Nucleic acid translator gates for protein measurement

Abstract

This disclosure relates to systems and methods for measurement of proteins in biological samples.

Description

NUCLEIC ACID TRANSLATOR GATES FOR PROTEIN MEASUREMENTCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of US Provisional Application No. 63 / 643,620, filed May 7, 2024, which is incorporated by reference herein in its entirety for any purpose.FIELD

[0002] This disclosure relates to systems and methods for measurement of proteins in biological samples.BACKGROUND

[0003] Conventionally, various attempts to evaluate genetic activity and / or decode biological processes, including disease processes or biological processes of pharmacological effect, have been focused on genomics. However, proteomics can provide further information about the biological function of cells and organisms. Proteomics includes qualitative and quantitative measurement of gene activity by detecting and quantifying the expression on a protein level rather than the genetic level. Proteomics also includes a study of events which are not coded genetically, such as a post-translational modification of proteins and interactions between proteins.

[0004] At present, it is possible to obtain an enormous volume of genome information. DNA chips have come into practical use as molecular arrays for this purpose and the price of direct DNA sequencing has continued to drop significantly. Likewise, there is an increasing demand for high throughput proteomics. Proteomics is far preferable to genomics for the monitoring of health, as the genome is static, indicating only medical potential, while the proteome varies dynamically with a patient’s medical state, and may even be said to define their medical state. However, detecting and quantitating proteins can be difficult, while detecting and quantitating nucleic acids is relatively easy, at least in part because proteins are more complicated and more variable in biological functions than DNA. This has motivated many efforts to measure mRNA (messenger RNA) concentrations as a proxy for protein concentrations. However, mRNA concentrations have been shown not to correlate well with protein concentrations. There remains a need for improved methods of detecting proteins.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Fig. 1 is a schematic diagram depicting an illustrative translator gate in accordance with aspects of the present teachings.

[0006] Fig. 2 is a schematic diagram depicting the translator gate of Fig. 1 after having captured an aptamer.

[0007] Fig. 3 is a schematic diagram depicting an illustrative reporter strand of a translator gate in accordance with aspects of the present teachings.

[0008] Fig. 4 is a schematic diagram depicting an illustrative reporter-free gate and an illustrative translator gate having a reporter strand at a 3’ end of an aptamer-capture strand, in accordance with aspects of the present teachings.

[0009] Fig. 5 is a schematic diagram of an illustrative translator gate having a reporter strand at a 5’ end of an aptamer-capture strand, in accordance with aspects of the present teachings.

[0010] Fig. 6 is a schematic diagram depicting an illustrative readable translator gate and an illustrative unreadable translator gate having reporter strands bound to 3’ ends of aptamer-capture strands in accordance with aspects of the present teachings.

[0011] Fig. 7 is a schematic diagram depicting an illustrative readable translator gate and an illustrative unreadable translator gate having reporter strands bound to 5’ ends of aptamer-capture strands in accordance with aspects of the present teachings.

[0012] Fig. 8 is a schematic diagram depicting an illustrative trimolecular complex in accordance with aspects of the present teachings.

[0013] Fig. 9 is a flowchart depicting steps of an illustrative method for obtaining quantitative information about proteins in a sample, in accordance with aspects of the present teachings.

[0014] Fig. 10 shows the results of detecting two different aptamers with exemplary translator gates.DETAILED DESCRIPTION

[0015] Various aspects and examples of systems and methods for protein measurement involving nucleic acid translator gates are described below and illustrated in the associated drawings. Unless otherwise specified, a system for protein measurement in accordance with the present teachings, and / or its various components, may contain at least one of the structures, components, functionalities, and / or variations described, illustrated, and / or incorporated herein. Furthermore, unless specifically excluded, the process steps, structures, components, functionalities, and / or variations described, illustrated, and / or incorporated herein in connection with the present teachings may be included in other similar devices and methods, including being interchangeable between disclosed embodiments. The following description ofvarious examples is merely illustrative in nature and is in no way intended to limit the disclosure, its application, or uses. Additionally, the advantages provided by the examples and embodiments described below are illustrative in nature and not all examples and embodiments provide the same advantages or the same degree of advantages.

[0016] The following definitions are used herein unless otherwise indicated.

[0017] “Comprising,” “including,” and “having” (and conjugations thereof) are used interchangeably to mean including but not necessarily limited to, and are open-ended terms not intended to exclude additional, unrecited elements or method steps.

[0018] Terms such as “first”, “second”, and “third” are used to distinguish or identify various members of a group, or the like, and are not intended to show serial or numerical limitation.

[0019] In this disclosure, one or more publications, patents, and / or patent applications may be incorporated by reference. However, such material is only incorporated to the extent that no conflict exists between the incorporated material and the statements and drawings set forth herein. In the event of any such conflict, including any conflict in terminology, the present disclosure is controlling.Overview

[0020] One way to detect and quantify the presence of specific proteins in a biological sample is through the use of protein-binding nucleic acid aptamers, such as SOMAmer® (Slow Off-rate Modified Aptamer) reagents. In some embodiments, an aptamer may comprise one or more chemically modified nucleotides that improve the stability and / or binding affinity of the aptamer for the protein target. In some embodiments, an assay using aptamers may measure native proteins in complex matrices by transforming each individual protein concentration into a corresponding aptamer concentration, which can be quantified by, e.g., standard DNA techniques such as microarrays or PCR.

[0021] Aptamers are typically single stranded DNA-based protein affinity reagents that may include chemically modified nucleotides. In some embodiments, an aptamer comprises one or more chemically modified nucleotides that mimic amino acid side chains, expanding the chemical diversity of standard DNA aptamers and enhancing the specificity and affinity of protein-nucleic acid interactions. These modified nucleotides are incorporated into nucleic acid libraries used for iterative selection and amplification of aptamers to a target, using for example, a process called SELEX (SystematicEvolution of Ligands by Exponential enrichment). In some embodiments, the inclusion of certain chemically modified nucleotides allows for the generation of aptamers capable of binding proteins that had been resistant to selection with nucleic acid slacking similar chemical modifications. Aptamers can be selected according to the desirable properties of specificity and slow off-rate, under the assay conditions in which the reagents will be used.

[0022] In aptamer-based assays, the presence of proteins in a sample is transformed into a specific aptamer-based DNA reporter. An aptamer-protein binding step is followed by a series of partitioning and wash steps that converts relative protein concentrations into measurable nucleic acid reporters that are quantified using DNA detection technology, such as by hybridization of fluorophore-labeled aptamers to custom DNA microarrays. Upon laser scanning the microarray, the readout in relative fluorescent units (RFU) is directly proportional to the amount of target protein in the initial sample.

[0023] In some embodiments, the present disclosure describes illustrative systems and methods that involve capturing target molecules (e.g., proteins) from a sample using aptamers, isolating the aptamers that captured one of the target molecules in an eluate, using nucleic acid translator gates to capture the aptamers of the eluate and release reporter strands (also called reporter molecules), and detecting or identifying the reporter strands using next-generation sequencing techniques and / or any other suitable techniques. Data obtained by detecting the reporter strands can be analyzed and / or otherwise processed to determine information about the target molecules in the original sample. In some embodiments, systems and methods according to aspects of the present teachings are configured to alleviate potential drawbacks associated with the very large dynamic range of types of target molecules that may be present in a sample.

[0024] In some embodiments, systems and methods according to aspects of the present teachings do not include use of microarrays. In other embodiments, systems and methods disclosed herein include use of microarrays. For example, the translator gate output is in some examples quantified on microarray surfaces.Examples, Components, and Alternatives

[0025] The following sections describe selected aspects of illustrative systems and methods involving translator gates. The examples in these sections are intended for illustration and should not be interpreted as limiting the scope of the present disclosure.Each section may include one or more distinct embodiments or examples, and / or contextual or related information, function, and / or structure.A. Illustrative Translator Gate

[0026] This section describes an illustrative translator gate 102 configured to “translate” or “convert” an aptamer into a reporter strand by capturing an aptamer (e.g., from an aptamer-containing eluate) and, based on capturing the aptamer, releasing a reporter molecule.

[0027] Fig. 1 schematically depicts an illustrative translator gate 102 in accordance with aspects of the present teachings. Unless otherwise specified, the drawings of the present disclosure are schematic and not necessarily to scale with respect to, e.g., strand length or bead size. Gate 102 comprises a capture strand 104 and a reporter strand 106. In the depicted example, capture strand 104 is attached to a magnetic bead 108; as described below, the bead is optional and may be omitted. In some embodiments, capture strand 104 is attached to another type of solid support, such as a location on an array.

[0028] Capture strand 104 is an oligonucleotide complementary to a particular type of aptamer. Strand 104 may comprise ribonucleic acid (RNA), deoxyribonucleic acid (DNA), locked nucleic acid (LNA), peptic nucleic acid (PNA), any suitable nucleic acid analog, and / or any suitable combination of the foregoing.

[0029] A first portion 110 of capture strand 104 is bound to, and is a complement of, a first portion 112 of reporter strand 106. Another portion 114 of capture strand 104, referred to as a toehold and / or toehold domain, is not directly bound to reporter strand 106 and is available as a binding site for an aptamer 116, of which strand 104 is a complement. Accordingly, if gate 102 is suitably exposed to aptamer 116, the aptamer binds to toehold portion 114. The bound aptamer 116 displaces reporter strand 106, for example, by branch migration, such that the bound aptamer is bound to toehold portion 114 and also to at least some of first portion 110, releasing reporter strand 106 from the capture strand. In some examples, toehold domain 114 comprises the entirety of the portion of capture strand 104 that is not directly bound to reporter strand 106. In other examples, capture strand 104 includes at least one portion that is not part of toehold domain 114 and is also not bound to reporter strand 106; such a portion may, e.g., act as a spacer that spaces bead 108 from other parts of the capture strand 104.

[0030] Freed reporter strand 106 can be detected and / or identified by nextgeneration sequencing technique(s) and / or any other suitable method(s), indicating the presence and capture of aptamer 116 by the capture strand 104. Reporter strand 106may have any suitable structure for being bound to a portion of the capture strand, displaced by the captured aptamer, and read out by a suitable process; Section B describes an example.

[0031] Fig. 1 depicts gate 102 prior to capturing aptamer 116 from an eluate; at this point, reporter strand 106 is bound to first portion 110 of capture strand 104 and toehold 114 is available for binding. Fig. 2 depicts the situation after capture strand 104 has captured aptamer 116 and the aptamer has fully displaced reporter strand 106, such that the reporter strand is unbound from capture strand 104; accordingly, the gate has translated an unbound aptamer into an unbound reporter strand.

[0032] In the depicted example, capture strand 104 is a full complement to aptamer 116 (i.e. , an entire complement of the aptamer is contained in the capture strand).Accordingly, the interaction domain between the aptamer and the capture strand may be a relatively large number of nucleotides, such as 20-100, 20-90, 20-80, 20-70, 20- 60, 20-50, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides, which allows the complex comprising the bound-together aptamer and complement to have a high thermal stability and long bond lifetime. In other examples, however, the capture strand may contain less than an entire complement of the aptamer, but in some embodiments, comprises at least 20, at least 25, or at least 30 nucleotides complementary to the aptamer.

[0033] Toehold domain 114 may have any length suitable for facilitating capture of the aptamer and displacement of the reporter strand by the captured aptamer. In general, the bond lifetime of the aptamer-toehold complex should be long enough to permit initiation of the branch migration process. The bond lifetime is a direct function of the number of base pairs that engage in the complex, so the toehold length is generally selected to be long enough to allow for branch migration to begin. A longer toehold length generally improves bond lifetime at least up to a threshold length that is sufficient to engage branch migration.

