectable probes. For example, SNAP 5850 may be a nucleic acid fold, or may be replaced with fluorescent particles. Polypeptide 5840 can be covalently or non-covalently attached to SNAP 5850. Polypeptide 5840 may be contacted with a detectable probe having SNAP-based retention component 5810, one or more labeling components 5820, and one or more binding components 5830. Optionally, SNAP 5810 may be replaced or modified with a retention component set forth herein in the context of an affinity reagent and a detectable probe. SNAP 5810 may be a nucleic acid fold, for example, or may be replaced with fluorescent particles. Because polypeptide 5840 is a binding partner for one or more of binding components 5830, a complex is formed. The complex includes SNAP 5850 attached to polypeptide 5840 and SNAP 5810 attached to one or more binding components 5830, at least one of which binds to polypeptide 5840, SNAP 5810 also attached to at least one marker component 5820. Optionally, SNAP 5850 may be attached to a solid support, such as a site on an array. SNAP may be attached to a solid support using any of a variety of covalent or non-covalent chemical methods, including but not limited to those set forth herein in the context of attaching components of an affinity reagent or a detectable probe to each other.

In some configurations, the polypeptide assay may include contacting a detectable probe or affinity reagent with a plurality of polypeptides, wherein each polypeptide of the plurality of polypeptides is bound to a solid support at a unique, optically observable spatial location, such as at a site in a polypeptide array. Detection of a signal at a given spatial location may prove that a detectable probe or affinity reagent has bound to a polypeptide at that spatial location, thereby indicating the presence of a particular epitope or target moiety at that spatial location in the polypeptide. The predicted or determined presence or absence of one or more epitopes or target moieties predicts or determines the characteristics of the polypeptide bound at a given spatial location. Furthermore, the amount of a particular polypeptide in a sample may be predicted or determined based on the intensity of the signal from a given spatial location and/or based on the number of sites in the array that generate a signal indicative of the particular probe (or series of probes) having bound.

The multiplex binding reaction is shown in FIG. 58B. The reaction is exemplified by a polypeptide analyte, but may also be performed with other analytes known in the art or set forth herein. Polypeptide array 5800 includes four different polypeptides 5841 to 5844. Each of the polypeptides is attached to SNAP 5851. The attachment may be covalent or non-covalent. The array 5800 is in contact with a plurality of detectable probes. Each of the probes includes SNAP-based retention component 5811 attached to at least one labeling component and at least one binding component. Three different detectable probes are shown, including a first probe having SNAP 5811 attached to at least one labeling portion 5821 and at least one binding portion 5831, a second probe having SNAP 5811 attached to at least one labeling portion 5822 and at least one binding portion 5832, and a third probe having SNAP 5811 attached to at least one labeling portion 5823 and at least one binding portion 5833. The product of the binding reaction is the binding of the first and third probes to the corresponding polypeptides on array 5800. Because polypeptide 5841 is a binding partner for one or more of binding components 5831, a first complex is formed. The first complex includes SNAP 5851 attached to polypeptide 5841 and SNAP 5811 attached to one or more binding components 5831, at least one of which binds to polypeptide 5841, SNAP 5811 also attached to at least one marker component 5821. Because polypeptide 5843 is a binding partner for one or more of binding components 5833, a second complex is formed on array 5800. The first complex includes SNAP 5851 attached to polypeptide 5843 and SNAP 5811 attached to one or more binding components 5833, at least one of which binds to polypeptide 5843, SNAP 5811 also attached to at least one marker component 5823. In this example, polypeptides 5842 and 5844 remain unbound because they are not binding partners to any detectable probes. Moreover, the third detectable probe of SNAP 5811, which includes at least one binding component 5832 and at least one labeling component 5822 attached thereto, has no binding partner on array 5800 and remains unbound. The first and second detectable probes may be detected on the array, wherein they are spatially resolved, and wherein they may be further distinguished and confirmed based on their different label types. Thus, based on knowledge of the binding characteristics of each detectable probe and the type of label associated with each detectable probe, the signal detected from the array site can be used to confirm the polypeptide.

In the example of fig. 58B, the polypeptides, while separated into separate sites on array 5800, are also attached to SNAP having a common structure. For example, SNAP 5851 may be a nucleic acid fold, and the polypeptides may be attached to folds that have the same scaffold fold as each other. The subset of one or more staples in the fold may be different between folds, for example to accommodate attachment of different polypeptides. The use of a common SNAP structure or other retention component for this purpose may provide for convenient loading of polypeptides on an array. For example, the array may be configured with uniform sites of interaction with uniform structural elements of SNAP to effect attachment of the polypeptides to the array. The common retention component structure may also be used for a plurality of different detectable probes or a plurality of different affinity reagents. As illustrated in fig. 58B, the three detectable probes differ in the label component and the binding component attached to SNAP 5811. However, the three affinity probes have substantially the same SNAP structure. For example, SNAP 5811 may be a nucleic acid fold with the same scaffold fold for each of the three detectable probes. The subset of one or more staples in the fold may differ between different detectable probes, for example to accommodate the attachment of different label components or different binding components. While fig. 58B is illustrated with a common SNAP attached to different analytes and a common SNAP attached to different detectable probes, it will be appreciated that the SNAP may instead differ in structure. Additionally, SNAP may include sequence tags that may detect differences between probes or between polypeptides. The tag may be used to identify individual probes in the mixture or to identify individual polypeptides in the array.

The array may be used for multiplex processing of analytes, whereby multiple different types of analytes are processed or detected in parallel. Particularly useful analytes include, but are not limited to, binding partners for the affinity reagents set forth herein, such as polypeptides, nucleic acids, and the like. Parallel processing may provide cost savings, time savings, and uniformity of conditions, although one or more steps of the methods set forth herein may also be employed to continuously process different types of analytes. The array may comprise at least 2, 10, 100, 1000, 1x 10 ⁴ 、1x 10 ⁵ 、1x 10 ⁶ 、1x 10 ⁷ 、1x 10 ⁸ 、1x 10 ⁹ One or more different analyte sites. Alternatively or additionally, the array may comprise up to 1x 10 ⁹ 、1x 10 ⁸ 、1x10 ⁷ 、1x 10 ⁶ 、1x 10 ⁵ 、1x 10 ⁴ 1000, 100, 10, 2 or fewer different analyte sites. The different analytes may optionally be attached to the site via a structured nucleic acid particle or a retention component having a common structure. As such, the sites of the array may be attached to structured nucleic acid particles or retention components having substantially the same structure as each other, and each of the structured nucleic acid particles or retention components in the array may be attached to a different analyte.

The array may be attached to the inner surface of the flow cell wall or to a solid support inside the flow cell. The flow cell or solid support may be made of any of a variety of materials for analytical biochemistry. Suitable materials may include glass, polymeric materials, silicon, quartz (fused silica), boron float glass, silica-based materials, carbon, metals, optical fibers or bundles, sapphire or plastic materials. The materials may be selected based on the characteristics desired for a particular application. For example, materials transparent to the desired radiation wavelength may be used in analytical techniques that will utilize radiation at that wavelength. Instead, it may be desirable to select a material that does not pass radiation of a certain wavelength (e.g., opaque, absorptive, or reflective). Other material properties that may be utilized are the ease of inertness or reactivity or handling of certain reagents used in downstream processes or low cost of manufacture as set forth herein.

The polypeptide or other analyte may be attached to the support in a manner that provides detection at the single molecule level. For example, a plurality of different polypeptides may be attached to a solid support in such a way that individual detectable probes or affinity reagents that bind to individual polypeptide sites on the support are distinguishable from all adjacent sites on the array, even though adjacent sites bind to detectable probes or affinity reagents. In this manner, one or more different polypeptides (or other binding partners of the detectable probes or affinity reagents set forth herein) may be attached to a solid support in a form in which each individual polypeptide molecule is physically separated and detected in such a way that the individual molecule is distinguishable from all other molecules on the solid support. Alternatively, the methods of the present disclosure may be practiced on one or more pools, which are populations of substantially the same type of analyte, such as populations of nucleic acids or polypeptides having a common sequence.

Fig. 22 shows the configuration of the polypeptide assay. The plurality of polypeptides are optionally bound to solid support 2210 via an anchor group 2230 (e.g., SNAP or a chemical linker). The detectable probes 2240 are contacted with a plurality of polypeptides, allowing the detectable probes 2240 to bind to an available binding target. After contact, the detectable probe 2240 has bound to the first polypeptide 2221 and the fourth polypeptide 2224, and has not bound to the second polypeptide 2222 or the third polypeptide 2223. If viewed by a suitable detection system, such as a fluorescence microscope, a detectable signal can be observed on solid support 2210 at a spatial location corresponding to the location of first polypeptide 2221 and fourth polypeptide 2224.

The polypeptides may be of natural or synthetic origin. The polypeptide may contain one or more post-translational modifications. Alternatively or additionally, one or more post-translational modifications may not be present in the polypeptide. In some cases, the polypeptide may be treated to remove, reverse, or alter post-translational modifications. For example, the polypeptide assays set forth herein can be performed to detect one or more polypeptides before and after removal, reversal, or alteration of post-translational modifications. Comparing the results before and after may provide benefits, such as increasing the confidence in identifying the number or type of post-translational modifications of the polypeptide. In some cases, the polypeptide may be treated to produce a post-translational modification. For example, the polypeptide assays set forth herein can be performed to detect one or more polypeptides before and after post-translational modification. Instead, the polypeptide assays set forth herein can be performed to detect one or more polypeptides before and after removal of the post-translational modification. Comparing the results before and after may provide benefits such as confirming the number or type of post-translational or post-synthetic modifications to which the polypeptide is susceptible. In some cases, the polypeptide may be proteolytic to remove at least a portion of the polypeptide. For example, the polypeptide assays set forth herein can be performed to detect one or more polypeptides before and after proteolysis. Comparing the results before and after may provide benefits, such as locating an epitope or target portion in the polypeptide relative to the position of the proteolytic site in the polypeptide. A further benefit of comparing the results before and after may be to more easily identify the number or type of post-translational modifications in the polypeptide susceptible to proteolysis.

