Method for detecting sense and antisense strands in an oligonucleotide duplex - Patents.com

Abstract

Described herein are methods for detecting oligonucleotides in a sample, specifically, methods for detecting the sense and antisense strands of an oligonucleotide duplex in a sample.

Description

[Technical field]

[0001] The present disclosure relates to methods for detecting or quantifying oligonucleotides in a sample, and in particular, methods for detecting or quantifying the sense and antisense strands of an oligonucleotide duplex. [Background technology]

[0002] Nucleic acid-based therapeutic agents constitute a class of promising candidates for drug therapy, especially for biological targets that cannot be accessed by conventional therapeutic agents such as small molecule, protein-based, or antibody-based therapeutic agents. Nucleic acid-based therapeutic agents include single-stranded or double-stranded oligonucleotide molecules that inhibit DNA or RNA expression, for example, to reduce or prevent the production of abnormal proteins associated with disease. Several nucleic acid-based therapeutic agents have been approved by the US Food and Drug Administration, and many more are being investigated in clinical trials for the treatment of various diseases.

[0003] Nucleic acids are large molecules that are highly charged and rapidly degraded and cleared from the body, which can result in poor pharmacological properties. Stoddard et al. (2018) "Editorial: Nucleic Acids Research and Nucleic Acid Therapeutics." Nuc. Acids Res. 46(4): 1563-1564. As a result, pharmacokinetics, tissue targeting, and tissue accumulation are all important considerations when developing nucleic acid-based therapeutics. Sensitive and quantitative assays are needed to characterize nucleic acid pharmacokinetics. Thayer et al. (2020) "POE Immunoassay: Plate-based oligonucleotide electrochemiluminescent immunoassay for the quantification of nucleic acids in biological matrices." Scientific Reports. 10(1): 10425 (doi.org / 10.1038 / s41598-020-66829-6).

[0004] A variety of polymerase chain reaction (PCR)-based, size-exclusion chromatography (SEC), and liquid chromatography-mass spectrometry (LC-MS) methods exist for characterizing nucleic acid therapeutics, but these methods are limited by assay sensitivity and time-consuming extraction steps. Id. The unique biophysical properties of oligonucleotide therapeutics can result in atypical absorption, distribution, metabolism and elimination (ADME) processes, as well as pharmacokinetics-pharmacodynamics (PKPD) dissociations. Difficulties in understanding these relationships can lead to inefficiencies throughout the drug development process, including increased animal use, and increased risk in human clinical trials. Id. Quantification of both strands of an oligonucleotide therapeutic can be important for understanding the stability and metabolic pathways of an oligonucleotide therapeutic, and for developing models that elucidate the pharmacology of the therapeutic. Id.

[0005] Thus, there remains a need for sensitive assays for the detection or quantification of oligonucleotide therapeutic agents, for example, from samples obtained from patients. Summary of the Invention

[0006] Described herein are methods for detecting or quantifying sense and antisense strands of an oligonucleotide duplex in a sample. In one aspect, the method comprises: (a) contacting a sample with a composition comprising a set of probes, the set of probes comprising: (i) a sense probe comprising a first single-stranded oligonucleotide tag that is complementary to at least a portion of a first capture oligonucleotide immobilized on a support surface, a sense binding moiety capable of hybridizing to a nucleotide sequence of a sense strand of an oligonucleotide duplex, and a first label; and (ii) an antisense probe comprising a second single-stranded oligonucleotide tag that is complementary to at least a portion of the second capture oligonucleotide immobilized on the support surface, an antisense binding moiety capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label; the sense binding portion of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand; contacting, wherein the antisense binding portion of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand; (b) incubating the probe with a sample; (i) a sense complex comprising a sense probe hybridized to the sense strand of an oligonucleotide duplex; and (ii) forming a hybridization mixture containing a hybridization complex, the hybridization complex including an antisense complex that includes an antisense probe hybridized to the antisense strand of the oligonucleotide duplex; (c) contacting the support surface with a hybridization mixture under conditions in which the first and second oligonucleotide tags of the hybridization complex hybridize to the first and second capture oligonucleotides immobilized on the support surface, and contacting the hybridization mixture with a single-strand specific nuclease; (d) detecting or quantifying the sense and antisense strands of the oligonucleotide duplex based on the presence of the label immobilized on the support surface.

[0007] In one embodiment, (c) is (i) contacting the support surface with a hybridization mixture under conditions in which the first and second oligonucleotide tags of the hybridization complex hybridize to the first and second capture oligonucleotides on the support surface to immobilize the hybridization complex on the support surface; (ii) contacting the immobilized hybridization complex with a single-strand specific nuclease.

[0008] In one embodiment, (c) is (i) contacting the hybridization mixture with a single-strand specific nuclease to form a reaction mixture; (ii) contacting the support surface with the reaction mixture of (i) under conditions in which the first and second oligonucleotide tags of the sense and antisense probes hybridize to the first and second capture oligonucleotides immobilized on the support surface.