[0034] In some examples, toehold domains in the length range of 5-10 nucleotides have been shown to facilitate high-fidelity strand displacement. In at least some circumstances, longer toeholds perform better kinetically than shorter toeholds.Accordingly, in some examples of the present teachings, toehold domain 114 has a length in the range of 5-12, or 5-15, or 5-20 nucleotides (inclusive), which may be kinetically better able to facilitate high-fidelity strand displacement compared to shorter lengths. In some examples, a longer length toehold may improve initial binding of the aptamer to the capture strand in these examples.

[0035] In the depicted example, capture strand 104 is immobilized to a magnetic bead 108. As shown in Fig. 2, strand 104 remains immobilized to bead 108 after capturing aptamer 116, such that after the aptamer has displaced reporter strand 106 by branch migration, the complex comprising the bound aptamer and complement remains attached to the bead. This facilitates separation of the complexes from the unbound reporter strands so that the reporter strands can be used for next-generation sequencing and / or another suitable identification process.

[0036] Alternatively, or additionally, capture strand 104 may initially not be attached to any bead or other solid support. In some examples, a plurality of gates 102 (comprising capture strand 104 and reporter strand 106 and no magnetic bead) are prepared in a suitable solution. The solution is incubated with the aptamer-containing eluate that is output by an assay. Similar to the bead-based implementation described above, aptamers of the eluate are captured by toehold domains of the gates and displace the associated reporter strands, which become unbound. In some such embodiments, the capture strand 104 and / or the aptamer may comprise biotin. The solution, now containing the unbound reporter strands, is incubated with streptavidin beads or other solid support comprising streptavidin. As a result, the complexes comprising the bound capture strands and aptamers are attached to beads, facilitating separation of the complexes from the unbound reporter strands.

[0037] Irrespective of whether gate 102 is initially attached to a bead or other solid support, any suitable method(s) may be used to facilitate and / or enhance hybridization between capture strand 104 and the cognate aptamer in the aptamer-containing eluate. In some examples, a suitable substance is added to the solution phase to increase the rate of hybridization. Nonlimiting exemplary such substances include cationic-comb type copolymers, the presence of which can accelerate hybridization. Additionally, or alternatively, crowding reagent(s) and / or variation in salt concentration may be used to accelerate hybridization.

[0038] In general, the reporter strand of a translator gate may be bound to the capture strand of the gate at the 3’ end of the capture strand or at the 5’ end of the capture strand, such that the toehold domain is at the 5’ end or 3’ end of the capture strand respectively. In a process in which a plurality of translator gates are used to capture aptamers, all the translator gates may have reporter strands bound to 3’ ends of their capture strands, all the translator gates may have reporter strands bound to 5’ ends of their capture strands, or the plurality of translator gates may include some translator gates having reporter strands bound to 3’ ends of their capture strands andsome translator gates having reporter strands bound to 5’ ends of their capture strands. The concentrations of each of these types of gates, and / or the ratio between them, may be selected based on properties of the aptamers to be captured and / or on any other suitable factors. For example, some aptamers have 3’ ends that are not readily accessible (e.g., based on a secondary structure of the aptamer) and these aptamers therefore do not easily bind to capture strands having toeholds at their 5’ ends.Similarly, some aptamers have 5’ ends that are not readily accessible and therefore do not bind easily to capture strands having toeholds at their 3’ ends. Accordingly, using a mixture of gates having 3’-bound reporter strands and gates having 5’-bound reporter strands may be beneficial in cases where certain aptamers expected to be present cannot easily be bound at a particular end.B. Illustrative Reporter Strand

[0039] This section describes an illustrative reporter strand architecture, which is a non-limiting example of an architecture suitable for reporter strand 106, described above.

[0040] Fig. 3 schematically depicts an illustrative translator gate 122 in accordance with aspects of the present teachings. Gate 122 is similar in respects to gate 102, described above. Accordingly, gate 122 comprises a capture strand 124 and a reporter strand 126, and in this example capture strand 124 is attached to a magnetic bead 128. A first portion 130 of capture strand 124 is bound to a first portion 132 of reporter strand 126. A toehold domain 134 of capture strand 124 is not bound to reporter strand 126 and is available as a binding site for an aptamer of which strand 124 is a complement.

[0041] First portion 132 of reporter strand 126 is also referred to as an aptamer- complement-complementary region, or ACR region, because it is complementary to first portion 130 of capture strand 124. ACR region 132 may have any suitable length. In some examples, the length of ACR region 132 is determined primarily or entirely by the choice of length of toehold 134. In some examples, ACR region 132 has a length in the range of 10-50, 20-50, 10-40, 20-40, 10-35, 10-30, 10-25, or 10-20 nucleotides and toehold region 134 has a length in the range of 5-12, or 5-15, or 5-20nucleotides.

[0042] ACR region 132 is located at one end of reporter strand 126. Adjacent ACR region 132 is a forward primer 135, adjacent forward primer 135 is a barcode domain 136, adjacent barcode domain 136 is a unique molecular identifier (UMI) region 138, and adjacent UMI region 138 is a reverse primer 140. Forward primer 135 and reverse primer 140 may each have any suitable lengths for facilitating reading barcode domain 136. For example, primers 135, 140 may each have a length in the range 6-20, 6-15, or6-12 nucleotides, which may be suitable for typical commercial next-generation sequencing platforms.

[0043] Barcode domain 136 may comprise any domain suitable for facilitating identification (e.g., when sequenced). Barcode domain 136 may have any suitable length. In general, a barcode domain length of n nucleotides allows a library of 4nunique barcodes. In some examples, barcode domain 136 has a length of 15 nucleotides, allowing a library of 415= 1 ,073,741 ,824 barcodes.

[0044] UMI region 138 comprises a unique or practically unique domain that facilitates identification and correction of errors associated with sequencing and / or other processing of reporter strand 126. UMI region 138 may have any suitable length. In some examples, UMI region 138 is five nucleotides long, which is a length of some commonly available commercial products. However, other lengths may be used where appropriate; for example, lengths longer than five nucleotides do not compromise performance in at least some examples.C. Illustrative Systems and Methods for Range Compression

[0045] This section describes illustrative systems and methods configured for efficient processing of an eluate in which the respective abundances of different aptamers vary over a large range. For example, in some cases target proteins in a sample could have concentrations in the range of fM-pM (i.e., spanning around nine orders of magnitude), resulting in eluate aptamer concentrations that span five or more orders of magnitude. Performing an assay on such an eluate may lead to overcounting of more abundant aptamers and corresponding undercounting of less abundant aptamers. Accordingly, it may be desirable to level, or compress, the range of abundances of aptamers before sequencing and counting. However, it is possible and is within the scope of the present teachings to perform NGS or another suitable process on an aptamer-containing eluate without utilizing any range-compression techniques. a. Illustrative Missing Reporter Strand Schema

[0046] This subsection describes an illustrative schema in accordance with aspects of the present teachings, in which range compression is achieved by replacing a desired fraction of translator gates with aptamer-capture strands to which no reporter strands are bound. Accordingly, if an aptamer-containing eluate would normally be incubated with (and / or otherwise exposed to) N translator gates, in this schema range compression is achieved by incubating the eluate with (1 - a)N translator gates and aN aptamer-capture strands having no reporter strands (or put another way, with(1 - a)N translator gates and aN translator gates that are missing their reporter strands).

[0047] As an example, to reduce the number of expected reads of a given aptamer type by 50%, half the translator gates having capture strands to that aptamer could be replaced by translator gates having no reporter strand. When a translator gate having no reporter strand (i.e., a -capture strand to which no reporter is bound) captures an aptamer from the eluate, there is no reporter strand to be released and read in a subsequent process. Accordingly, on average the number of reporter strands released per aptamer of that type is reduced by 50%, reducing by 50% the number of counts of that aptamer type that will ultimately be performed. As another example, a ratio of translator gates lacking a reporter strand to translator gates having a reporter strand could be 10:1 for a given aptamer, such that only ten percent of aptamers of that type in the aptamer-containing eluate yield a released reporter strand. This would lower the number of counts of that aptamer type in the NGS output by an order of magnitude compared to an assay in which no translator gates for that aptamer lack a reporter strand.

[0048] This schema is illustrated schematically in Fig. 4, which depicts a translator gate 152 and a reporter-free capture strand 162. Translator gate 152 is similar to gates 102 and 122, described above. Accordingly, gate 152 comprises a capture strand 154 and a reporter strand 156, with the capture strand 154 including a toehold domain 158. In contrast, reporter-free capture strand 162 comprises a capture strand 164 (which is a complement to the same aptamer to which strand 154 is a complement) and has no reporter strand. Accordingly, strands 154 and 164 are both capable of binding to the same aptamer, but a reporter strand is released only if capture strand 154 is captures the aptamer. Because half of capture strands depicted in Fig. 4 are incapable of releasing a reporter strand, the schema of Fig. 4 reduces by half the translated signal for that aptamer. While Fig. 4 depicts only one translator gate and one reporter-free strand, in general carrying out the illustrated schema includes exposing the aptamercontaining eluate to a large number, such as hundreds or thousands, of gates and / or reporter-free strands.

[0049] In this example, translator gate 152 is attached to a magnetic bead 166, and reporter-free capture strand 162 is attached to a magnetic bead 168. In various embodiments, the translator gate 152 and reporter-free capture strand 162 may be attached to the same bead or the same location on a solid support. Accordingly, implementing the schema illustrated in Fig. 4 may include preparing a magnetic beadsor other solid support with the desired number of capture strands and hybridizing reporter strands to only half of those capture strands. Ratios other than 50% may be utilized, for example, ratios of capture strand to report strand of between about 2:1 to about 10,000,000:1 , or between about 10:1 to about 1 ,000,000:1 , or between about 10:1 to about 100,000:1 , or between about 100:1 to about 1 ,000,000:1 , or between about 100:1 to about 100,000:1 , as described herein. As described above with reference to Figs. 1-2, in some examples the gates are not immobilized to a solid support, and in such embodiments, a solution comprises an excess of capture strands versus reporter strands.

[0050] In the example depicted in Fig. 4, capture strand 154 of translator gate 152 has a 5’ end 170 and a 3’ end 172, with reporter strand 156 being bound to strand 154 at 3’ end 172. Accordingly, toehold domain 158 of strand 154 is configured to bind to a 3’ end of a aptamer (not pictured). In some use cases, however, certain aptamers expected to be present in the eluate have a 3’ end that is not readily accessible (based, e.g., on a secondary structure of the aptamer). Unmodified gate 152 may have a high likelihood of failing to capture a aptamer with an obscured 3’ end. To alleviate this problem, unmodified translator gates that are bound to reporter strands at the 5’ end (and thus have a toehold domain at the 3’ end rather than the 5’ end) may be included along with translator gate 152.

[0051] Fig. 5 depicts an example translator gate 174 having a capture strand 176 having a 3’ end 178, which in this example is immobilized to a magnetic bead 180. In other examples, the magnetic bead may be omitted, or the translator gate may be bound to another solid support, such as a location in an array. A reporter strand 182 is bound to strand 176 adjacent a 5’ end 184 of strand 176. A toehold domain 186 of strand 176 is at 3’ end 178 of strand 176 and thus configured to bind to a 5’ end of a aptamer that is complementary to strand 176. Including a plurality of translator gates 174 along with (or instead of) gates 152 thus allows such a aptamer to be captured even if the 3’ end of that aptamer is inhibited by the aptamer structure or other factors from binding.