Post-translational modifications may include myristoylation, palmitoylation, prenylation, farnesylation, geranylgeranylation, lipidylation, flavin moiety attachment, heme C attachment, phosphopantetheinyl (phosphopanthetenation), retinoid formation, bisxylylenediamine formation, ethanolamine phosphoglycerol attachment, homolysine, beta-lysine addition, acylation, acetylation, deacetylation, formylation, alkylation, methylation, C-terminal amidation, arginylation, polyglutinylation, butyrylation, gamma-carboxylation, glycosylation, saccharification, polysialization, malonyl, hydroxylation, iodination nucleotide addition, phosphate formation, phosphoramidate formation, phosphorylation, adenylation, uridylation, propionylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, S-sulfinylation, S-sulfonylation, succinylation, sulfation, saccharification, carbamylation, carbonylation, isopeptidation bond formation, biotinylation, carbamylation, oxidation, reduction, pegylation, ISG, SUMO, ubiquitination, pupation (puplation), citrullination, deamination, ethylamino (elminylation), disulfide bridge formation, proteolytic cleavage, isoaspartic acid formation, racemization, and protein splicing. Particularly interesting and useful post-translational modifications are proteolysis, which may be site-specific (i.e. occur at specific amino acid residues or amino acid sequences) or non-site specific. Post-translational modifications may be reversed or removed from the polypeptide using biological enzymes, chemical reagents, or physical treatments as known to those of skill in the art. The polypeptides may be post-translationally modified using biological enzymes, chemical reagents, or physical treatments such as those known to those skilled in the art.

The polypeptide characterized may have a particular size. The polypeptide can be at least about 0.1 daltons (Da), 0.5Da, 1Da, 5Da, 10Da, 50Da, 100Da, 200Da, 300Da, 400Da, 500Da, 600Da, 700Da, 800Da, 900Da, 1 kilodaltons (kDa), 1.5kDa, 2kDa, 2.5kDa, 3kDa, 3.5kDa, 4kDa, 4.5kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 200kDa, 300kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 1200kDa, 1400kDa, 1600kDa, 1800kDa, 2000kDa, 2500kDa, 3000kDa, 3500kDa, 4000kDa or more. Alternatively or additionally, the polypeptide may be no greater than about 4000kDa, 3500kDa, 3000kDa, 2500kDa, 2000kDa, 1800kDa, 1600kDa, 1400kDa, 1200kDa, 1000kDa, 900kDa, 800kDa, 700kDa, 600kDa, 500kDa, 400kDa, 300kDa, 200kDa, 100kDa, 90kDa, 80kDa, 70kDa, 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa, 15kDa, 10kDa, 9kDa, 8kDa, 7kDa, 6kDa, 5kDa, 4.5kDa, 4kDa, 3.5kDa, 3kDa, 2.5kDa, 2kDa, 1.5kDa, 1kDa, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, 100Da, 50Da, 10Da, 5Da, 1Da, 0.5Da or less. The polypeptide may contain a minimum or maximum number of amino acid residues. The polypeptide may contain at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000 or more amino acid residues. Alternatively or additionally, the polypeptide may contain no more than about 40000, 30000, 20000, 15000, 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 250, 200, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or less amino acid residues.

Polypeptide assays may involve one or more fluid transfer operations, including fluids containing detectable probes or affinity reagents and fluids that mediate binding interactions involving detectable probes or affinity reagents (e.g., wash fluids, removal reagents). Fluid transfer operations may occur when fluid is transferred into or out of a fluid device such as a flow cell or chip. Polypeptide characterization assays may require that multiple polypeptides bound to a solid support are always in contact with a fluid medium. The fluid transfer operation may displace the first fluid medium from the flow cell with the second fluid medium to ensure that there is always fluid in contact with the solid support comprising the plurality of polypeptides.

The sample may be provided to a polypeptide detection system or method. The sample may be provided as a fraction comprising a plurality of polypeptides or other analytes, for example. The sample may be provided as a fraction comprising a plurality of polypeptide conjugates or other analyte conjugates. Analyte conjugates can include an analyte (e.g., a polypeptide analyte) attached to a solid support either directly or via a linker moiety such as a structured nucleic acid particle, polymer, or protein moiety. The sample may be provided to the detection system or method in a liquid medium (e.g., aqueous, pH buffered medium). The sample may be provided to the detection system or method as a solid (e.g., a lyophilized, precipitated, or crystallized sample). The solid may be dissolved or suspended in the liquid medium before, during or after being added to the detection system or method. The sample may be stored in the detection system after being provided to the system.

Samples comprising polypeptides or other analytes may be bound to a solid support in a fluidic device. The fluid comprising the sample may be pumped or flowed through the fluidic system to a detection chamber, such as a flow cell or well, comprising a solid support within the fluidic device. The fluid comprising the sample may be contacted with the solid support for a time sufficient to attach the analyte to the solid support. Optionally, the analyte may be conjugated to a substance that mediates attachment of the analyte to the solid support, such as a structured nucleic acid particle. In some cases, additional fluid may be mixed with the sample fluid to facilitate binding of the analyte to the solid support. If the sample includes a polypeptide, the solid support may first be contacted with a fluid that includes polypeptide linking groups (e.g., structured nucleic acid particles, polymers, proteins) for a time sufficient to deposit the polypeptide linking groups on the surface of the solid support. After the solid support has been deposited with polypeptide linking groups, the fluidic medium comprising the sample may be pumped or flowed into a detection chamber containing the solid support in a fluidic device. The attachment reaction between the polypeptide and the polypeptide linking group may be allowed to occur long enough to attach the polypeptide to the linking group. One or more reagents may be added to the fluidic device to facilitate the polypeptide attachment reaction, such as reactants and/or catalysts. In some cases, the polypeptide and polypeptide linking group may be configured to react via a spontaneous attachment reaction, such as a "click" reaction. In some cases, the polypeptide may be attached directly to the solid support, for example, by a bond between a functional group on the polypeptide and a functional group on the solid support. After the attachment reaction, the solid support will include one or more polypeptides bound to the solid support.

After the analyte (e.g., polypeptide) has been attached to the solid support, e.g., within the fluidic device, the detection chamber of the fluidic device may be rinsed one or more times. The flushing process may utilize one or more wash or rinse reagents that are pumped or flowed into the fluid device together or sequentially. The volume of the flushing fluid provided to the fluidic device may exceed the total volume of the detection chamber including the solid support. The rinsing step may remove some or all of the unbound polypeptide, polypeptide conjugate, or other agent. The flow rate of the flushing fluid to the fluidic device may be limited to prevent expulsion of the attached analyte from the solid support.

Optionally, the attached analyte (e.g., conjugated polypeptide) may be subjected to one or more processes that alter the structure of the analyte. For example, the polypeptide analyte may be partially or completely denatured, such as by adding a denaturing fluid to the fluidic device. The denaturing fluid may be contacted with the solid support for a time sufficient to promote complete denaturation of the polypeptide, or may be contacted with the solid support briefly to cause partial denaturation of the polypeptide. The polypeptide may also be altered by applying an altering agent such as an enzyme (ligase, kinase, glycosylase, phosphatase, protease, etc.), an oxidizing agent or a reducing agent to the solid support. The fluidic device may undergo a rinsing process after the addition of the structure-altering reagent, thereby removing residual reagent from the fluidic device. In some cases, the solid support may remain in contact with the altering agent (e.g., denaturing agent) until the affinity reagent is introduced into the fluidic device. In some cases, the polypeptide may be partially or fully refolded after the structure change process.

The affinity reagent may be contacted with a solid support comprising a plurality of polypeptides (e.g., an array of polypeptides) or other analytes, for example, in a fluidic device. In some configurations, the affinity reagent may be provided as a detectable probe or as an affinity reagent. Affinity reagents such as detectable probes may be provided in a fluid medium that is pumped or flowed into the fluidic device. The fluid medium may comprise a single kind of affinity reagent or detectable probe, or may comprise a mixture of different kinds of affinity reagents or detectable probes. For example, the fluidic medium may include at least two detectable probes or affinity reagents having different epitope binding specificities. Alternatively, the fluid medium may comprise at least two detectable probes or affinity reagents, which are different types of detectable probes or affinity reagents, e.g. 1 aptamer probe and 1 antibody probe. The affinity reagent or detectable probe may be contacted with the solid support in a fluidic device for a time sufficient to facilitate binding of the affinity reagent or detectable probe to an epitope or target moiety (e.g., an amino acid sequence epitope in a polypeptide) present in the at least one analyte.

Affinity reagents or binding components utilized in binding assays can be distinguished by their nature of binding specificity. In particular, the affinity reagent may have epitope binding specificity that has been characterized in a probabilistic manner. The affinity reagents of the present disclosure can be characterized by a variety of binding probabilities, rather than in a binary manner (e.g., affinity reagent a binds to epitope X, not to epitope Y). For example, of 8000 possible trimeric amino acid sequences (20 x20x 20), the non-zero binding probability of an affinity reagent may be known, measured or estimated for some or all 8000 sequences. In some cases, an affinity agent may be used in the present disclosure if it has a high known, measured or estimated probability of binding (e.g., greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999% or higher) to a first epitope and a low known, measured or estimated probability of binding (e.g., less than 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or lower) to a second epitope. In some cases, affinity reagents may include a high known, measured or estimated binding probability (e.g., greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999% or higher binding probability) for a family of epitopes and a low known, measured or estimated binding probability (e.g., less than 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or lower binding probability) for other epitopes. The family of epitopes can include epitopes that are related by amino acid sequence (e.g., amino acid sequence AXA, where a is alanine and X can be any amino acid; amino acid sequence αaaa beta, where α and beta are independently any possible amino acid flanking sequences) or can be related by chemical properties (e.g., nonpolar, polar, positively charged, negatively charged, post-translational modification, etc.).

After administration of an affinity reagent, such as a detectable probe, for a sufficient time to bind to an analyte (e.g., a polypeptide analyte) in a fluidic device, the detection chamber of the device may undergo one or more washes or rinses to remove any unbound affinity reagent. Polypeptide characterization assays can minimize the likelihood of disruption of analyte-affinity reagent interactions using only a single wash step after affinity reagent binding. The affinity reagent rinse fluid may be displaced from the fluidic device after the rinsing process by a medium configured for performing physical measurements, such as an optical imaging buffer.

The analyte: affinity reagent or analyte: probe interaction may be measured after the affinity reagent or detectable probe has been contacted with a plurality of analytes bound to the solid support. Detection of the labeled component of a physical measurement, such as a detectable probe, may be performed under stationary fluid conditions. By isolating all of the inlet and outlet ports, for example by closing a valve, the fluid may remain static in the fluid. The fluid may be held in the fluid device for a time sufficient to allow imaging of one, some or all of the unique, optically observable spatial locations.

After binding and/or physical measurement of analyte: affinity reagent or analyte: probe interactions has been performed, the bound affinity reagent or detectable probe may be stripped from the analyte by adding a disruption medium to the fluidic device. The disruption medium may include a denaturing agent, chaotrope, or other chemical entity that may disrupt analyte: affinity reagent or analyte: probe interactions. The destruction medium may be contacted with the analyte under stationary or flowing conditions. After the disruption medium has been contacted with the analyte, the fluidic device may be rinsed or washed one or more times with a rinsing medium to remove any unbound affinity reagent or unbound detectable probes from the fluidic device.

In some cases, the analyte (e.g., a polypeptide analyte) may be removed from the surface of the solid support during or after the binding measurement. For example, multiple polypeptides may be characterized to determine which polypeptides are glycosylated, followed by release of all non-glycosylated polypeptides from the solid support. In some cases, all analytes may be released from the solid support after completion of the characterization assay to allow reuse of the fluidic device. Analyte or analyte-conjugate may be released from the solid support by addition of a stripping fluid. The stripping fluid may provide a more stringent wash than other wash media utilized during characterization assays, such that it results in displacement of the analyte from the solid support. Displacement of the analyte from the solid support may also utilize physical conditions such as heat or light, for example by cleaving a photoactivatable linker between the analyte and the solid support.

During or after displacement of the analyte from the solid support, a flushing medium may be flowed through the fluidic device to remove the displaced analyte. The solid support can then be observed by physical measurement methods to determine or confirm the displacement of the analyte from the surface.

Regardless of the specificity described above, in some cases, a particular polypeptide analyte may be identified based on a series of observations of different probes that bind or do not bind to the protein. These series can be evaluated using software algorithms that use probabilistic modeling to evaluate the probability that a given series identifies a given protein or polypeptide. In a multiple use configuration, binding and non-binding patterns of a plurality of different polypeptides can be observed. For example, the proteins may be displayed in an array consisting of individually distinguishable sites, each site having one of the polypeptides. A series of observations can be made for each site to see if different probes bind. The identity of the polypeptide at each site can be determined from a series of observations at each site. The overall pattern of a series of observations at each site can be used to identify multiple polypeptides in an array. Examples of software algorithms, methods and compositions that can be used to identify proteins are described, for example, in published international patent application No. WO 2019/133892, U.S. patent No. 10,473,654, or U.S. patent application publication No. 2020/0286584A1, each of which is incorporated herein by reference.

In some cases, detection, validation or characterization of the polypeptide can utilize the affinity reagents set forth herein. The affinity reagents of the present disclosure may be hybrid or broad spectrum affinity reagents that have the potential to interact (e.g., bind) with more than one polypeptide in a sample. In some cases, the affinity reagent may have the potential to interact with two or more unique, structurally distinct proteins in the sample. For example, based on structurally similar regions within otherwise distinct proteins, affinity reagents may bind with near equal probability to a particular membrane protein and a particular cytoplasmic protein. In some cases, binding affinity reagents may have the potential to bind to a particular amino acid epitope or family of epitopes, regardless of the sequence context (e.g., the amino acid sequence up-chain and/or down-chain from the epitope).

The affinity reagents of the present disclosure can be characterized such that they have a probability-based binding profile that is confirmed, determined, or assessed. The affinity reagent can have a property that binds to the first polypeptide with a binding probability of greater than about 50% (e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or greater than about 99.999%) that is confirmed, determined, or assessed and binds to a second structurally different polypeptide with a binding probability of less than about 50% (e.g., no greater than about 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, or less than about 0.001%) that is confirmed, determined, or assessed. In certain cases, the observed difference in the probability of binding of the affinity reagent to the first and second polypeptides may be due to the presence, absence or inaccessibility of a particular epitope or family of epitopes in the first or second polypeptide. The probabilistic affinity binding profile may be determined or validated by in vitro measurements or computer predictions.

Alternative and additional embodiments and examples

The skilled artisan will readily recognize the usefulness of the detectable probes and affinity reagents of the present disclosure, including, for example, their tunable affinity and observability features. The detectable probes or affinity reagent compositions of the present disclosure can be used to provide high resolution spatial characterization in microscale as well as single molecule systems and environments. The described detectable probes or affinity reagents may also provide useful features in biological contexts, such as diagnostic and therapeutic applications.

The detectable probes or affinity reagents of the present disclosure can facilitate immunohistochemistry for purposes such as elucidation of cells and tissue structures. The detectable probes or affinity reagents can be attached to a plurality of binding components with high specificity for a particular cellular component (e.g., surface biomarker, polysaccharide, structural protein, organelle protein, etc.). The detectable probe or affinity reagent can be contacted directly with the cell (e.g., microorganism) or tissue sample (e.g., tissue slice, frozen tissue sample, formalin-fixed paraffin-embedded sample) and incubated for a time sufficient to allow binding interaction between the detectable probe or affinity reagent and the cell or tissue-related target, thereby forming a treated cell or tissue sample. The treated cell or tissue sample may be directly observed by any suitable method (depending on the probe-e.g. fluorescence microscopy, scintillation counting, etc.). The detectable probes or affinity reagents may be advantageous for immunohistochemical applications due to their high binding affinity and high observability. Analysis of the binding data may provide high resolution spatial data regarding the number and distribution of biomarkers or biomolecules in the cell and/or tissue sample.

Detectable probes or affinity reagents may be utilized as pull-down or capture reagents. The high binding affinity may facilitate capturing and retaining the targeted substance from a heterogeneous mixture of substances. The affinity reagent may be free in solution or may be attached to a solid support (e.g., a bead, chip or chromatographic resin) during or after binding to the binding partner. Contacting the affinity reagent with a heterogeneous mixture that may include a binding partner may allow separation of the binding partner from at least one other component of the mixture. For example, an affinity reagent attached to a solid support may be contacted with the sample to allow binding partners to bind to the solid support via the affinity reagent, the solid support may be separated from the sample, and the solid support may optionally be washed to remove residual sample components from the solid support. In another example, an affinity reagent may be contacted with the sample to allow binding of the binding partner to the affinity reagent, the resulting affinity reagent-binding partner complex may be attached to a solid support, the solid support may be separated from the sample, and the solid support may optionally be washed to remove residual sample components from the solid support. In either instance, subsequent collection and separation of the affinity reagent or release of the binding partner may enable separation of the binding partner from the sample (e.g., heterogeneous mixture). Optionally, one or more binding partners captured by the methods described above may then be detected in a detection assay set forth elsewhere herein. For example, a subset of different polypeptides may be captured from a sample, such that other polypeptides are removed from the sample, and then the subfractions of the polypeptides may be quantified or characterized using the polypeptide assays set forth herein.

Detectable probes or affinity reagents may be used as components within a separation system such as an affinity chromatography column. The affinity chromatography system may be configured to attach a plurality of detectable probes or affinity reagents having a specific affinity for one or more binding partners, epitopes or target moieties within a porous matrix or resin. With the polypeptides as exemplary analytes, a mixture comprising polypeptides (e.g., cell lysates, samples of synthetic proteins or peptides, non-biological samples containing possible biological contamination, etc.) may be applied to a column comprising detectable probes or affinity reagents to affect separation between the targeted polypeptides (e.g., those comprising binding partners, epitopes, or target moieties) and the non-targeted polypeptides. The detectable probes or affinity reagents affinity chromatography system may comprise a resin or matrix to which the detectable probes or affinity reagents are permanently or irreversibly conjugated, allowing for reuse of the system. The captured polypeptide may be eluted from the column after the non-targeted fraction of the mixture has passed through the column. The detectable probe or affinity reagent affinity chromatography system may comprise a resin or matrix that irreversibly attaches the detectable probe or affinity reagent to the resin or matrix, thereby allowing release of the detectable probe or affinity reagent comprising the captured target from the system. In some cases, the affinity chromatography system may include a detection system (e.g., fluorescence measurement, IR, UV) that allows detection of a signal from a detectable probe or affinity reagent as the probe elutes from the system. The detection system may allow for measurement of background or unintended release of the detectable probe or affinity agent from the resin or matrix and monitoring of the intended release of the detectable probe or affinity agent from the matrix or resin. After capturing the target polypeptide from the mixture, the polypeptide may be eluted from the system by releasing the detectable probe or affinity reagent. In some cases, the released detectable probe-polypeptide complex (or affinity reagent-polypeptide complex) may be collected and then deposited on a solid support for use in a polypeptide assay, such as a polypeptide characterization assay or a binding ligand assay.