[0052] In general, in examples in which the capture strands are initially immobilized to beads or other solid support, the ability of a reporter-free strand to bind to a aptamer is independent of, or at least not significantly affected by, which end of the reporter-free strand is immobilized to a solid support because the opposite end of the reporter-free strand is not obscured by a reporter strand and is thus available for capturing the corresponding end of the aptamer. Accordingly, in such examples, it is generally notnecessary to include reporter-free strands that are immobilized to a solid support at a 5’ end and also reporter-free strands immobilized to a solid support at a 3’ end. However, in some cases both types of reporter-free strands are included, e.g., in cases where including both types facilitates more convenient preparation of the correct number of reporter-free strands. For example, in some cases the solid support is prepared for exposure to the eluate by attaching the capture strands to the support (at 3’ ends and / or at 5’ ends, as appropriate) without knowing in advance which capture strands will become unmodified translator gates and which will remain reporter-free strands. In this situation, most likely some reporter-free strands will be attached to the solid support at the 3’ end and some will be attached at the 5’ end.

[0053] Accordingly, translating aptamers to reporter strands may include exposing a aptamer-containing eluate to a combination of gates 152 and / or gates 174 and / or reporter-free strands 164 suitable to capture aptamers from the eluate at the desired range compression ratio. Any suitable combination of gates having toeholds at the 5’ end and gates having toeholds at the 3’ end may be included as appropriate for any given aptamer, and the ratio of unmodified gates to reporter-free strands for a given aptamer may be selected based on the expected abundance of that aptamer and / or on any other suitable factors.

[0054] In some examples, for a given aptamer type, the number of 3’-toehold gates provided and the number of 5’ -toehold gates provided are at least approximately equal, and the number is large enough that there is a very high probability that a suitable fraction of aptamers of that type will be captured if present, even if the aptamers of that type have a very low probability of being captured by one particular end (e.g., if the aptamer is only readily capturable by its 5’ end and has a low chance of being captured by its 3’ end, or vice versa). The determination of how many gates to include also accounts for the desired range compression ratio and associated number of reporter- free strands provided.

[0055] In examples in which all translator gates have a toehold region at the 5’ end or all have a toehold region at the 3’ end, the method comprises two types of strand per aptamer (i.e. , a capture strand and a reporter strand). In examples in which both 5’- toehold gates and 3’ -toehold gates are included for each type of aptamer to be captured, the method comprises four DNA strands per aptamer (i.e., the two types of capture strand and two corresponding types of reporter strand, each type of reporter strand being configured to bind to one of the types of capture strands).

[0056] In the examples described above and depicted in Figs. 4-5, the translator gates and reporter-free strands are attached to magnetic beads. However, as described above with reference to Fig. 1 , in some examples the gates and reporter-free strands are attached to another type of solid support, such as a location in an array, or are prepared in solution unattached to a solid support. The solution is incubated with the aptamer-containing eluate such that aptamers of the eluate are captured by toehold domains of the gates or by reporter-free strands. The resulting solution contains unbound reporter strands freed from the gates, complexes made up of capture strands from the gates bound with captured aptamers, and complexes made up of reporter-free capture strands bound to captured aptamers. In some embodiments, the capture strands and / or aptamers comprise biotin. In some such embodiments, the solution is incubated with streptavidin beads or other solid support, facilitating separation of the complexes from the unbound reporter strands. b. Illustrative Null-Barcode Schema

[0057] This subsection describes another illustrative schema in accordance with aspects of the present teachings, in which range compression is achieved by having the barcode domains of a desired fraction of reporter strands associated with a particular aptamer type be “null” barcodes that are configured not to be read during NGS sequencing (or other suitable processing). Accordingly, a fraction of aptamers release reporter strands that are not read during the NGS process, while the remaining fraction of aptamers release reporter strands that can be read.

[0058] A null barcode may be configured in any suitable manner to be unreadable in a particular NGS process, and different barcodes used in the same assay may be configured in different manners. In some examples, a null barcode comprises a barcode that is readable in principle (e.g., that is not physically incapable of being read) but for which the subsequent process lacks suitable reagents to read. For example, the sequencing flow cell surface may be configured to lack any cognate partners for the barcode, such that the barcode cannot bind to any of the oligonucleotides present on the sequencing flow cell surface. In general, any suitable method(s) of configuring the barcode to be unreadable in subsequent processing, and / or configuring the subsequent processes to be unable to read the barcode, may be used. This may, for example, allow for a testing process in which an NGS test run does include molecular machinery configured to read the null barcode. The output of the NGS test run can be analyzed to assess whether the schema worked as intended (e.g., by confirming, based on NGScounts of the null and unmodified barcodes and on the number and / or ratio of modified and unmodified gates, that the NGS results are consistent with conservation of mass).

[0059] The null-barcode schema is illustrated schematically in Fig. 6, which depicts a readable translator gate 202, which is similar to gates 102 and 122 described above, and an unreadable translator gate 204. Gates 202, 204 have respective capture strands 206, 208 that are complementary to the same aptamer. Readable gate 202 has a barcode domain 210 that is readable in one or more particular readout processes, such as NGS, and unreadable gate 204 has a null barcode domain 212 configured to be unreadable in the one or more particular readout processes. Unreadable gate 204 may also be referred to as a dummy gate. Because half the translator gates depicted in Fig. 6 have null barcode domains and half have readable barcode domains, the example depicted in Fig. 6 reduces by 50% the signal from the particular aptamer being measured. Any suitable ratio may be achieved by adjusting the ratio of unreadable to readable gates.

[0060] As described above with reference to Figs. 4-5, though only one readable and one unreadable gate are depicted in Fig. 6, in general carrying out the illustrated schema includes exposing the aptamer-containing eluate to a large number, such as hundreds or thousands, of readable and / or unreadable gates.

[0061] In the depicted example, capture strands 206, 208 are each immobilized to a respective magnetic bead 214, 216; in other examples, the magnetic beads are omitted, and replaced with another solid support or the capture strands may be in solution. Strands 206, 208 have respective toehold regions at respective 5’ ends 218, 220. Accordingly, gates 202, 204 are configured to capture the 3’ ends of an aptamer and, as described above with reference to Figs. 4 - 5, may fail to capture a given aptamer if the 3’ end of that aptamer is not readily accessible. Accordingly, for some or all types of aptamers to be captured from the eluate, gates including capture strands having toeholds at 3’ ends may be provided in addition to gates including capture strands having toeholds at 5’ ends.

[0062] Fig. 7 depicts an illustrative translator gate 222 having a capture strand 224 with a toehold at a 3’ end 226 and an illustrative modified gate 228 having a capture strand 230 with a toehold at a 3’ end 232. The number of unmodified gates 202 provided may be equal to the number of unmodified gates 222 provided, the number of modified gates 204 provided may be equal to the number of modified gates 228, and the ratio of modified to unmodified gates may be selected to achieve a desired compression ratio. The total number of modified and unmodified gates provided for agiven type of aptamer may be selected to be large enough that there is a very high probability that a suitable fraction of aptamers of that type that are present in the eluate will be captured even if the aptamers of that type have a very low probability of being captured by one particular end (e.g., if the aptamer is only readily capturable by its 5’ end and has a low chance of being captured by its 3’ end, or vice versa).

[0063] In examples in which all readable and unreadable translator gates have a toehold region at the 5’ end (or all have a toehold region at the 3’ end), the present method comprises three strand types per aptamer (i.e. , the capture strand, the readable reporter strand, and the unreadable reporter strand). In examples in which both 5’- toehold gates and 3’ -toehold gates are included for each type of aptamer to be captured, the method may comprise twice as many DNA strands per aptamer, i.e., six DNA strands per aptamer (i.e., the two types of capture strand and, for each of those two types, corresponding readable and unreadable reporter strands).D. Illustrative Systems and Methods Including Trimolecular Complexes

[0064] U.S. Patent Application Serial No. 18 / 148,350, incorporated herein by reference in its entirety, describes proteomics systems and methods in which a postassay aptamer (i.e., an aptamer remaining in an aptamer-containing eluate after exposure to a biological sample, such that the presence of the aptamer in the postassay eluate signifies the presence of a corresponding target protein in the sample) is bound to a pair of probes to form a trimolecular complex. Fig. 8 depicts an example trimolecular complex 250 comprising a post-assay aptamer 252 bound to a first probe 254 and a second probe 256. First probe 254 has a hybridization region Hi complementary to a left-hand portion of aptamer 252 in Fig. 8. Second probe 256 has a hybridization region H2 complementary to a right-hand portion of aptamer 252 in Fig. 8, and further has common primer regions P1 and P2 and a unique aptamer identification sequence Is corresponding to aptamer 252.

[0065] As described in U.S. Patent Application Serial No. 18 / 148,350, the aptamer identification sequences of a plurality of trimolecular complexes formed in a post-assay eluate can be sequenced (e.g., in a next-generation sequencing process) to obtain information about concentrations in the original sample of the target molecules corresponding to the associated aptamer. By comparison, in the translator-gate methods according to aspects of the present teachings, the reporter strands that become unbound from the translator gates are sequenced to provide information about target molecule concentrations.

[0066] In some examples, however, a post-assay eluate is exposed to translator gates and also to capture probes Hi and H2 configured to form a trimolecular complex with a post-assay aptamer. Accordingly, some aptamers of the eluate are captured by translator gates and converted into reporter strands, and some aptamers of the eluate bind to capture probes Hi and H2 to form trimolecular complexes. The reporter strands and the aptamer identification sequences of the H2 probes can be sequenced to obtain target molecule information (in some cases after one or more suitable preparation steps, such as dissociating the H2 probes from the trimolecular complexes).

[0067] In some examples, the output of the translator gates (i.e. , the freed reporter strands) and the output of the trimolecular complexes (e.g., the dissociated H2 probes or portions thereof) are sequenced separately. Separate sequencing of the translator gate output and the trimolecular complex output can be beneficial because in at least some cases, the trimolecular assay output is collected using a hydroxide wash that would effectively release of reporter strands from translator gates that had not already released their reporter strands by capturing an aptamer. As a result of this reporter strand release in the absence of aptamer, sequencing the mixed output would artificially yield the same high count value for all types of aptamer for which translator gates had been included. Separately sequencing the translator gate output and trimolecular complex output requires more sequencing runs compared to a process that uses only trimolecular complexes and no translator gates, but requires no extra assay steps.

[0068] In some examples, however, the translator gate output and trimolecular complex output are sequenced together rather than separately. Examples of this process may include releasing the unreleased reporter strands and removing the corresponding released reporter strands after collecting the correct gate outputs (i.e., after collecting the reporter strands freed from a triggered gate that did capture a postassay aptamer) but before denaturation and collection of the trimolecular assay outputs. This involves two additional assay steps but enables the outputs of the gates and the trimolecular complexes to be recombined and sequenced in a single run, rather than in two separate runs.

[0069] Alternatively or additionally to releasing the unreleased reporter strands prior to denaturing the trimolecular complexes as described above, different barcoded PCR steps can be performed on the translator gate outputs and the trimolecular complex outputs (e.g., with the barcoded PCR used for the trimolecular complex outputs being configured to fail to work for reporter strands of the translator gates, based on primer identity and / or other suitable mechanisms). This allows the translator gate output andthe trimolecular complex output to be distinguished from each other and thus facilitates sequencing the translator gate output and the trimolecular complex output together in a single sequencing run.E. Illustrative Aptamers

[0070] Any nucleic-acid based aptamer that binds a protein target may be used in the present methods.