FIGS. 17A-17C depict schemes that utilize affinity reagents as capture agents. As shown in fig. 17A, an affinity reagent 1710 is provided, wherein the affinity reagent includes a capture handle 1720 and a plurality of binding components having high binding specificity for a target binding partner 1730. The capture handle may comprise any suitable handle, such as a capture tag (e.g., biotin), a nucleic acid, or a functional group (e.g., a click functional group). Affinity reagent 1710 is contacted with a heterogeneous mixture comprising target binding partner 1730 and a plurality of non-target substances 1735, thereby facilitating binding of affinity reagent 1710 to target binding partner 1730. Optionally, there may be a solid support 1750 comprising capture sites 1740 configured to bind to capture handle 1720. The capture sites may be produced on a solid support, for example as a patterned array or a random array. As shown in fig. 17B, the heterogeneous mixture is separated from the affinity agent 1710-target binding partner 1730 complex. If solid support 1750 is present, affinity reagent 1710 and target binding partner 1730 may bind to the solid support at anchor point 1745 resulting from the binding of capture handle 1720 at capture site 1740. Deposition of affinity reagent 1710 and target binding partner 1730 on solid support 1750 can produce a random or patterned array of captured target binding partners (e.g., target polypeptides). Alternatively, the affinity reagent 1710-target binding partner 1730 complex may be separated from the free solution by separation methods such as affinity chromatography, size exclusion chromatography, gravity settling, filtration, magnetic capture (of a magnetic or paramagnetic solid support), or centrifugation.

Fig. 17C depicts an optional final step. Captured target binding partners 1730 may be characterized or quantified using detectable probes 1760 as an assay reagent. One or more detectable probes 1760 can be contacted with the captured target binding partners 1760 to characterize the presence or absence of binding interactions between the target binding partners 1730 and the detectable probes 1760. Observing the presence or absence of a detectable signal from the detectable probes 1760 may indicate the probability of the presence of a binding partner, epitope, or target moiety at the observed location on the solid support 1750.

Detectable probes or affinity reagents can be used in a co-binding assay. A co-binding assay may refer to any assay in which multiple affinity reagents and/or detectable probes are simultaneously contacted with a binding partner, epitope, or target moiety to simultaneously determine the presence of two or more features (e.g., epitopes) in a binding target. Co-binding assays can be utilized, for example, to determine the simultaneous presence of two different epitopes in a polypeptide, thereby yielding a high confidence prediction of polypeptide trait.

The detectable probes or affinity reagents may be formulated for co-binding assays by combining two or more unique species of detectable probes or affinity reagents, each distinguished by a unique detection signature or fingerprint (e.g., first probe use 488, second Probe use->647 fluorescent dye). Simultaneous detection of a unique marker or fingerprint (or lack thereof) at a spatial location may provide evidence of whether one or more detectable probes or affinity reagents have bound to a binding partner, epitope or target moiety at that spatial location.

Alternatively, a co-binding assay using a detectable probe or affinity reagent may be performed with a detectable probe or affinity reagent that includes a nucleic acid barcode. FIGS. 18A-18C depict schemes for performing a co-binding assay using a bar code detection signal. Fig. 18A depicts binding partners 1890 in contact with a first detectable probe 1810 and a second detectable probe 1840. First detectable probe 1810 is configured to bind to first epitope or target portion 1870 and includes first linker 1820 having barcode sequence 1838 and terminal priming sequence 1830. Second detectable probe 1840 is configured to bind to second epitope or target portion 1880 and includes a second adaptor 1850 having terminal complementary priming sequence 1860. If a binding interaction occurs between first detectable probe 1810 and first epitope or target portion 1870 and a binding interaction occurs between second detectable probe 1840 and second epitope or target portion 1880, terminal priming sequence 1830 and complementary priming sequence 1860 may be sufficiently close to hybridize by nucleic acid base pairing. As shown in fig. 18B, contacted polymerase 1865 may bind to the hybridized nucleic acid at the priming sequence, allowing an extension reaction to occur. As shown in fig. 18C, after the extension reaction, second detectable probe 1840 may dissociate from binding partner 1890 after complementary barcode sequence 1868 has been added to complementary priming sequence 1860. Subsequent analysis of the detectable probe barcodes will detect the transcribed barcodes, thereby indicating that both detectable probes bind to the binding partner 1890 simultaneously.

The detectable probes or affinity reagents of the present disclosure may be used for medical diagnostic purposes, such as diagnostic assays. The detectable probes or affinity reagents may be suitable for use in vitro and in vivo systems. The detectable probes or affinity reagents may be adapted for common in vitro assays, such as western blots and ELISA, where the detectable probes or affinity reagents replace antibodies commonly used in such assays. The detectable probes or affinity reagents may also be used as capture or pull-down reagents for the localization of proteins or other biomarkers from fluids or other biological samples. For example, the detectable probe or affinity reagent may be contacted with blood or plasma to isolate a blood-borne biomarker. The detectable probes or affinity reagents may be used in vivo assays, such as PET scans. In some configurations, the in vivo detectable probe or affinity reagent may be attached to a radiolabel (e.g., ¹⁵ O、 ¹⁸ F、 ⁶⁸ Ga、 ⁸⁹ Zr、 ⁸² rb) to enable the detectable probe or affinity reagent to act as a radiotracer. The detectable probe or affinity reagent may provide high resolution, high signal data for viewing tissue displaying the biomarker, the detectable probe or affinity reagent configured to have high affinity for the biomarker. Also, a detectable probe or parent And reagents may be applied for real-time prediction or diagnosis of medical procedures. For example, detectable probes or affinity reagents with high affinity for tumor surface biomarkers can be used to monitor radiation therapy or surgical treatment in real time. High resolution fluorescence data regarding the presence of surface biomarkers can provide feedback to the surgeon regarding the clearance of cancer markers from the surgical incision and the progress of radiation therapy or surgical treatment (fluorescence extinction will be related to destruction of residual tumor tissue).

Affinity reagents or detectable probes may be used as therapeutic agents for medical applications. High affinity reagents or probes may constitute logical disruptors or facilitators of in vivo signaling and/or binding processes. Affinity reagents or detectable probes may be attached to multiple binding ligands (e.g., hormones, cytokines, etc.) or to targets (e.g., surface receptors) to facilitate interaction with in vivo signaling or binding systems. The high affinity reagents or detectable probes may be provided to the in vivo system in sufficient quantity to partially or completely block signaling or bind to the receptor system, thereby reducing or increasing cellular responses. The high affinity reagents or detectable probes may be provided to the in vivo system in sufficient quantity to partially or fully capture one or more signaling or binding ligands, thereby reducing or increasing the biological response. The high affinity reagents or detectable probes may be used as antimicrobial or antiviral compositions. For example, an affinity reagent or detectable probe may be configured to bind to a receptor system used by a microorganism or virus to initiate its reproductive cycle. Alternatively, the affinity reagent or detectable probe may be configured to bind to a microorganism or virus (e.g., via a viral spike protein), thereby inhibiting the ability of the microorganism or virus to bind to a cellular target.

Detectable probes or affinity reagents comprising alternative binding members may be used in therapeutics, diagnostics or drug development. A detectable probe or affinity reagent comprising a plurality of attached affinity reagent chimeras may be used for therapeutic or drug development purposes. The affinity agent chimera may comprise any complex, including an affinity agent coupled to a secondary molecule such as a small molecule, nucleic acid, peptide, or protein. Chimeras may include secondary molecules having specific biological functions, such as antisense nucleic acids, exons, introns, transcriptional repressors, transcriptional promoters, receptor binding ligands, enzyme substrates, and the like. Affinity reagent chimeras may include aptamer-siRNA chimeras, antibody-siRNA chimeras, aptamer-ligand chimeras or antibody-ligand chimeras. Detectable probes or affinity reagents, including chimeras of affinity reagents, can be used to localize targets of secondary molecules, such as binding target molecules to deliver antisense RNA to the target molecules. In some configurations, a detectable probe or affinity reagent may include a plurality of affinity reagents coupled to the probe and a further plurality of secondary molecules coupled to the detectable probe or affinity reagents.

The detectable probes or affinity reagents can be used as drug delivery platforms. The increased binding affinity of the detectable probes or affinity reagents provided herein will allow for high binding to membrane receptors or other targetable systems for cellular uptake. The detectable probe or affinity agent may be associated with a drug delivery formulation, such as a colloidal particle or a coated drug formulation. FIGS. 38A-38D depict cellular drug delivery using a detectable probe or affinity reagent. Fig. 38A shows a combination of a detectable probe or affinity reagent comprising a hydrophobic surface modifying group 3810 and colloidal drug particles 3820 comprising a drug formulation 3830. As shown in fig. 38B, the hydrophobic interactions between the colloidal particles 3820 and the hydrophobic modification groups of the detectable probes or affinity reagents 3810 form targeted drug delivery complexes. Fig. 38C depicts interactions between membrane-associated protein 3845 (e.g., a cell receptor) and targeted drug delivery complexes embedded within the cell membrane 3842 of cells 3840 due to binding interactions between membrane-associated protein 3845 and targeted drug delivery complexes. Fig. 38D depicts uptake of the drug formulation 3830 into the cell 3830 after the targeted drug delivery complex has interacted with the targeted membrane-associated protein 3835, thereby releasing the drug formulation 3830 into the cell 3840. The detectable probes or affinity reagents 3810 may be released or absorbed into the cells during uptake of the colloidal particles 3820.

Detectable probes or affinity reagents can be used to characterize non-biological materials. The high affinity reagents or probes may be configured to bind to non-biological targets (e.g., nanoparticles, polymer structures, etc.). The detectable probes or affinity reagents can enable high resolution imaging of structures in non-biological materials. For example, detectable probes or affinity reagents having affinity for certain nanoparticle structures can be used to study the structure, degradation, and poisoning processes of composite catalyst materials. Also, detectable probes or affinity reagents having affinity for certain charged surface active sites can be used to provide high resolution data regarding the distribution and availability of reactive sites on the surface of a material. The detectable probes or affinity reagents can be used to target and localize microscale degradation and/or destruction in polymeric systems such as synthetic and natural fabrics. The resolution provided by the high affinity probes may allow features that are not apparent to typical optical inspection processes to be identified in a variety of materials.

The detectable probes or affinity reagents may be used in additional methods of polypeptide characterization. The detectable probes or affinity reagents may be configured to implement single molecule detection of the ademan degradation technique. In general, edman degradation can produce sequence reads for multiple peptides by stepwise removal and detection of a single amino acid residue from each peptide. Single molecule methods that facilitate edman degradation can be used to detect terminal amino acids or terminal amino acid sequences by using detectable probes or affinity reagents. FIGS. 19A-19D depict possible methods of Edemann degradation using a detectable probe to increase the signal intensity of each sequence read. Fig. 19A depicts a solid support 1910 having a plurality of polypeptides bound to the solid support 1910. The first polypeptide 1920 has a terminal amino acid residue with a side chain R1, and the second polypeptide 1930 has a terminal amino acid residue with a side chain R2. FIG. 19B depicts the contacting of a plurality of peptides with a detectable probe composition comprising a plurality of probe species (e.g., comprising at least 20 unique probe species) having high binding specificity for each natural terminal amino acid residue. The first polypeptide 1920 is bound by a detectable probe 1940 configured to have binding specificity for the side chain R1. The second polypeptide 1930 is bound by a detectable probe 1950 configured to have binding specificity for the side chain R2. Each detectable probe of the detectable probe composition has a unique detection signature or fingerprint, allowing for unique identification of each binding event. FIG. 19C depicts a typical intermediate step in the Edman degradation process after each peptide of the plurality of polypeptides has reacted with an isothiocyanate compound to form a cleavable product. The first polypeptide 1925 has a terminally modified amino acid residue with a side chain R1 and the second polypeptide 1935 has a modified terminal amino acid residue with a side chain R2. FIG. 19D depicts the contacting of a plurality of modified peptides with a detectable probe composition comprising a plurality of probe species (e.g., comprising at least 20 unique probe species) having high binding specificity for each modified natural terminal amino acid residue. The first modified polypeptide 1925 is bound by a detectable probe 1945 configured to have binding specificity for the side chain R1. The second modified polypeptide 1935 is bound by a detectable probe 1955 configured to have binding specificity for the side chain R2. Each detectable probe of the detectable probe composition has a unique detection signature or fingerprint, allowing for unique identification of each binding event. In some configurations, the detectable probe composition can be contacted with the detectable probe composition before and after modification of the terminal amino acid residues to increase the confidence of the sequence reads.

The skilled artisan will readily recognize that many of the embodiments discussed may be readily configured to utilize other techniques discussed above, such as using tandem barcodes as depicted in fig. 15A-15D, or methods for forming weak secondary binding interactions such as those depicted in fig. 8, 9A, and 9B.

Examples

Example 1: design and synthesis of affinity reagents with folded paper tiles as retention components

Affinity reagents based on folded paper tiles were designed using the cadno software with internal python script attached. The tiles are designed as nucleic acid folds of approximately square shape, with a single layer. The tile was designed with 20 DNA aptamer binding sites on the top surface of the tile and 44 dye molecule attachment sites evenly distributed along the sides of the tile molecule. FIG. 23 depicts a schematic of a tile-based affinity reagent 2310, where points 2330 represent aptamer attachment locations and circles 2320 represent dye attachment locations. The tile is designed to have a side length of about 83 nanometers (nm)

Paper folding tiles were prepared using a scaffold strand containing a 7249 nucleotide M13 single stranded circular DNA strand. 217 single stranded oligonucleotides were designed and synthesized for hybridization to the scaffold strand as "staples" to allow assembly of tile-based affinity reagents.

The paper folding tile was assembled by combining the scaffold strand with 217 oligonucleotides. The DNA strand was incubated at 90℃with 5mM Tris-HCl, 5mM NaCl, 1mM EDTA, 12.5mM MgCl at pH8.0 ₂ Is combined in the buffer of (2). The DNA strand mixture was cooled to a temperature of 20℃and annealed for 1.5 hours.

The annealed tile-based affinity reagent was purified via HPLC size exclusion chromatography or 100kDa size cut-off spin filter. The purified tile-based affinity reagent was resuspended at pH8.0 containing 5mM Tris-HCl, 200mM NaCl, 1mM EDTA, 11mM MgCl ₂ And stored at 4 ℃.

Example 2: image analysis of folded paper tiles

Affinity reagents based on folded paper tiles were prepared as described in example 1. The tile probe was imaged via Transmission Electron Microscopy (TEM) to confirm the assembly method was successful. The tile probe solution was spotted onto glow discharge carbon deposited copper grids and negative staining was performed using uranyl formate.

The tile probe was imaged on a FEI Tecnai T12 transmission electron microscope at an accelerating voltage of 120 kilovolts (kV) at a magnification of 30,000 times. As predicted, the tile probe appears to be approximately square in shape, with sides of about 83nm.

Example 3: design and synthesis of tile-based affinity reagents with antibody-based binding components

Antibody tile detectable probes were designed using cadno software with internal python script attached. The folded paper tile is designed to be approximately square in shape, with a single layer. The tile was designed with 6 antibody binding sites (2 on the top surface and 1 along each side of the tile) and 40 dye molecule attachment sites evenly distributed along the sides of the tile molecule. FIG. 25 depicts a schematic of tile-based affinity reagent 2510 with antibody component 2520 at respective attachment locations, and circle 2530 represents the attachment location of the labeling component. The tile is designed to have a side length of about 83 nanometers (nm)

Paper folding tiles were prepared using a scaffold strand containing a 7249 nucleotide M13 single stranded circular DNA strand. 217 single stranded oligonucleotides were designed and synthesized for hybridization to the scaffold strand to allow assembly of the tile probe. 6 of the oligonucleotides were synthesized with trans-cyclooctene modified nucleotides to enable attachment of antibody components via a click reaction.

The tiles were assembled by combining the scaffold strand with 217 oligonucleotides. The DNA strand was incubated at 90℃with 5mM Tris-HCl, 5mM NaCl, 1mM EDTA, 12.5mM MgCl at pH8.0 ₂ Is combined in the buffer of (2). The DNA strand mixture was cooled to a temperature of 20℃and annealed for 1.5 hours.

The annealed tiles were purified via HPLC size exclusion chromatography or 100kDa size cut-off spin filters. The purified tile was resuspended in pH 8.0 containing 5mM Tris-HCl, 200mM NaCl, 1mM EDTA, 11mM MgCl ₂ Is contained in the buffer solution of (2). The antibody was attached to the tile via a trans-cyclooctene (TCO) -methyltetrazine (mTz) click reaction. Fig. 26 depicts an exemplary reaction scheme for attaching antibodies (or other binding components) to a folded paper tile. Purified leaf tile with TCO modified nucleotides was combined with mTz modified 10x anti-His tag antibody at a temperature of 20 ℃ and allowed to react for 6 to 10 hours. After the click reaction, the tile probe was purified via HPLC size exclusion chromatography.

FIG. 27 shows SDS-page gels of purified antibody-DNA oligonucleotide conjugates produced via TCO-mTz click reaction. Lane a contains antibody-DNA oligonucleotide conjugates. Lane B contains a mTz modified antibody negative control. Lane C contains an unmodified antibody control. The uppermost black band was observed confirming that the antibody was successfully attached to the DNA oligonucleotide.

Example 4: binding of tile-based affinity reagents with aptamer-based binding components

Four different kinds of aptamer tile probes were prepared according to the method of example 1. Each baseThe affinity reagent on the tile contains different aptamers with different binding affinities to the terminal histidine tag. The four aptamers used were: b1 (highest affinity), A1 (medium affinity), D1 (low affinity) and SC2 (no affinity). With 20 aptamers and 40647 dye molecules assemble each tile-based affinity reagent.

Each aptamer was screened for binding to a single molecular array of his-tagged ubiquitin. Protein arrays were prepared by depositing a 10 nanogram/microliter solution of ubiquitin conjugate (ubiquitin attached to structured nucleic acid particles) on a fluidic chip. Each fluidic chip contained 3 lanes, each lane having a glass surface covered with a 3-aminopropyl triethoxysilane (APTMS) coating. Three different types of protein arrays were prepared, 1 per lane on each chip: ubiquitin with his tag; ubiquitin with flag tag (negative control); and unlabeled ubiquitin (negative control). Each deposited protein conjugate contains488 dye molecule. Protein conjugate positions in the array were measured via fluorescence microscopy at 488nm excitation. The fluidic chip was blocked for 60 minutes with a solution containing 1% BSA, 2nM unlabeled DNA tile and 100mg/ml dextran sulfate in buffer containing, prior to affinity reagent binding.

To test binding, 15 μl of 20nM affinity reagent solution was flowed into the fluidic chip. The probes were allowed to bind for 10 minutes. After the affinity reagent is bound, the fluidic chip is rinsed with a rinsing solution. The binding was imaged on the surface using a ThorLab microscope at 20x magnification and 647nm excitation. The microscopic image at 647nm was compared to the image at 488nm to determine the fraction of protein bound by the applied affinity reagent. All four affinity reagent species were tested for each of the 3 arrays. Each group was run in duplicate 12 experiments.