[0071] In some embodiments, an aptamer that binds a desired target is developed using “Systematic Evolution of Ligands by Exponential enrichment,” or SELEX. The SELEX process is a method for the in vitro evolution of nucleic acid molecules for a certain desired activity, such as specific binding to target molecules, such as proteins. In some embodiments, a SELEX-identified nucleic acid capture reagent is a specific ligand of a given target molecule, such as a protein. Molecules of any size or composition can serve as targets.

[0072] The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and stepwise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, and then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule. In this manner, aptamers suitable for binding to virtually any target protein can be discovered.

[0073] SOMAmers are protein-binding aptamers that have a rate of dissociation (ti / 2) generally between 30 and 240 minutes, this being the average time it takes for half of the protein-aptamer complexes to dissociate. SOMAmers generally comprise modified nucleosides that improve protein binding, affinity, and / or off rate. In some embodiments, the modifications comprise chemical groups that are attached to the 5-position of the pyrimidine bases. By functionalizing the 5-position with amino acid-like moieties (e.g., benzyl, 2-napthyl, etc.), the chemical diversity of oligonucleotides is expanded, allowinghigh affinity binding with a wider range of target molecules. Nonlimiting exemplary 5- position modified pyridine nucleobases include 5-(N-benzylcarboxamide) (“Bn”)- cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)-cytosine, 5-(N-3- phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1 -naphthylmethylcarboxamide) (“Nap”)- cytosine, 5-(N-2-naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1-naphthyl-2- ethylcarboxamide) (“NE”)-cytosine, 5-(N- tyrosylcarboxamide) (”Tyr”)-cytosine, Bn- uracil, Nap-uracil, PE-uracil, 5-(N-isobutylcarboxamide) (“lb”)-uracil, 5-(N-4-fluoro- benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4- methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5-(N-3- benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3-benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3-phenylpropylcarboxamide) (“PP”)-uracil, 5-(N- morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3-indole-2- ethylcarboxamide) (“Trp”)-uracil, 5-(n-(R)-2-hydrozypropylcarboxamide) (“Thr”)-uracil, 5-(N-((1 , 1 ’-biphenyl)-4-yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4- phenylbenzylcarboxamide) (“PBn”)-uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)- uracil, 5-(N-3,3-diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3- phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)- uracil, BPE-cytosine, PBn-cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine. Nonlimiting exemplary modified nucleosides that may be included in aptamers, such as SOMAmers, include 5-position modified pyridine nucleosides. Exemplary 5-position modified pyridine nucleosides that may be used in aptamers, such as SOMAmers, include, but are not limited to, BndC, 2’-OMe-Bn-C, PEdC, 2’- OMe-PE-C, PPdC, 2’-OMe-PP-C, NapdC, 2’-OMe-Nap-C, 2NapdC, 2’-OMe-2Nap-C, NEdC, 2’-OMe-NE-C, 2NEdC, 2’-OMe-2NE-C, TyrdC, 2’-OMe-Tyr-C, BndU, 2’-0Me- Bn-U, NapdU, 2’-OMe-Nap-U, PEdll, 2’-OMe-PE-U, IbdU, 2’-OMe-lb-U, FBndll, 2’- OMe-FBn-U, 2Napdll, 2’-OMe-2Nap-U, NEdll, 2’-OMe-NE-U, MBndll, 2’-0Me-MBn-U, BFdll, 2’-OMe-BF-U, BTdll, 2’-OMe-BT-U, PPdU, 2’-OMe-PP-U, MOEdll, 2’-0Me- MOE-U, Tyrdll, 2’-OMe-Tyr-U, Trpdll, 2’-OMe-Trp-U, Thrdll, and 2’-OMe-Thr-U, BPEdll, 2’-OMe-BPE-U, 2’-F-BPE-U, PBndll, 2’-OMe-PBn-U, 2’-F-PBn-U, POPdU, 2’- OMe-POP-U, 2’-F-POP-U, DPPdU, 2’-OMe-DPP-U, 2’-F-DPP-U, DBMdll, 2’-0Me- DBM-U, 2’-F-DBM-U, BHdll, 2’-OMe-BH-U, 2’-F-BH-U, BPEdC, 2’-OMe-BPE-C, 2’-F- BPE-C, PBndC, 2’-OMe-PBn-C, 2’-F-PBn-C, POPdC, 2’-OMe-POP-C, 2’-F-POP-C, DPPdC, 2’-OMe-DPP-C, 2’-F-DPP-C, DBMdC, 2’-0Me-DBM-C, 2’-F-DBM-C, BHdC, 2’- OMe-BH-C, and 2’-F-BH-C. See, e.g., PCT Publication Nos. WO 2022 / 0221241 and WO 2018 / 0005974.

[0074] Assays directed to the detection and quantification of physiologically significant molecules in biological samples and other samples are important tools in scientific research and in the health care field. Aptamers are capable of binding to a target molecule in the sample in a highly specific manner and with very high affinity. After appropriate washing and partitioning steps to first remove unbound proteins and then to remove unbound aptamers, aptamers are eluted from the resultant aptamerprotein complexes. The eluted aptamers may then be detected, qualitatively or quantitatively, using the methods provided herein, thereby enabling a determination of the absence, presence, amount, and / or concentration of the target molecules in the sample.F. Illustrative Method

[0075] With reference to Fig. 9, this section describes an illustrative method 300 for obtaining qualitative and / or quantitative information about proteins in a sample. In general, a method for obtaining qualitative and / or quantitative information includes capturing target proteins from the sample with aptamers, exposing an eluate containing the aptamers to a plurality of translator gates, and detecting the released reporter strands as a basis for identifying and / or quantifying proteins in the sample. Method 300 is an example of such a method.

[0076] At step 302, method 300 includes capturing target proteins by exposing a biological sample to a plurality of aptamers, including aptamers each configured to bind to a specific protein. Exposing the sample to a library of many aptamers allows for detecting a large number of target protein species in a single assay. In some examples, the aptamers are SOMAmers. SOMAmers have slower off-rates than typical aptamers, which in some embodiments allows the SOMAmer reagents to remain bound to the corresponding proteins during execution of additional assay steps such as multiple washes, improving assay performance.

[0077] At step 304, method 300 includes isolating the aptamers that captured a target protein in an aptamer-containing eluate. Step 304 may include, e.g., performing a SomaScan Assay (SomaLogic, Inc.), which may include binding the aptamers to assay beads, capturing proteins with the aptamers, washing away unbound proteins, tagging the bound proteins with biotin, releasing the aptamers from the beads, capturing the tagged protein / aptamer complexes to new beads, removing unbound aptamers, denaturing the aptamers from the captured complexes, and separating the aptamers into an eluate.

[0078] At step 306, method 300 includes exposing the aptamer-containing eluate produced at step 304 to a plurality of translator gates, which may include one or more of the translator gates described herein. At least a first subset of the translator gates are each configured to capture one of the aptamers from the eluate and to release a reporter strand configured to be detected using an appropriate method, such as a nextgeneration sequencing method. In this manner, the plurality of translator gates are configured to “convert” aptamers in the eluate into easily readable nucleic acid strands, in the sense that aptamers from the eluate are captured by the gates and replaced by nucleic acid reporter strands that are freed from the gates in response to the gates capturing the aptamers. After the nucleic acid reporter strand is released from a translator gate, the captured aptamer remains in a complex with the capture strand.

[0079] The translator gates of the first subset each include a capture strand that is complementary to the aptamer the gate is configured to capture, and a reporter strand bound to the capture strand, such that a toehold domain of the capture strand is available for capturing the aptamer.

[0080] In some examples, a second subset of the translator gates are configured not to release a reporter strand configured to be read out. For example, the second subset of translator gates may have a reporter strand that is configured to be unreadable by the process to be used to read the reporter strands of the first subset of translator gates (e.g., a dummy reporter strand). Alternatively, or additionally, the second subset of translator gates may lack a reporter strand altogether, in which case the second subset of gates are simply aptamer capture strands each configured to capture an aptamer without releasing a reporter strand. Accordingly, the second subset of gates capture aptamers of the type(s) corresponding to the second subset of gates without converting the captured aptamers into a readable nucleic acid.

[0081] The second subset of gates facilitates dynamic range compression of the assay output. For example, the second subset of translator gates may be configured to capture aptamers of one or more types expected to be relatively abundant in the aptamer-containing eluate. By including translator gates lacking reporter strands or comprising dummy reporter strands, the signal from more abundant aptamers may be dampened, thus compressing the dynamic range of the assay to facilitate detection of reporter strands released by less abundant aptamers. A ratio of the readable translator gates to the unreadable translator gates (comprising either no reporter strand or an unreadable reporter strand) may be controlled to control the range compression of one or more types of aptamer (e.g., to control a proportionality of the conversion of thosetypes of aptamer into released readable reporter strands). In various embodiments, the ratio of capture strands to readable reporter strands may be between about 2:1 to about 10,000,000:1 , or between about 10:1 to about 1 ,000,000:1 , or between about 10:1 to about 100,000:1 , or between about 100:1 to about 1 ,000,000:1 , or between about 100: 1 to about 100,000: 1 . In this way, a portion of the more abundant aptamers may be “soaked up” by capture strands without releasing readable reporter strands.

[0082] In some examples, two or more subsets of gates configured not to produce a readable reporter strand are included, and the two or more subsets may be configured in different manners not to produce readable reporter strands (e.g., with one subset lacking reporter strands and one subset including dummy reporter strands, and / or any other suitable combination).

[0083] At step 308, method 300 includes separating the reporter strands freed from the gates from the complexes comprising the capture strands and aptamers. The separation may be performed in any suitable manner and may include and / or be preceded, accompanied, and / or followed by any suitable processing steps, such as washing to remove uncaptured aptamers.

[0084] In some examples, separating the reporter strands from the complexes includes removing the eluate comprising the reporter strands from the complexes bound to solid supports, such as magnetic beads or an array. The complexes may be bound to the solid support in any suitable manner, typically through the capture strand, and may be bound to the solid support at any suitable step of the method. In some examples, the capture strands are bound to the solid support before the aptamercontaining eluate is exposed to the translator gates at step 306; e.g., the translator gates may have been prepared and bound to the solid support prior to the exposure. In some examples, the capture strands of the capture strand / aptamer complexes are bound to a solid support after the aptamer-containing eluate is exposed to the translator gates at step 306; e.g., the solution of complexes and freed reporter strands may be incubated with a solid support configured to bind to the capture strands. In general, the capture strands may be bound to solid supports in any suitable manner; for example, the solid support may comprise streptavidin and the capture strands may be biotinylated.

[0085] At step 310, method 300 includes preparing the freed reporter strands for readout, which may include amplifying the reporter strands (or portions thereof), attaching adapter sequences, demultiplexing barcode sequences, and / or performingany other suitable steps. Step 310 may be omitted if no preparation is needed or desired.

[0086] At step 312, method 300 includes reading out the reporter strands, e.g., by sequencing the strands, using next-generation sequencing and / or any other suitable techniques. The data obtained by reading out the reporter strands may be used to determine qualitative and / or quantitative information about the proteins of the original sample, such as protein abundances.