Fig. 28A-28D depict the occupancy rate of the bond. Occupancy is determined as the ratio of the locations where the detected binding is observed to the total locations with known protein conjugates. FIG. 28A shows the occupancy of B1 aptamer reagent compared to the occupancy of A1 aptamer reagent. Although binding affinity is low, A1 aptamer reagent has been shown to have almost as high occupancy as B1 aptamer reagent. Fig. 28B shows the occupancy of B1 aptamer reagent compared to the occupancy of D1 aptamer reagent. Fig. 28C and 28D compare the occupancy of B1 compared to SC2 and D1 compared to SC2, respectively. As shown in fig. 28B and 28D, despite the low binding affinity for his-tagged proteins, the occupancy of D1 aptamer reagent was observed to be nearly half that of B1 aptamer reagent and significantly higher than the negative control SC2 aptamer reagent, indicating that the detectable probes can significantly increase the binding affinity observed for weak-junction aptamers by the avidity effect.

Example 5 binding of tile-based affinity Agents with antibody-based binding Components

Tile-based affinity reagents with antibody-based binding components were prepared according to examples 2 and 3. Six 10x anti-His antibodies and 40 were used647 dye molecules each affinity reagent was prepared. Binding was tested using a 0.5nM probe solution via the method described in example 4.

Fig. 29A-29C show fluorescence microscope images of reagent binding. FIGS. 29A and 29B show binding of reagents to unlabeled and flag-tagged ubiquitin arrays, respectively. Figure 29C shows binding of reagents to his-tagged ubiquitin arrays. Higher levels of probe binding to the his-tagged array were observed.

FIG. 30 shows the occupancy of affinity reagents for 3 different protein arrays on 6 different chips. High occupancy on his-tagged protein arrays was observed for all 6 chips, while much lower occupancy was observed for other his-tagged proteins on each fluidic chip.

EXAMPLE 6 multiplex binding assay

A detectable affinity reagent comprising a type 1 or type 2 fluorophore is assembled. Useful fluorophores have emission at red, yellow, green, or blue wavelengths (R, Y, G, B). Each fluorophore is named to indicate the total number of each fluorophore on the detectable probe. For example, a detectable affinity reagent having 10 red fluorophores and 10 yellow fluorophores will be designated as "R10Y10". Each affinity reagent having a unique combination of fluorophores is attached to a unique class of binding components having a unique binding specificity for the target moiety. Two distinct fluorophore pools were formed, with the composition of each pool listed in table 1 below.

The pool of affinity reagents is contacted with a first binding partner. Fluorescence intensity was measured at wavelengths corresponding to four possible fluorophores. The measured fluorescence intensity correlates with the number of fluorophores that may be available. Table 2 shows the fluorophore counts measured based on fluorescence intensity, as well as the combination of bound probes from each cell, which will yield a unique combination of observed fluorescence intensities.

TABLE 2

Based on the observed fluorescence intensity, the available target moiety for the binding partner can be predicted.

The pool of affinity reagents is contacted with a second binding partner. Fluorescence intensity was measured at wavelengths corresponding to four possible fluorophores. The measured fluorescence intensity correlates with the number of fluorophores that may be available. Table 3 shows the fluorophore counts measured based on fluorescence intensity, and the combination of bound affinity reagents from each cell, which will yield a unique combination of observed fluorescence intensities.

Based on the observed fluorescence intensity, the available target moiety for the binding partner can be predicted. Rational engineering of multiple pools of detectable affinity reagents can yield unique binding signatures that predict the simultaneous presence of multiple target moieties within the binding partner.

EXAMPLE 7 affinity chromatography

Three affinity chromatography columns were prepared. The first chromatography column comprises a first chromatography resin comprising an affinity reagent covalently attached to a resin. The affinity reagent in the first column has a general binding affinity for the polypeptide, regardless of the amino acid sequence. The second chromatography column comprises a second chromatography resin comprising an affinity reagent covalently attached to a resin. The affinity reagent in the second column has binding affinity for a polypeptide comprising within its amino acid sequence a lysine-arginine-alanine (KRA) amino acid trimer sequence. The third chromatography column comprises a third chromatography resin comprising an affinity reagent coupled to the resin by oligonucleotide hybridization. The affinity reagent in the third column has binding affinity for a polypeptide comprising within its amino acid sequence a lysine-glutamic acid-asparagine (KEN) amino acid trimer sequence.

The three affinity chromatography columns are arranged in sequence. The crude cell lysate is applied to a first universal separation column. The polypeptide is captured from the crude cell lysate, while the non-polypeptide fraction of the lysate is passed through the column and discarded. After discarding the non-polypeptides of the lysate, the polypeptide fraction is eluted by applying an elution buffer to the first column. The eluted fraction was captured and applied to a second KRA specific chromatography column. A first fraction of the polypeptide is captured on the KRA-specific resin, while a second fraction is passed through the column and captured. After the second fraction is collected from the end of the column, the first fraction of the polypeptide is eluted from the column by the elution buffer and collected at the end of the column.

The first collected polypeptide fraction from the KRA specific chromatography column was added to the third KEN specific chromatography column. A first fraction of the polypeptide is captured on the KEN-specific resin, while a second fraction is passed through the column and captured. After the second fraction is collected from the end of the column, the first fraction of the polypeptide is eluted from the column by heating the column to melt the oligonucleotide hybridization, thereby releasing the detectable probe from the column.

The KEN-specific column was regenerated by supplementing the affinity reagent released from the first fraction separation with a new batch of KEN-specific detectable affinity reagent. The separation was then repeated for the second collected fraction from the KRA specific column. The first pass fraction was collected followed by the second captured fraction. After separation of the two fractions from the KRA-specific column, a total of four fractions were formed. FIG. 40 shows a schematic representation of the chromatographic process from the initial cell lysate to the final four polypeptide fractions, with the initially defined features of the four fractions listed at the bottom. Preliminary characterization of the four fractions was evaluated based on the chromatographic behavior of each fraction. Characterization is considered preliminary due to the likelihood of false negative or false positive capture interactions.

Two fractions of the polypeptide captured on the KEN-specific column were collected as affinity reagent-polypeptide complexes. Each of the affinity reagent-bound polypeptide fractions is applied in turn to a patterned silicon solid support comprising a plurality of attachment sites, wherein each site is configured to capture and bind an affinity reagent. After applying each affinity reagent-bound fraction to the chip, the position of each occupied attachment site on the solid support was measured and recorded using fluorescence microscopy.

Two non-captured fractions from the KEN-specific column are attached to the structured nucleic acid particles. Each non-captured fraction was applied to a silicon solid support and the occupied attachment sites were observed by fluorescence microscopy as the captured fraction. After all four fractions were bound to the solid support, a patterned array of polypeptides had been generated for use in polypeptide assays.

Example 8 FluoSphere based ^TM Synthesis of affinity reagents for (a)

The detectable affinity reagent is prepared using highly fluorescent organic nanoparticles that include modifiable surface chemistry. FIG. 41 shows a process for preparing FluoSphere-based ^TM Is a probe of the above-mentioned type. Modification of 40nm or 200nm carboxylate functionalized FluoSpheres with polyethylene glycol moieties ^TM (Thermo-Fisher) to in FluoSphere ^TM A passivation layer is formed around the particles. Activation of FluoSpheres by NHS-EDC activation prior to functionalization with PEG groups ^TM . FluoSpheres functionalized with carboxylic esters ^TM With NH ₂ -PEG and NH ₂ The 95:5 mixture of PEG-azides is combined to give FluoSphere containing azide functions ^TM A pegylated surface coating was formed around. PEGylated FluoSpheres ^TM Incubated with a 3' -Dibenzocyclooctene (DBCO) capped oligonucleotide to covalently attach the oligonucleotide to an azide-capped PEG moiety on the surface of the pegylated layer. Incubation was performed in phosphate buffer (pH 7.4) at about 24 ℃ overnight.

In attaching oligonucleotides to FluoSpheres ^TM Thereafter, an attachment site for coupling an affinity reagent is provided. FluoSpheres for functionalizing oligonucleotides ^TM Combined with an oligonucleotide comprising a complementary sequence to the functionalized oligonucleotide handle. The mixture was heated to 95 ℃ for 5 minutes, then annealed at 70 ℃ for 10 minutes, and then cooled to room temperature. Alternatively, the complementary oligonucleotide may be annealed to the DBCO functionalized oligonucleotide before attaching to FluoSpheres ^TM . The sequences of the oligonucleotides are listed in Table 4.

TABLE 4 oligonucleotide attachment handle sequences

Example 9 Synthesis of Quantum dot based affinity reagents

The detectable affinity reagent is prepared using highly fluorescent inorganic nanoparticles that include modifiable surface chemistry. 15nm carboxylated quantum dots (Thermo Fisher) were partially modified with polyethylene glycol to form a non-adherent layer around the quantum dot particles. Combining quantum dots with NH ₂ -PEG and NH ₂ The 75:25 mixture of PEG-azides is combined to give an azide-containing mixtureThe quantum dots of functional groups form a pegylated surface coating around. The pegylated quantum dots are incubated with a 3' -DBCO-capped oligonucleotide to covalently attach the oligonucleotide to the azide-capped PEG moiety on the surface of the pegylated layer. Incubated overnight at about 24℃in phosphate buffer (pH 7.4).

After the oligonucleotides are attached to the quantum dots, attachment sites for coupling affinity reagents are provided. The oligonucleotide functionalized quantum dots are combined with oligonucleotides comprising complementary sequences of functionalized oligonucleotide handles. The mixture was heated to 95 ℃ for 5 minutes, then annealed at 70 ℃ for 10 minutes, and then cooled to room temperature. Alternatively, the complementary oligonucleotide may be annealed to the DBCO functionalized oligonucleotide before being attached to the quantum dot. The sequences of the oligonucleotides are listed in Table 4.

Example 10 FluoSphere based ^TM Optimization of affinity reagents of (c)

The examination is based on FluoSphere ^TM Various configurations of detectable affinity reagents to determine the optimal pegylation strategy. Carboxylated FluoSpheres according to the method described in example 8 ^TM PEGylation is performed. Variables tested included PEG size, PEG type, and PEG surface density.

Characterization of PEGylated FluoSpheres by measuring zeta potential to determine the extent of surface passivation ^TM . Zeta potential measurements were performed in 2% Phosphate Buffered Saline (PBS). FIG. 42A shows the use of PEG-NH ₂ PEG-OH and NHS activated FluoSpheres ^TM FluoSpheres for PEGylation ^TM Measured zeta potential. When combined with PEG-NH ₂ Upon reaction, the zeta potential was reduced in magnitude, indicating FluoSphere ^TM PEGylation and passivation of the surface were successful. Fig. 42B shows zeta potential as a function of estimated surface density of PEG groups. FluoSpheres were prepared by the method of example 8 ^TM Wherein varying PEG-NH ₂ At a concentration of 0.5mM, 5.9mM or 59mM PEG-NH ₂ . The magnitude of zeta potential was shown to decrease as the estimated PEG surface density increased.

PEGylated FluoSpheres ^TM Coupled to the oligonucleotide to provide an attachment site for coupling the binding component. Example 8Strategies for providing attachment sites are described. FIG. 42C shows that when an oligonucleotide and an attachment oligonucleotide are added to FluoSphere ^TM When to PEGylated FluoSpheres ^TM Measured zeta potential. It was found that the magnitude of zeta potential increased with the addition of nucleic acid, confirming the oligonucleotide to FluoSpheres ^TM The coupling was successful. FIG. 42D shows FluoSpheres with aptamer components coupled to oligonucleotides ^TM And (3) coupling. FluoSpheres ^TM Coupled to the poly-T oligonucleotide to form an attachment site configured to couple an aptamer component that displays a poly-a annealing sequence. The average total number of annealed oligonucleotides was calculated by qPCR after annealing of the aptamer components. FIG. 42D shows successful coupling of the poly-A containing aptamer component, and little binding of the poly-T containing aptamer was observed.

Preparation of PEGylated FluoSpheres with varying sizes of PEG groups ^TM . FIG. 42E shows FluoSphere PEGylated with mPEG-12, mPEG-24, mPEG-36, mPEG-45 or mPEG-112 ^TM Measured zeta potential. It can be seen that the magnitude of the zeta potential decreases as the size of the PEG group increases. FIG. 42F shows PEGylated FluoSpheres as a function of PEG size ^TM Polydispersity index (PDI). PDI was measured by dynamic light scattering. An increase in PDI was observed over the PEG size of mPEG-36, indicating a decrease in colloidal stability.

EXAMPLE 11 FluoSphere based ^TM Binding characterization of affinity reagents of (c)

The test is based on FluoSphere ^TM To assess their target binding characteristics. FluoSphere based was prepared by the method described in example 8 ^TM Is contained in the composition. The aptamer was pre-annealed with the attachment oligonucleotide at 95℃for 5 min and then coupled to FluoSpheres ^TM 。FluoSpheres ^TM The following aptamers were used for the preparation: P7-B1-P5 (target histidine tag), P7-H3T-P5 (target histidine tag), and P7-VEGF aptamer 89 (target VEGF).

Peptide targets for binding studies were prepared by fixing the biotinylated trimeric amino acid sequence to a streptavidin coated plate. FIG. 43A shows various aptamer formulations (mid-target: P7-B1-P5-FS is FluoSphere) ^TM Probe, streptavidin-Alexa-for P7-B1-P5-strep647; and (3) off-target: her2-FS is FluoSphere ^TM Probe, her2-strep with streptavidin Alexa->647) against binding of various peptide trimer targets. FluoSphere containing P7-B1-P5 was observed ^TM The probe has a similar binding profile to P7-B1-P5-Strep, while having a different binding profile to Her 2-specific affinity reagents.

The measurement is based on FluoSphere ^TM Dissociation constants for the various targets. The targets were prepared by coupling biotinylated targets (proteins or peptides) to streptavidin plates. FIG. 43B shows FluoSphere containing the P7-B1-P5 aptamer ^TM Binding measurement of affinity reagent against histidine-tagged Her2 protein target. EC50 of 6.5nM was measured, indicating FluoSphere based ^TM The P7-B1-P5 affinity reagent of (C) was able to successfully bind to the histidine tag of the Her2-His conjugate. FIG. 43C shows the measurement of binding of the P7-H3T-P5 aptamer to HH trimeric peptide. EC50 of 3.4nM was measured, indicating FluoSphere based ^TM Is capable of binding to a trimeric target. FIG. 43D shows FluoSphere for P7-VEGF-aptamer 89-based ^TM The binding of the affinity reagent to the VEGF target (mid-target) and myoglobin (off-target) measurements was a function of probe concentration. For binding to VEGF, the measured fluorescence intensity was much higher, as a function of affinity reagent concentration, indicating FluoSphere-based ^TM Has binding affinity for its intended target.

Example 12 FluoSphere based ^TM Stability characterization of affinity reagents of (c)

FluoSphere based test preparation ^TM To determine the binding activity of the affinity reagent after prolonged storage in PBS buffer at 4 ℃. A detectable affinity reagent was prepared by the method described in example 8. With P7-B1-P5 aptamer (target histidine tag)Preparing a detectable affinity reagent. Also for the fluorescent labelled streptavidin-Alexa available commerciallyStability measurements were performed on individual P7-B1-P5 aptamers coupled to 647 conjugates (Thermo Fisher) and streptavidin-APC (Thermo Fisher). Figures 44A-44C show affinity measurements for each detectable affinity reagent at the indicated time points. At each time interval, the affinity of the detectable affinity reagent was evaluated as described in example 11. Based on FluoSphere compared to streptavidin conjugates ^TM Shows a stronger affinity for the target for a longer period of time. Surprisingly, in comparison with commercial fluorescent alternatives, based on P7-B1-P5 FluoSphere ^TM Has significantly improved stability.

EXAMPLE 13 FluoSphere based ^TM Binding characterization of affinity reagents of (c)

FluoSphere based measurement of P7-B1-P5 coupling compared to the binding affinity of the single aptamer P7-B1-P5-fluorophore conjugate ^TM Binding affinity of the affinity reagent of (c). Preparation of P7-B1-P5 coupled FluoSphere based according to the procedure of example 8 ^TM Is a detectable affinity reagent of (a). Also prepared are the following fluorogenic single aptamer P7-B1-P5 conjugates: streptavidin-Alexa647 (Thermo Fisher), streptavidin-APC (Thermo Fisher) and SureLight ^TM APC (Columbia Biosciences). Binding curves were measured for histidine (HHH) peptides using various concentrations of probes or aptamers. The dissociation constant (EC 50) is derived from the binding measurement data.

FIGS. 45A-45D show FluoSphere based ^TM Affinity reagent of AlexaAptamer, APC aptamer, and surehight ^TM Dissociation constant map of APC aptamer. Based on FluoSphere ^TM Has been measured to have an affinity reagent of about 2.2nEC50, alexa->And APC aptamers have EC50 measurements of 28.0nM and 29.9nM, respectively. SureLight ^TM The EC50 measured for APC aptamer was 2.3nM. Based on FluoSphere ^TM It appears that the affinity reagent of (c) binds to histidine peptide targets quite or better, depending on the exact detectable probe system utilized.

Example 14 binding characterization of Quantum dot-based affinity Agents

A detectable affinity reagent containing a quantum dot fluorophore as a retention component was prepared according to the method of example 9. The quantum dot-based affinity reagent was conjugated to the P7-B1-P5 aptamer (target histidine tag). Binding measurements were performed to determine the binding characteristics of quantum dot-based affinity reagents.

FIGS. 24A and 24B show binding measurement data for P7-B1-P5-quantum dot based affinity reagents. FIG. 24A shows fluorescence measurements of quantum dot based P7-B1-P5 affinity reagents in contact with his-tagged Her2 protein (mid-target binding) and myoglobin (off-target control). For Her2-his targets, an increase in fluorescence signal was observed with increasing concentration of affinity reagent. When the affinity reagent was contacted with the myoglobin control, little change in fluorescence signal was observed, indicating that the quantum dot-based P7-B1-P5 aptamers were able to distinguish and bind their intended targets. FIG. 24B shows EC50 measurement data for quantum dot-based P7-B1-P5 affinity reagents against HHH peptides. EC50 was measured to be 60nM, indicating that quantum dot-based affinity reagents bind to their intended targets.

EXAMPLE 15 FluoSphere based ^TM Synthesis of affinity reagents for (a)

Preparation of FluoSphere-based polymers having different ratios of functional groups to nonfunctional groups ^TM Is used as an affinity reagent. The test is based on FluoSphere ^TM To evaluate the optimum amount of functionalization of the functionalization groups on the surface of the affinity reagent. FluoSphere based preparation of mPEG-amine to amine-PEG-azide ratios of 95:5, 99.5:0.5, 99.95:0.05 and 99.995:0.005 ^TM Is used as an affinity reagent. After the manufacture, the product is prepared,affinity reagents were attached to B1 aptamers as described in example 8 and evaluated in plate-based binding studies. The 95:5 ratio shows a signal that is about 1.5 times higher than the 99.5:0.5 ratio (see fig. 46). When fully made, an increase in the amount of available azide groups (attachment groups for attaching the aptamer annealed oligonucleotides) increases the amount of mid-target binding of the affinity reagent.