[0087] In some examples, steps 306, 308, and optionally 310 are performed as a separate method, indicated in Fig. 9 as method 350, of converting aptamers from an existing aptamer-containing eluate into a plurality of reporter strands to be read out. As described above, in some examples method 350 includes compressing the range of the readout by capturing certain subset(s) of the aptamers and not translating them into reporter strands and / or translating them into unreadable nucleic acids.Illustrative Advantages, Features, and Benefits

[0088] Various embodiments and examples of the translator gates and associated methods described herein provide advantages over known solutions for determining protein concentrations based on aptamer concentrations, though it is not necessarily the case that all embodiments and examples described herein provide the same advantages or the same degree of advantage.

[0089] For example, illustrative embodiments and examples described herein allow capture of aptamers from an aptamer-containing eluate without the need for barcodes or other linkers attached to the aptamers. Accordingly, there is no need to attach barcodes or linkers to the aptamers prior to using the aptamers to capture proteins.

[0090] Additionally, and among other benefits, illustrative embodiments and examples described herein allow hybridization to occur over a relatively long domain length (e.g., at least 25, at least 30, at least 35, at least 40, at least 45, etc., nucleotides), which avoids a problem that in some cases affects shorter hybridization domains. Complexes formed by shorter hybridization domains (e.g., a complex comprising an aptamer and one or more probes bound to the aptamer by hybridization over a shorter length) in some cases have a low melting temperature and are therefore prone to denaturing, leading the aptamer to “drop out” of the process before it can be counted. In contrast, the performance of translator gates is generally less dependent on sequence, because the capture strand of the translator gate is in at least some examples a full complement to the aptamer. This allows aptamers of various lengthsand sequence to be converted to reporter strands approximately equally well, with less variation between types of aptamers than is the case with shorter hybridization domains.Example 1 : Translator Gate Performance

[0091] Two translator gate complexes were prepared by annealing a biotinylated first capture strand (CS1 ) and a biotinylated second capture strand (CS2) with their corresponding fluorescently-labeled first reporter strand (RS1 ) and fluorescently-labeled second reporter strand (RS2), in a 1 :2 ratio. Each mixture was then incubated with streptavidin-coated MyOne C1 beads for 2 hours at room temperature. The beads were then washed 12x to remove non-immobilized capture strands and reporter strands. The bound translator gates (TG2 and TG3) were then incubated with 100 pM of their respective cognate aptamers ( Somamer2 or Somamer 3) overnight.Somamer2 comprised 2Napdll nucleoside modifications and Somamer3 comprised Trpdll nucleoside modifications. The resulting supernatants comprising released reporter strands were removed and evaluated using a fluorescent plate reader. Control reactions did not include aptamers.

[0092] The results are shown in Fig. 10. High efficiency of aptamer to reporter signal was observed. The greater output concentration compared to the input concentration (100 pM) is believed to be due to background, as evidenced by the controls signals. Further, the translator gates showed equivalent performance regardless of the aptamer nucleoside modifications.Conclusion

[0093] The disclosure set forth above may encompass multiple distinct examples with independent utility. Although each of these examples has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the example(s) includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein.

[0094] Certain combinations and subcombinations regarded as novel and nonobvious are particularly pointed out throughout this disclosure. Other combinations and subcombinations of features, functions, elements, and / or properties may be claimed, with or without variation in scope, in applications claiming priority from this or a related application.

[0095] Explicit reference is hereby made to all examples, embodiments, inventions, labels, terms, descriptions, and illustrative measurements shown in the drawings and / or in any included appendices, whether or not described further herein. To the extent that section headings are used within this disclosure, such headings are for organizational purposes only.

Claims

CLAIMS1 . A composition comprising: a) a first capture oligonucleotide; b) a first reporter oligonucleotide, wherein the first reporter oligonucleotide comprises a portion that is complementary to a portion of the first capture oligonucleotide; c) a second capture oligonucleotide; and d) a second reporter oligonucleotide, wherein the second reporter oligonucleotide comprises a portion that is complementary to a portion of the second capture oligonucleotide; wherein the amount of the first capture oligonucleotide in the composition is about equal to the amount of the first reporter oligonucleotide in the composition; wherein the amount of the second capture oligonucleotide in the composition is greater than the amount of the second reporter oligonucleotide in the composition; and wherein the sequence of the first capture oligonucleotide and the second capture oligonucleotide are different from one another, and the sequence of the first reporter oligonucleotide and the second reporter oligonucleotide are different from one another.

2. The composition of claim 1 , wherein the first capture oligonucleotide is bound to a first solid support and the second capture oligonucleotide is bound to a second solid support.

3. The composition of claim 2, wherein the first solid support and the second solid support are different locations on an array.

4. The composition of claim 2, wherein the first solid support and the second solid support are separate microbeads.

5. The composition of any one of claims 1 -4, wherein the first capture oligonucleotide and the second capture oligonucleotide each comprises a first member of a binding pair.

6. The composition of claim 5, wherein the first capture oligonucleotide and the second capture oligonucleotide each comprises biotin.

7. The composition of any one of claims 1 -6, wherein the ratio of the amount of the first capture oligonucleotide in the composition to the amount of the first reporter oligonucleotide in the composition is about 1 :1.

8. The composition of any one of claims 1 -7, wherein the ratio of the amount of the second capture oligonucleotide in the composition to the amount of the secondreporter oligonucleotide in the composition is between about 2:1 to about 10,000,000:1 , or between about 10:1 to about 1 ,000,000: 1 , or between about 10:1 to about 100,000:1 , or between about 100:1 to about 1 ,000,000:1 , or between about 100:1 to about 100,000:1.

9. The composition of any one of claims 1 -8, wherein the amount of the second capture oligonucleotide in the composition is greater than the amount of the first capture oligonucleotide in the composition.

10. The composition of any one of claims 1 -9, wherein the ratio of the amount of the first capture oligonucleotide to the amount of the second capture oligonucleotide is between about 1 :10 to about 1 :10,000,000, or between about 1 :10 to about1 : 1 ,000,000, or between about 1 : 100 to about 1 : 1 ,000,000, or between about 1 : 100 to about 1 :100,000.11 . The composition of any one of claims 1 -10, wherein the amount of the second reporter oligonucleotide in the composition is greater than the amount of the first reporter oligonucleotide in the composition.

12. The composition of any one of claims 1-11 , wherein the ratio of the amount of the first reporter oligonucleotide to the amount of the second reporter oligonucleotide is between about 1 :1 to about 1 :10,000, or between about 1 :1 and about 1 : 1 ,000, or between about 1 : 1 and about 1 : 100.

13. The composition of any one of claims 1 -12, wherein the portion of the first reporter oligonucleotide that is complementary to the portion of the first capture oligonucleotide comprises 10-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

14. The composition of any one of claims 1 -13, wherein the portion of the first reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the first capture oligonucleotide, optionally wherein the portion of the first reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the first capture oligonucleotide.

15. The composition of any one of claims 1 -14, wherein the portion of the second reporter oligonucleotide that is complementary to the portion of the second capture oligonucleotide comprises 10-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

16. The composition of any one of claims 1 -15, wherein the portion of the second reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the second capture oligonucleotide, optionally wherein the portion of the second reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the second capture oligonucleotide.

17. The composition of any one of claims 1 -16, wherein the first reporter oligonucleotide comprises a first detectable label and the second reporter oligonucleotide each comprise a second detectable label.

18. The composition of claim 17, wherein the first detectable label and the second detectable label are the same or different.

19. The composition of claim 17 or claim 18, wherein the first detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

20. The composition of any one of claims 17-19, wherein the second detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.21 . The composition of any one of claims 17-20, wherein the first detectable label is a first barcode and the second detectable label is a second barcode, wherein the sequence of the first barcode and the sequence of the second barcode are different.

22. The composition of any one of claims 1 -21 , further comprising a first aptamer comprising a portion that is complementary to a portion of the first capture oligonucleotide and a second aptamer comprising a portion that is complementary to a portion of the second capture oligonucleotide.

23. The composition of claim 22, wherein the portion of the first aptamer that is complementary to the first capture oligonucleotide overlaps with and has a higher melting temperature than the portion of the first reporter oligonucleotide that is complementary to the first capture oligonucleotide, and wherein the portion of the second aptamer that is complementary to the second capture oligonucleotide overlaps with and has a higher melting temperature than the portion of the second reporter oligonucleotide that is complementary to the second capture oligonucleotide.

24. The composition of claim 22 or claim 23, wherein the composition comprises a complex of the first capture oligonucleotide hybridized to the first aptamer and a complex of the second capture oligonucleotide hybridized to the second aptamer.

25. The composition of any one of claims 22-24, wherein the amount of the second aptamer in the composition is greater than the amount of the first aptamer in the composition.

26. The composition of any one of claims 22-25, wherein the composition comprises: a) the first capture oligonucleotide hybridized to the first aptamer;b) the first reporter oligonucleotide in solution; c) the second capture oligonucleotide hybridized to the second aptamer; d) the second reporter oligonucleotide in solution; wherein the amount of the first capture oligonucleotide hybridized to the first aptamer in the composition is about the same as the amount of the first reporter oligonucleotide in the composition; and wherein the amount of the second capture oligonucleotide hybridized to the second aptamer in the composition is greater than the amount of the second reporter oligonucleotide in the composition.

27. The composition of any one of claims 22-26, wherein the portion of the first aptamer that is complementary to the first capture oligonucleotide comprises 15-60, 15-50, 15-40, 20-60, 20-50, 20-40, 30-60, 30-50, or 30-40 nucleobases.

28. The composition of any one of claims 22-27, wherein the portion of the first aptamer is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the portion of the first capture oligonucleotide.

29. The composition of any one of claims 22-28, wherein the portion of the second aptamer that is complementary to the second capture oligonucleotide comprises 15-60, 15-50, 15-40, 20-60, 20-50, 20-40, 30-60, 30-50, or 30-40 nucleobases.

30. The composition of any one of claims 22-29, wherein the portion of the second aptamer is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the portion of the second capture oligonucleotide.31 . The composition of any one of claims 22-30, wherein the first aptamer and the second aptamer each independently comprise at least one, at least two, at least three, at least four, or at least five C-5 modified nucleobases.

32. The composition of claim 31 , wherein the modified nucleobase is selected from 5-(N-benzylcarboxamide) (“Bn”)-cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)- cytosine, 5-(N-3-phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1- naphthylmethylcarboxamide) (“Nap”)-cytosine, 5-(N-2-naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1-naphthyl-2-ethylcarboxamide) (“NE”)-cytosine, 5-(N- tyrosylcarboxamide) (”Tyr”)-cytosine, Bn-uracil, Nap-uracil, PE-uracil, 5-(N- isobutylcarboxamide) (“lb”)-uracil, 5-(N-4-fluoro-benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4-methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5- (N-3-benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3- benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3-phenylpropylcarboxamide)(“PP”)-uracil, 5-(N-morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3- indole-2-ethylcarboxamide) (“Trp”)-uracil, 5-(n-(R)-2-hydrozypropylcarboxamide) (“Thr”)-uracil, 5-(N-((1 ,1’-biphenyl)-4-yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4- phenylbenzylcarboxamide) (“PBn”)-uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)- uracil, 5-(N-3,3-diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3- phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)- uracil, BPE-cytosine, PBn-cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine.

33. The composition of any one of claims 22-32, wherein the first aptamer comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30- 60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

34. The composition of any one of claims 22-33, wherein the second aptamer comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30- 60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

35. The composition of any one of claims 1 -34, wherein the first capture oligonucleotide comprises 15-100, 20-100, 20-90, 20-80, 20-70, 20-60, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

36. The composition of any one of claims 1 -35, wherein the second capture oligonucleotide comprises 15-100, 20-100, 20-90, 20-80, 20-70, 20-60, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

37. The composition of any one of claims 1 -36, wherein the first reporter oligonucleotide comprises 10-200, 10-150, 20-200, 20-150, 30-200, 30-250, 40-200, 40-150, 50-200, 50-150, 60-200, or 60-150 nucleotides.

38. The composition of any one of claims 1-37, wherein the second reporter oligonucleotide comprises 10-200, 10-150, 20-200, 20-150, 30-200, 30-250, 40-200, 40-150, 50-200, 50-150, 60-200, or 60-150 nucleotides.

39. The composition of any one of claims 1 -38, wherein the composition further comprises a third capture oligonucleotide and a third reporter oligonucleotide, wherein the third reporter oligonucleotide comprises a portion that is complementary to a portion of the third capture oligonucleotide, wherein the sequence of the third capture oligonucleotide is different from the sequence of the first and second capture oligonucleotides and the sequence of the third reporter oligonucleotide is different from the sequence of the first and second reporter oligonucleotides.

40. The composition of claim 39, wherein the amount of the third capture oligonucleotide in the composition is about equal to the amount of the third reporter oligonucleotide in the composition.41 . The composition of claim 39, wherein the amount of the third capture oligonucleotide in the composition is greater than the amount of the third reporter oligonucleotide in the composition.

42. The composition of any one of claims 39-41 , wherein the third capture oligonucleotide is bound to a third solid support.

43. The composition of claim 42, wherein the third solid support is a different location on an array from the first solid support and the second solid support.

44. The composition of claim 42, wherein the third solid support is a microbead.

45. The composition of any one of claims 39-44, wherein the third capture oligonucleotide comprises a first member of a binding pair.

46. The composition of claim 55, wherein the third capture oligonucleotide comprises biotin.

47. The composition of any one of claims 39-46, wherein the ratio of the amount of the third capture oligonucleotide in the composition to the amount of the third reporter oligonucleotide in the composition is about 1 :1 ; or wherein the ratio of the amount of the third capture oligonucleotide in the composition to the amount of the third reporter oligonucleotide in the composition is between about 2:1 to about 10,000,000:1 , or between about 10:1 to about 1 ,000,000: 1 , or between about 10:1 to about 100,000:1 , or between about 100:1 to about 1 ,000,000:1 , or between about 100:1 to about 100,000:1.

48. The composition of any one of claims 39-47, wherein the portion of the third reporter oligonucleotide that is complementary to the portion of the third capture oligonucleotide comprises 10-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

49. The composition of any one of claims 39-48, wherein the portion of the third reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the third capture oligonucleotide, optionally wherein the portion of the third reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the third capture oligonucleotide.

50. The composition of any one of claims 39-49, wherein the third reporter oligonucleotide comprises a third detectable label.51 . The composition of claim 50, wherein the third detectable label is the same or different from the first detectable label and the second detectable label.

52. The composition of claim 50 or claim 51 , wherein the third detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

53. The composition of any one of claims 50-52, wherein the third detectable label is a third barcode, wherein the sequence of the third barcode is different from the sequences of the first barcode and the second barcode.

54. The composition of any one of claims 39-53, further comprising a third aptamer comprising a portion that is complementary to a portion of the third capture oligonucleotide.

55. The composition of claim 54, wherein the portion of the third aptamer that is complementary to the third capture oligonucleotide overlaps with and has a higher melting temperature than the portion of the third reporter oligonucleotide that is complementary to the third capture oligonucleotide.

56. The composition of claim 54 or 55, wherein the composition comprises a complex of the third capture oligonucleotide hybridized to the third aptamer.

57. The composition of any one of claims 54-56, wherein the composition comprises: a) the third capture oligonucleotide hybridized to the third aptamer; and b) the third reporter oligonucleotide in solution.

58. The composition of any one of claims 54-57, wherein the portion of the third aptamer that is complementary to the third capture oligonucleotide comprises 15- 60, 15-50, 15-40, 20-60, 20-50, 20-40, 30-60, 30-50, or 30-40 nucleobases.

59. The composition of any one of claims 54-58, wherein the portion of the third aptamer is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the portion of the third capture oligonucleotide.

60. The composition of any one of claims 54-59, wherein the third aptamer comprises at least one, at least two, at least three, at least four, or at least five C-5 modified nucleobases.61 . The composition of claim 60, wherein the modified nucleobase is selected from 5-(N-benzylcarboxamide) (“Bn”)-cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)- cytosine, 5-(N-3-phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1- naphthylmethylcarboxamide) (“Nap”)-cytosine, 5-(N-2-naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1-naphthyl-2-ethylcarboxamide) (“NE”)-cytosine, 5-(N-tyrosylcarboxamide) (”Tyr”)-cytosine, Bn-uracil, Nap-uracil, PE-uracil, 5-(N- isobutylcarboxamide) (“lb”)-uracil, 5-(N-4-fluoro-benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4-methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5- (N-3-benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3- benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3-phenylpropylcarboxamide) (“PP”)-uracil, 5-(N-morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3- indole-2-ethylcarboxamide) (“Trp”)-uracil, 5-(n-(R)-2-hydrozypropylcarboxamide) (“Thr”)-uracil, 5-(N-((1 ,1’-biphenyl)-4-yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4- phenylbenzylcarboxamide) (“PBn”)-uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)- uracil, 5-(N-3,3-diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3- phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)- uracil, BPE-cytosine, PBn-cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine.

62. The composition of any one of claims 54-61 , wherein the third aptamer comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30- 60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

63. The composition of any one of claims 39-62, wherein the third capture oligonucleotide comprises 15-100, 20-100, 20-90, 20-80, 20-70, 20-60, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

64. The composition of any one of claims 39-62, wherein the third reporter oligonucleotide comprises 10-200, 10-150, 20-200, 20-150, 30-200, 30-250, 40-200, 40-150, 50-200, 50-150, 60-200, or 60-150 nucleotides.

65. An array comprising at least a first location and a second location, wherein: a) the first location comprises: i) a first capture oligonucleotide; and ii) a first reporter oligonucleotide, wherein the first reporter oligonucleotide comprises a portion that is complementary to a portion of the first capture oligonucleotide; and b) the second location comprises: i) a second capture oligonucleotide; and ii) a second reporter oligonucleotide, wherein the second reporter oligonucleotide comprises a portion that is complementary to a portion of the second capture oligonucleotide;wherein the amount of the first capture oligonucleotide is about equal to the amount of the first reporter oligonucleotide; wherein the amount of the second capture oligonucleotide is greater than the amount of the second reporter oligonucleotide; and wherein the sequence of the first capture oligonucleotide and the second capture oligonucleotide are different from one another, and the sequence of the first reporter oligonucleotide and the second reporter oligonucleotide are different from one another.

66. The array of claim 65, wherein the first capture oligonucleotide is bound to a first solid support at the first location of the array and the second capture oligonucleotide is bound to a second solid support at the second location of the array.

67. The array of claim 66, wherein the first solid support and the second solid support are the locations on the array.

68. The array of claim 66, wherein the first solid support and the second solid support are microbeads contained in the locations on the array.

69. The array of any one of claims 65-68, wherein the first capture oligonucleotide and the second capture oligonucleotide each comprises a first member of a binding pair.

70. The array of claim 69, wherein the first capture oligonucleotide and the second capture oligonucleotide each comprises biotin.71 . The array of any one of claims 65-70, wherein the ratio of the amount of the first capture oligonucleotide in the first location of the array to the amount of the first reporter oligonucleotide in the first location of the array is about 1 :1.

72. The array of any one of claims 65-71 , wherein the ratio of the amount of the second capture oligonucleotide in the second location of the array to the amount of the second reporter oligonucleotide in the second location of the array is between about 2: 1 to about 10,000,000: 1 , or between about 10:1 to about 1 ,000,000: 1 , or between about 10:1 to about 100,000:1 , or between about 100:1 to about 1 ,000,000:1 , or between about 100:1 to about 100,000:1.

73. The array of any one of claims 65-72, wherein the amount of the second capture oligonucleotide in the second location of the array is greater than the amount of the first capture oligonucleotide in the first location of the array.

74. The array of any one of claims 65-73, wherein the ratio of the amount of the first capture oligonucleotide to the amount of the second capture oligonucleotide is between about 1 :10 to about 1 :10,000,000, or between about 1 :10 to about1 : 1 ,000,000, or between about 1 : 100 to about 1 : 1 ,000,000, or between about 1 : 100 to about 1 :100,000.

75. The array of any one of claims 65-74, wherein the amount of the second reporter oligonucleotide at the second location of the array is greater than the amount of the first reporter oligonucleotide at the first location of the the array.

76. The array of any one of claims 65-75, wherein the ratio of the amount of the first reporter oligonucleotide to the amount of the second reporter oligonucleotide is between about 1 :1 to about 1 :10,000, or between about 1 :1 and about 1 :1 ,000, or between about 1 :1 and about 1 :100.

77. The array of any one of claims 65-76, wherein the portion of the first reporter oligonucleotide that is complementary to the portion of the first capture oligonucleotide comprises 10-50, 20-50, 10-40, 20-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

78. The array of any one of claims 65-77, wherein the portion of the first reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the first capture oligonucleotide, optionally wherein the portion of the first reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the first capture oligonucleotide.

79. The array of any one of claims 65-78, wherein the portion of the second reporter oligonucleotide that is complementary to the portion of the second capture oligonucleotide comprises 10-50, 20-50, 10-40, 20-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

80. The array of any one of claims 65-79, wherein the portion of the second reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the second capture oligonucleotide, optionally wherein the portion of the second reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the second capture oligonucleotide.81 . The array of any one of claims 65-80, wherein the first reporter oligonucleotide comprises a first detectable label and the second reporter oligonucleotide each comprise a second detectable label.

82. The array of claim 81 , wherein the first detectable label and the second detectable label are the same or different.

83. The array of claim 81 or claim 82, wherein the first detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

84. The array of any one of claims 81-83, wherein the second detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

85. The array of any one of claims 81-84, wherein the first detectable label is a first barcode and the second detectable label is a second barcode, wherein the sequence of the first barcode and the sequence of the second barcode are different.

86. The array of any one of claims 65-85, further comprising a first aptamer comprising a portion that is complementary to a portion of the first capture oligonucleotide and a second aptamer comprising a portion that is complementary to a portion of the second capture oligonucleotide.

87. The array of claim 86, wherein the portion of the first aptamer that is complementary to the first capture oligonucleotide overlaps with and has a higher melting temperature than the portion of the first reporter oligonucleotide that is complementary to the first capture oligonucleotide, and wherein the portion of the second aptamer that is complementary to the second capture oligonucleotide overlaps with and has a higher melting temperature than the portion of the second reporter oligonucleotide that is complementary to the second capture oligonucleotide.

88. The array of claim 86 or claim 87, wherein the first location of the array comprises a complex of the first capture oligonucleotide hybridized to the first aptamer and the second location of the array comprises a complex of the second capture oligonucleotide hybridized to the second aptamer.