EXAMPLE 16 affinity reagent and FluoSphere-based ^TM Direct conjugation of affinity reagents of (a)

Preparation of FluoSphere-based via direct conjugation of binding component to functionalized nanoparticles ^TM Is used as an affinity reagent. Combining a B1 aptamer comprising a 5' -DBCO functional group with a functionalized FluoSpheres comprising surface-displayed mPEG-azide groups ^TM And (3) contact. A detectable affinity reagent is formed by the reaction of the azide moiety with the DBCO moiety. FluoSphere-based targets to be prepared by direct conjugation strategy against Her2-his tag targets ^TM Affinity reagents and FluoSphere-based prepared via oligonucleotide annealing ^TM Is compared with the binding of the affinity reagent of (see example 8). FIG. 47 shows the mid-target Her2-his binding data for directly conjugated and annealed affinity reagents and the binding of both affinity reagent types to myoglobin (off-target) controls. The binding observed for the directly conjugated affinity agent was observed to be lower than that of the annealed affinity agent, but significantly exceeded that seen for the negative control.

Example 17 enzymatic incorporation of fluorescent dUTP nucleotides

AlexaFluor-647dUTP nucleotides were enzymatically incorporated into the 3' end region of the aptamer as depicted in FIG. 49B. Fluorophores incorporated into the aptamer were visualized at 647nm, as shown in fig. 52, indicating successful fluorophore incorporation.

EXAMPLE 18 AlexaFluor-647 NHS ester conjugated to Aminoallyl dUTP

AAdUTP nucleotides were incorporated into the aptamer using the method described in fig. 49B. The resulting labeled aptamer was chemically conjugated to a fluorophore, run on a gel, treated with SYBR, and visualized at 488nm to reveal double stranded DNA (fig. 53A), and at 647nm to reveal the presence of the fluorophore (fig. 53B).

EXAMPLE 19 measurement of concentration of dsDNA-647 labeled aptamer

The concentration of dsDNA-647 labeled aptamer was measured using absorbance at 260nm and the results are shown in FIG. 54. To examine the conjugation efficiency of AlexaFluor-647 with the aptamer, the fluorophore concentration was determined using a fluorophore standard curve (fig. 55A) (fig. 55B). Fluorophore concentration measurements were used to determine the fluorophore to DNA ratio of the estimated degree of labeling, as shown in FIG. 56.

Example 20 target binding and imaging in dsDNA-647

To assess target binding and imaging of the labeled aptamer, experiments were performed using dsDNA-647 labeled aptamer specific for epitope HHH. HHH peptides were immobilized on the microplate surface and exposed to labeled aptamers, as shown in fig. 57A. FIG. 57B shows the binding of the labeled aptamer to the HHH peptide and FIG. 57C shows the lack of binding to the negative control peptide MetGluThr.

To evaluate imaging, the labeled aptamers were visualized at various concentrations on a custom 20x objective epifluorescence microscope, as shown in fig. 57D.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The invention is not intended to be limited to the specific embodiments provided within the specification. While the invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention will also cover any such alternatives, modifications, variations or equivalents. The following claims are intended to define the scope of the invention and their methods and structures within the scope of these claims and their equivalents are thereby covered.

Claims (46)

1. A detectable probe comprising:

(a) Retaining the components;

(b) A labeling component; and

(c) Two or more binding components attached to the retention component,

wherein the equilibrium dissociation constant of the detectable probe for the binding partner is less than the equilibrium dissociation constant of any of the two or more binding components for the binding partner, or wherein the dissociation rate constant of the detectable probe for the binding partner is less than the dissociation rate constant of any of the two or more binding components for the binding partner.

2. The detectable probe of claim 1, wherein the labeling component is coupled to the retention component.

3. The detectable probe of claim 1, wherein the retention component comprises a nucleic acid.

4. The detectable probe of claim 3, wherein the nucleic acid comprises a nucleic acid fold.

5. The detectable probe of claim 1, wherein the retention component comprises a fluorescent nanoparticle.

6. The detectable probe of claim 1 comprising two or more labeling components coupled to the retention component.

7. The detectable probe of claim 1, further comprising the binding partner, wherein the binding partner binds to the detectable probe via a binding component of the two or more binding components.

8. The detectable probe of claim 7, wherein the binding partner is attached to a solid support.

9. The detectable probe of claim 8, wherein the binding partner is attached to the solid support via a structured nucleic acid particle.

10. The detectable probe of claim 1, wherein the retention component restricts a first binding component of the two or more binding components from contacting a second binding component of the two or more binding components.

11. The detectable probe of claim 1, wherein the retention component comprises a three-dimensional structure having a first side offset from a second side, and wherein the two or more binding components are confined to the first side and are confined to not contact the second side.

12. The detectable probe of claim 11, wherein the labeling component is confined to the second side and is confined to not contact the first side.

13. The detectable probe of claim 1, wherein the detectable probe comprises two or more label components.

14. The detectable probe of claim 13, wherein the retention component comprises a three-dimensional structure having a first side offset from a second side, and wherein the two or more labeling components are confined to the first side and are confined to not contact the second side.

15. The detectable probe of claim 14, wherein the two or more binding components are confined to the second side and are confined to not contact the first side.

16. The detectable probe of claim 1, wherein the retention component comprises a bead.

17. The detectable probe of claim 16, wherein the labeling component comprises one or more fluorescent moieties within the bead.

18. A method of detecting an analyte, comprising:

(a) Contacting an analyte with the detectable probe of any of the preceding claims, wherein the analyte comprises the binding partner; and

(b) Signals from the labeled component are acquired to detect the analyte.

19. A method of detecting an analyte comprising

(a) Contacting an analyte with a first detectable probe comprising:

(i) A first one of the components of the retention composition,

(ii) A labeling component, and

(iii) A first set of two or more binding components attached to the retention component, wherein at least one of the binding components in the first set binds to a first epitope in the analyte;

(b) Obtaining a signal from the labeled component of the first detectable probe;

(c) Contacting the analyte with a second detectable probe comprising:

(i) A second retention component comprising substantially the same structure as the first retention component,

(ii) A labeling component, and

(iii) A second set of two or more binding components attached to the retention component, wherein at least one of the binding components in the second set binds to a second epitope in the analyte, the second epitope having a different chemical composition than the first epitope; and

(d) Obtaining a signal from the labeled component of the second detectable probe to detect the analyte.

20. The method of claim 19, wherein the first retention component and the second retention component comprise structured nucleic acid particles having substantially the same structure.

21. The method of claim 20, wherein the substantially identical structure is a nucleic acid paper folding scaffold.

22. The method of claim 19, wherein the labeling component of the first detectable probe comprises a structure that is substantially identical to the structure of the labeling component of the second detectable probe.

23. The method of claim 19, wherein the labeled component of the first detectable probe and the labeled component of the second detectable probe produce the same signal.

24. The method of claim 19, wherein the labeling component of the first detectable probe comprises a structure that is different from the structure of the labeling component of the second detectable probe.

25. The method of claim 19, wherein the labeling component of the first detectable probe and the labeling component of the second detectable probe produce different signals.

26. The method of claim 19, further comprising removing the first detectable probe from the analyte prior to step (c).

27. The method of claim 27, wherein the moving the first detectable probe away from the analyte comprises cleaving the first detectable probe.

28. A composition comprising:

(a) A probe comprising a first structured nucleic acid particle attached to a binding component; and

(b) An analyte comprising a second structured nucleic acid particle attached to an epitope of the binding component, wherein the probe is attached to the analyte via binding of the binding component to the epitope.

29. The composition of claim 28, wherein the probe further comprises a labeling component attached to the first structured nucleic acid particle.

30. The composition of claim 28, wherein the second structured nucleic acid particles are further attached to a solid support.

31. The composition of claim 28, wherein the second structured nucleic acid particles are further attached to a site in an array.

32. The composition of claim 28, wherein the first structured nucleic acid particle comprises a nucleic acid fold.

33. The composition of claim 32, wherein the second structured nucleic acid particle comprises a nucleic acid fold.

34. A composition comprising:

(a) A plurality of different probes, each of the different probes comprising a first structured nucleic acid particle attached to a binding member, each of the different probes comprising a different binding member; and

(b) A plurality of different analytes, each of the different analytes comprising a second structured nucleic acid particle attached to an epitope of a different binding component, wherein the different probes are attached to the different analytes via binding of the different binding components of the plurality of different probes to the epitopes of the plurality of different analytes.

35. The composition of claim 34, wherein the first structured nucleic acid particles are substantially identical for the different probes.

36. The composition of claim 34, wherein the second structured nucleic acid particles are substantially identical for the different analytes.

37. The composition of claim 34, wherein the first structured nucleic acid particle is substantially identical to the second structured nucleic acid particle.

38. The composition of claim 34, wherein the first structured nucleic acid particles are different for the different probes.

39. The composition of claim 38, wherein the first structured nucleic acid particle comprises a folded paper having a scaffold strand annealed to a plurality of oligonucleotides.

40. The composition of claim 38, wherein the scaffold strand is the same for the different probes and at least one of the oligonucleotides is different between the different probes.

41. The composition of claim 34, wherein the second structured nucleic acid particles are different for the different analytes.

42. The composition of claim 41, wherein the second structured nucleic acid particles comprise a paper break having a scaffold strand annealed to a plurality of oligonucleotides.

43. The composition of claim 42, wherein the scaffold strands are the same for the different analytes and at least one of the oligonucleotides is different between the different analytes.

44. The composition of claim 34, wherein the first structured nucleic acid particle is different from the second structured nucleic acid particle.

45. The composition of claim 44, wherein each of the first and second structured nucleic acid particles comprises a paper break having a scaffold strand annealed to a plurality of oligonucleotides.

46. The composition of claim 45, wherein the scaffold strand is the same for the first and second structured nucleic acid particles and at least one of the oligonucleotides is different between the first and second structured nucleic acid particles.