89. The array of any one of claims 86-88, wherein the amount of the second aptamer at the first location of the array is greater than the amount of the first aptamer at the second location of the array.

90. The array of any one of claims 86-89, wherein: a) the first location of the array comprises: i) the first capture oligonucleotide hybridized to the first aptamer; ii) the first reporter oligonucleotide in solution; and b) the second location of the array comprises: i) the second capture oligonucleotide hybridized to the second aptamer; ii) the second reporter oligonucleotide in solution; wherein the amount of the first capture oligonucleotide hybridized to the first aptamer is about the same as the amount of the first reporter oligonucleotide in the first location of the array; andwherein the amount of the second capture oligonucleotide hybridized to the second aptamer is greater than the amount of the second reporter oligonucleotide in the second location of the array.91 . The array of any one of claims 86-90, wherein the portion of the first aptamer that is complementary to the first capture oligonucleotide comprises 15-60, 15- 50, 15-40, 20-60, 20-50, 20-40, 30-60, 30-50, or 30-40 nucleobases.

92. The array of any one of claims 86-91 , wherein the portion of the first aptamer is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the portion of the first capture oligonucleotide.

93. The array of any one of claims 86-92, wherein the portion of the second aptamer that is complementary to the second capture oligonucleotide comprises 15-60, 15-50, 15-40, 20-60, 20-50, 20-40, 30-60, 30-50, or 30-40 nucleobases.

94. The array of any one of claims 86-93, wherein the portion of the second aptamer is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the portion of the second capture oligonucleotide.

95. The array of any one of claims 86-94, wherein the first aptamer and the second aptamer each independently comprise at least one, at least two, at least three, at least four, or at least five C-5 modified nucleobases.

96. The array of claim 95, wherein the modified nucleobase is selected from 5-(N-benzylcarboxamide) (“Bn”)-cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)- cytosine, 5-(N-3-phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1- naphthylmethylcarboxamide) (“Nap”)-cytosine, 5-(N-2-naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1-naphthyl-2-ethylcarboxamide) (“NE”)-cytosine, 5-(N- tyrosylcarboxamide) (”Tyr”)-cytosine, Bn-uracil, Nap-uracil, PE-uracil, 5-(N- isobutylcarboxamide) (“lb”)-uracil, 5-(N-4-fluoro-benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4-methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5- (N-3-benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3- benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3-phenylpropylcarboxamide) (“PP”)-uracil, 5-(N-morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3- indole-2-ethylcarboxamide) (“Trp”)-uracil, 5-(n-(R)-2-hydrozypropylcarboxamide) (“Thr”)-uracil, 5-(N-((1 ,1’-biphenyl)-4-yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4- phenylbenzylcarboxamide) (“PBn”)-uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)- uracil, 5-(N-3,3-diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3- phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)-uracil, BPE-cytosine, PBn-cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine.

97. The array of any one of claims 86-96, wherein the first aptamer comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

98. The array of any one of claims 86-97, wherein the second aptamer comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30- 60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

99. The array of any one of claims 65-98, wherein the first capture oligonucleotide comprises 15-100, 20-100, 20-90, 20-80, 20-70, 20-60, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

100. The array of any one of claims 65-99, wherein the second capture oligonucleotide comprises 15-100, 20-100, 20-90, 20-80, 20-70, 20-60, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

101. The array of any one of claims 65-100, wherein the first reporter oligonucleotide comprises 10-200, 10-150, 20-200, 20-150, 30-200, 30-250, 40-200, 40-150, 50-200, 50-150, 60-200, or 60-150 nucleotides.

102. The array of any one of claims 65-101 , wherein the second reporter oligonucleotide comprises 10-200, 10-150, 20-200, 20-150, 30-200, 30-250, 40-200, 40-150, 50-200, 50-150, 60-200, or 60-150 nucleotides.

103. The array of any one of claims 65-102, wherein the array comprises a third location comprising a third capture oligonucleotide and a third reporter oligonucleotide, wherein the third reporter oligonucleotide comprises a portion that is complementary to a portion of the third capture oligonucleotide, wherein the sequence of the third capture oligonucleotide is different from the sequence of the first and second capture oligonucleotides and the sequence of the third reporter oligonucleotide is different from the sequence of the first and second reporter oligonucleotides.

104. The array of claim 103, wherein the amount of the third capture oligonucleotide is about equal to the amount of the third reporter oligonucleotide.

105. The array of claim 104, wherein the amount of the third capture oligonucleotide is greater than the amount of the third reporter oligonucleotide.

106. The array of any one of claims 103-105, wherein the third capture oligonucleotide is bound to a third solid support.

107. The array of claim 106, wherein the third solid support is the third location of the array.

108. The array of claim 106, wherein the third solid support is a microbead contained in the third location of the array.

109. The array of any one of claims 103-108, wherein the third capture oligonucleotide comprises a first member of a binding pair.

110. The array of claim 109, wherein the third capture oligonucleotide comprises biotin.

111. The array of any one of claims 103-110, wherein the ratio of the amount of the third capture oligonucleotide in the third location of the array to the amount of the third reporter oligonucleotide in the third location of the array is about 1 :1 ; or wherein the ratio of the amount of the third capture oligonucleotide in the third location of the array to the amount of the third reporter oligonucleotide in the third location of the array is between about 2:1 to about 10,000,000:1 , or between about 10:1 to about1 ,000,000: 1 , or between about 10: 1 to about 100,000: 1 , or between about 100: 1 to about 1 ,000,000: 1 , or between about 100: 1 to about 100,000: 1 .

112. The array of any one of claims 103-111 , wherein the portion of the third reporter oligonucleotide that is complementary to the portion of the third capture oligonucleotide comprises 10-50, 20-50, 10-40, 20-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

113. The array of any one of claims 103-112, wherein the portion of the third reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the third capture oligonucleotide, optionally wherein the portion of the third reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the third capture oligonucleotide.

114. The array of any one of claims 103-113, wherein the third reporter oligonucleotide comprises a third detectable label.

115. The array of claim 114, wherein the third detectable label is the same or different from the first detectable label and the second detectable label.

116. The array of claim 114 or claim 115, wherein the third detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

117. The array of any one of claims 114-116, wherein the third detectable label is a third barcode, wherein the sequence of the third barcode is different from the sequences of the first barcode and the second barcode.

118. The array of any one of claims 114-117, further comprising a third aptamer comprising a portion that is complementary to a portion of the third capture oligonucleotide.

119. The array of claim 118, wherein the portion of the third aptamer that is complementary to the third capture oligonucleotide overlaps with and has a higher melting temperature than the portion of the third reporter oligonucleotide that is complementary to the third capture oligonucleotide.

120. The array of claim 118 or 119, wherein the third location of the array comprises a complex of the third capture oligonucleotide hybridized to the third aptamer.

121. The array of any one of claims 118-120, wherein the third location of the array comprises: a) the third capture oligonucleotide hybridized to the third aptamer; and b) the third reporter oligonucleotide in solution.

122. The array of any one of claims 118-121 , wherein the portion of the third aptamer that is complementary to the third capture oligonucleotide comprises 15-60, 15-50, 15-40, 20-60, 20-50, 20-40, 30-60, 30-50, or 30-40 nucleobases.

123. The array of any one of claims 118-122, wherein the portion of the third aptamer is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the portion of the third capture oligonucleotide.

124. The array of any one of claims 118-123, wherein the third aptamer comprises at least one, at least two, at least three, at least four, or at least five C-5 modified nucleobases.

125. The array of claim 124, wherein the modified nucleobase is selected from 5-(N-benzylcarboxamide) (“Bn”)-cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)- cytosine, 5-(N-3-phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1- naphthylmethylcarboxamide) (“Nap”)-cytosine, 5-(N-2-naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1-naphthyl-2-ethylcarboxamide) (“NE”)-cytosine, 5-(N- tyrosylcarboxamide) (”Tyr”)-cytosine, Bn-uracil, Nap-uracil, PE-uracil, 5-(N- isobutylcarboxamide) (“lb”)-uracil, 5-(N-4-fluoro-benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4-methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5- (N-3-benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3- benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3-phenylpropylcarboxamide) (“PP”)-uracil, 5-(N-morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3- indole-2-ethylcarboxamide) (“Trp”)-uracil, 5-(n-(R)-2-hydrozypropylcarboxamide)(“Thr”)-uracil, 5-(N-((1 ,1’-biphenyl)-4-yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4- phenylbenzylcarboxamide) (“PBn”)-uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)- uracil, 5-(N-3,3-diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3- phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)- uracil, BPE-cytosine, PBn-cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine.

126. The array of any one of claims 118-125, wherein the third aptamer comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30- 60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

127. The array of any one of claims 103-126, wherein the third capture oligonucleotide comprises 15-100, 20-100, 20-90, 20-80, 20-70, 20-60, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

128. The array of any one of claims 103-127, wherein the third reporter oligonucleotide comprises 10-200, 10-150, 20-200, 20-150, 30-200, 30-250, 40-200, 40-150, 50-200, 50-150, 60-200, or 60-150 nucleotides.

129. A method of determining the amount of a first aptamer and a second aptamer in a sample, comprising: a) contacting the sample comprising the first aptamer and the second aptamer with a composition comprising: i) a first complex comprising a first capture oligonucleotide hybridized to a first reporter oligonucleotide, wherein the first aptamer comprises a portion that is complementary to a portion of the first capture oligonucleotide; ii) a second complex comprising a second capture oligonucleotide hybridized to a second reporter oligonucleotide, wherein the second aptamer comprising a portion that is complementary to a portion of the second capture oligonucleotide; iii) single-stranded second capture oligonucleotide; under conditions suitable for hybridizing the first aptamer to the first capture oligonucleotide of the first complex and releasing the first reporter oligonucleotide and hybridizing the second aptamer to the single-stranded second capture oligonucleotide and to the second capture oligonucleotide of the second complex and releasing the second reporter oligonucleotide; andb) detecting the released first reporter oligonucleotide and the released second reporter oligonucleotide.

130. The method of claim 129, wherein the first capture oligonucleotide is bound to a first solid support and the second capture oligonucleotide is bound to a second solid support.

131. The method of claim 130, wherein the first solid support and the second solid support are different locations on an array.

132. The method of claim 130, wherein the first solid support and the second solid support are separate microbeads.

133. A method of determining the amount of a first aptamer and a second aptamer in a sample, comprising: a) Contacting an array with the sample comprising the first aptamer and the second aptamer, wherein the array comprises: i) a first location comprising:1 . a first complex comprising a first capture oligonucleotide hybridized to a first reporter oligonucleotide, wherein the first aptamer comprises a portion that is complementary to a portion of the first capture oligonucleotide; and ii) a second location comprising:1 . a second complex comprising a second capture oligonucleotide hybridized to a second reporter oligonucleotide, wherein the second aptamer comprising a portion that is complementary to a portion of the second capture oligonucleotide; and2. single-stranded second capture oligonucleotide; under conditions suitable for hybridizing the first aptamer to the first capture oligonucleotide of the first complex and releasing the first reporter oligonucleotide and hybridizing the second aptamer to the single-stranded second capture oligonucleotide and to the second capture oligonucleotide of the second complex and releasing the second reporter oligonucleotide; and b) detecting the released first reporter oligonucleotide and the released second reporter oligonucleotide.

134. The method of claim 133, wherein the first capture oligonucleotide is bound to the first location of the array and the second capture oligonucleotide is bound to the second location of the array.

135. The method of claim 133, wherein the first capture oligonucleotide is bound to microbeads at the first location of the array and the second capture oligonucleotide is bound to microbeads at the second location of the array.

136. The method of any one of claims 129-135, wherein the first capture oligonucleotide and the second capture oligonucleotide each comprises a first member of a binding pair.

137. The method of claim 136, wherein the first capture oligonucleotide and the second capture oligonucleotide each comprises biotin.

138. The method of any one of claims 129-137, wherein the ratio of the amount of the first capture oligonucleotide to the amount of the first reporter oligonucleotide is about 1 :1.

139. The method of any one of claims 129-138, wherein the ratio of the amount of the second capture oligonucleotide to the amount of the second reporter oligonucleotide is between about 2:1 to about 10,000,000:1 , or between about 10:1 to about 1 ,000,000:1 , or between about 10:1 to about 100,000:1 , or between about 100:1 to about 1 ,000,000: 1 , or between about 100: 1 to about 100,000: 1.

140. The method of any one of claims 129-139, wherein the amount of the second capture oligonucleotide in the composition or array is greater than the amount of the first capture oligonucleotide in the composition or array.

141. The method of any one of claims 129-140, wherein the ratio of the amount of the first capture oligonucleotide to the amount of the second capture oligonucleotide is between about 1 :10 to about 1 :10,000,000, or between about 1 :10 to about1 : 1 ,000,000, or between about 1 : 100 to about 1 : 1 ,000,000, or between about 1 : 100 to about 1 :100,000.

142. The method of any one of claims 129-141 , wherein the amount of the second reporter oligonucleotide in the composition or array is greater than the amount of the first reporter oligonucleotide in the composition or array.

143. The method of any one of claims 129-142, wherein the ratio of the amount of the first reporter oligonucleotide to the amount of the second reporter oligonucleotide is between about 1 :1 to about 1 :10,000, or between about 1 :1 and about 1 :1 ,000, or between about 1 :1 and about 1 :100.

144. The method of any one of claims 129-143, wherein the portion of the first reporter oligonucleotide that is complementary to the portion of the first capture oligonucleotide comprises 10-50, 20-50, 10-40, 20-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

145. The method of any one of claims 129-144, wherein the portion of the first reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the first capture oligonucleotide, optionally wherein the portion of the first reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the first capture oligonucleotide.

146. The method of any one of claims 129-145, wherein the portion of the second reporter oligonucleotide that is complementary to the portion of the second capture oligonucleotide comprises 10-50, 20-50, 10-40, 20-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

147. The method of any one of claims 129-146, wherein the portion of the second reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the second capture oligonucleotide, optionally wherein the portion of the second reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the second capture oligonucleotide.

148. The method of any one of claims 129-147, wherein the first reporter oligonucleotide comprises a first detectable label and the second reporter oligonucleotide comprises a second detectable label.

149. The method of claim 148, wherein the first detectable label and the second detectable label are the same or different.

150. The method of claim 148 or claim 149, wherein the first detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

151. The method of any one of claims 148-150, wherein the second detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

152. The method of any one of claims 148-151 , wherein the first detectable label is a first barcode and the second detectable label is a second barcode, wherein the sequence of the first barcode and the sequence of the second barcode are different.

153. The method of any one of claims 129-147, wherein detecting comprises contacting the released first reporter oligonucleotide with a first detector complex and contacting the released second oligonucleotide with a second detector complex; wherein the first detector complex comprises a first detector capture oligonucleotide hybridized to a first detector reporter oligonucleotide, wherein the first reporter oligonucleotide comprises a portion that is complementary to a portion of the first detector capture oligonucleotide; and wherein the second detector complex comprises a second detector capture oligonucleotide hybridized to a second detector reporter oligonucleotide, wherein the second reporter oligonucleotide comprises a portion that is complementary to a portion of the second detector capture oligonucleotide; under conditions suitable for hybridizing the first reporter oligonucleotide to the first detector capture oligonucleotide of the first detector copmlex and releasing the first detector reporter oligonucleotide and hybridizing the second reporter oligonucleotide to the second reporter capture oligonucleotide of the second detector complex and releasing the second detector reporter oligonucleotide; and detecting the released first detector reporter oligonucleotide and the released second detector reporter oligonucleotide.

154. The method of claim 153, wherein the first detector capture oligonucleotide is bound to the first location of a second array and the second detector capture oligonucleotide is bound to the second location of the second array.

155. The method of claim 154, wherein the first detector capture oligonucleotide is bound to microbeads at the first location of the second array and the second detector capture oligonucleotide is bound to microbeads at the second location of the second array.

156. The method of any one of claims 153-155, wherein the first detector capture oligonucleotide and the second detector capture oligonucleotide each comprises a first member of a binding pair.

157. The method of claim 156, wherein the first detector capture oligonucleotide and the second detector capture oligonucleotide each comprises biotin.

158. The method of any one of claims 153-157, wherein the portion of the first detector reporter oligonucleotide that is complementary to the portion of the first detector capture oligonucleotide comprises 10-50, 20-50, 10-40, 20-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

159. The method of any one of claims 153-158, wherein the portion of the first detector reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the first detector capture oligonucleotide, optionally wherein the portion of the first detector reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the first detector capture oligonucleotide.

160. The method of any one of claims 153-159, wherein the portion of the second detector reporter oligonucleotide that is complementary to the portion of the second detector capture oligonucleotide comprises 10-50, 20-50, 10-40, 20-40, 10-35, 10-30, 10-25, or 10-20 nucleobases.

161. The method of any one of claims 153-160, wherein the portion of the second detector reporter oligonucleotide is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the portion of the second detector capture oligonucleotide, optionally wherein the portion of the second detector reporter oligonucleotide is less than 95% or less than 90% complementary to the portion of the second detector capture oligonucleotide.

162. The method of any one of claims 153-161 , wherein the first detector reporter oligonucleotide comprises a first detectable label and the second detector reporter oligonucleotide comprises a second detectable label.

163. The method of claim 162, wherein the first detectable label and the second detectable label are the same or different.

164. The method of claim 162 or claim 163, wherein the first detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

165. The method of any one of claims 162-164, wherein the second detectable label is selected from a barcode, a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, and a quantum dot.

166. The method of any one of claims 162-165, wherein the first detectable label is a first barcode and the second detectable label is a second barcode, wherein the sequence of the first barcode and the sequence of the second barcode are different.

167. The method of any one of claims 153-166, wherein the first reporter oligonucleotide and second reporter oligonucleotide do not comprise a fluorescent label, an enzymatic label, a radiolabel, a colorimetric label, or a quantum dot.

168. The method of any one of claims 129-167, wherein the portion of the first aptamer that is complementary to the first capture oligonucleotide overlaps with and has a higher melting temperature than the portion of the first reporter oligonucleotide that is complementary to the first capture oligonucleotide, and wherein the portion of the second aptamer that is complementary to the second capture oligonucleotide overlaps with and has a higher melting temperature than the portion of the second reporter oligonucleotide that is complementary to the second capture oligonucleotide.

169. The method of any one of claims 129-168, wherein the amount of the second aptamer in the sample is greater than the amount of the first aptamer in the sample.

170. The method of any one of claims 129-169, wherein the ratio of the amount of the first aptamer to the amount of the second aptamer in the sample is between about 1 :10 to about 1 :10,000,000, or between about 1 :10 to about 1 :1 ,000,000, or between about 1 : 100 to about 1 : 1 ,000,000, or between about 1 : 100 to about 1 : 100,000.

171. The method of any one of claims 129-170, wherein the portion of the first aptamer that is complementary to the first capture oligonucleotide comprises 15-60, 15- 50, 15-40, 20-60, 20-50, 20-40, 30-60, 30-50, or 30-40 nucleobases.

172. The method of any one of claims 129-171 , wherein the portion of the first aptamer is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the portion of the first capture oligonucleotide.

173. The method of any one of claims 129-172, wherein the portion of the second aptamer that is complementary to the second capture oligonucleotide comprises 15-60, 15-50, 15-40, 20-60, 20-50, 20-40, 30-60, 30-50, or 30-40 nucleobases.

174. The method of any one of claims 129-173, wherein the portion of the second aptamer is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the portion of the second capture oligonucleotide.

175. The method of any one of claims 129-174, wherein the first aptamer and the second aptamer each independently comprise at least one, at least two, at least three, at least four, or at least five C-5 modified nucleobases.

176. The method of claim 175, wherein the modified nucleobase is selected from 5-(N-benzylcarboxamide) (“Bn”)-cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)- cytosine, 5-(N-3-phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1- naphthylmethylcarboxamide) (“Nap”)-cytosine, 5-(N-2-naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1-naphthyl-2-ethylcarboxamide) (“NE”)-cytosine, 5-(N-tyrosylcarboxamide) (”Tyr”)-cytosine, Bn-uracil, Nap-uracil, PE-uracil, 5-(N- isobutylcarboxamide) (“lb”)-uracil, 5-(N-4-fluoro-benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4-methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5- (N-3-benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3- benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3-phenylpropylcarboxamide) (“PP”)-uracil, 5-(N-morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3- indole-2-ethylcarboxamide) (“Trp”)-uracil, 5-(n-(R)-2-hydrozypropylcarboxamide) (“Thr”)-uracil, 5-(N-((1 ,1’-biphenyl)-4-yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4- phenylbenzylcarboxamide) (“PBn”)-uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)- uracil, 5-(N-3,3-diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3- phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)- uracil, BPE-cytosine, PBn-cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine.

177. The method of any one of claims 129-176, wherein the first aptamer comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30- 60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

178. The method of any one of claims 129-177, wherein the second aptamer comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30- 60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

179. The method of any one of claims 129-178, wherein the first capture oligonucleotide comprises 15-100, 20-100, 20-90, 20-80, 20-70, 20-60, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

180. The method of any one of claims 129-179, wherein the second capture oligonucleotide comprises 15-100, 20-100, 20-90, 20-80, 20-70, 20-60, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40-70, or 40-60 nucleotides.

181. The method of any one of claims 129-180, wherein the first reporter oligonucleotide comprises 10-200, 10-150, 20-200, 20-150, 30-200, 30-250, 40-200, 40-150, 50-200, 50-150, 60-200, or 60-150 nucleotides.

182. The method of any one of claims 129-181 , wherein the second reporter oligonucleotide comprises 10-200, 10-150, 20-200, 20-150, 30-200, 30-250, 40-200, 40-150, 50-200, 50-150, 60-200, or 60-150 nucleotides.

183. The composition, array, or method of any one of claims 1 -182, wherein the first reporter oligonucleotide, the second reporter oligonucleotide, and if present, the third reporter oligonucleotide, each comprises a unique molecular identifier (UMI).

184. The composition, array, or method of any one of claims 1 -183, wherein the first reporter oligonucleotide, the second reporter oligonucleotide, and if present, the third reporter oligonucleotide, each comprises a forward primer (FP) hybridization sequence and a reverse primer (RP) hybridization sequence.

185. The composition, array, or method of any one of claims 1 -184, further comprising a dummy oligonucleotide, wherein the dummy oligonucleotide comprises a portion that is complementary to the same portion of the second capture oligonucleotide to which the second reporter oligonucleotide is complementary, but wherein the dummy oligonucleotide does not comprise a detectable label.

186. The composition, array, or method of claim 185, wherein the amount of the dummy oligonucleotide is about equal to the amount of the second capture oligonucleotide minus the amount of the second reporter oligonucleotide.

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