[0009] In one embodiment, the oligonucleotide tag of the antisense probe hybridizes to a capture oligonucleotide immobilized on a support surface. In one embodiment, the oligonucleotide tag of the antisense probe that is part of an antisense complex hybridizes to a capture oligonucleotide immobilized on a support surface. In one embodiment, the oligonucleotide tag of the antisense probe is not part of an antisense complex. In one embodiment, the oligonucleotide tag of the antisense probe is part of a hybridization complex. In one embodiment, the hybridization complex does not include the antisense strand of the oligonucleotide duplex. In one embodiment, the hybridization complex is a probe-probe complex. In one embodiment, the probe-probe complex includes a single-stranded overhang.

[0010] In one embodiment, the oligonucleotide tag of the sense probe hybridizes to a capture oligonucleotide immobilized on a support surface. In one embodiment, the oligonucleotide tag of the sense probe that is part of a sense complex hybridizes to a capture oligonucleotide immobilized on a support surface. In one embodiment, the oligonucleotide tag of the sense probe is not part of a sense complex. In one embodiment, the oligonucleotide tag of the sense probe is part of a hybridization complex. In one embodiment, the hybridization complex does not include the sense strand of the oligonucleotide duplex. In one embodiment, the hybridization complex is a probe-probe complex. In one embodiment, the probe-probe complex includes a single-stranded overhang.

[0011] In one embodiment, the sense and antisense strands of the oligonucleotide duplex each individually comprise from about 8 to about 50 nucleotides. In one embodiment, the sense and antisense strands of the oligonucleotide duplex each individually comprise from about 16 to about 30 nucleotides.

[0012] In one embodiment, the sense strand of the oligonucleotide duplex comprises DNA. In one embodiment, the sense strand of the oligonucleotide duplex comprises RNA. In one embodiment, the antisense strand of the oligonucleotide duplex comprises DNA. In one embodiment, the antisense strand of the oligonucleotide duplex comprises RNA.

[0013] In one embodiment, the oligonucleotide duplex comprises a DNA / DNA duplex. In one embodiment, the oligonucleotide duplex comprises an RNA / RNA duplex. In one embodiment, the oligonucleotide duplex comprises a DNA / RNA heteroduplex.

[0014] In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands of the oligonucleotide duplex individually comprise one or more modified nucleic acids. In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands of the oligonucleotide duplex individually comprise a 5'-conjugate or a 3'-conjugate. In one embodiment, the conjugate comprises polyethylene glycol (PEG), N-acetylgalactosamine (GalNAc), a cell penetrating peptide (CPP), α-tocopherol, an aptamer, an antibody, cholesterol, squalene, a fatty acid, or a nucleolipid.

[0015] In one embodiment, the sense and antisense strands of the oligonucleotide duplex comprise a nucleic acid sequence of a microorganism. In one embodiment, the sense and antisense strands of the oligonucleotide duplex comprise a nucleic acid sequence of a microorganism that is a component of the human microbiome. In one embodiment, the microorganism is a bacterium, a fungus, a protozoan, or a virus. In one embodiment, the microorganism is a bacterium. In one embodiment, the sense and antisense strands of the oligonucleotide duplex comprise 16S rRNA or rDNA from a bacterium.

[0016] In one embodiment, the sense binding length of the sense probe is at least one nucleotide shorter than the sense strand length of the sense strand. In one embodiment, the sense binding length is about 10 to about 16 nucleotides in length. In one embodiment, the sense binding portion of the sense probe has a 5' end that aligns with the 3' end of the sense strand of the oligonucleotide duplex.

[0017] In one embodiment, the antisense binding length of the antisense probe is at least one nucleotide shorter than the antisense strand length of the antisense strand. In one embodiment, the antisense binding length is about 10 to about 16 nucleotides in length. In one embodiment, the antisense binding portion of the antisense probe has a 5' end that aligns with the 3' end of the antisense strand of the oligonucleotide duplex.

[0018] In one embodiment, the first oligonucleotide tag has a first oligonucleotide tag length, the first capture oligonucleotide has a first capture oligonucleotide length, and the first oligonucleotide tag length is the same as the first capture oligonucleotide length. In one embodiment, the first oligonucleotide tag has a first oligonucleotide tag length, the first capture oligonucleotide has a first capture oligonucleotide length, and the first oligonucleotide tag length is shorter than the first capture oligonucleotide length.

[0019] In one embodiment, the second oligonucleotide tag has a second oligonucleotide tag length, the second capture oligonucleotide has a second capture oligonucleotide length, and the second oligonucleotide tag length is the same as the second capture oligonucleotide length.In one embodiment, the second oligonucleotide tag has a second oligonucleotide tag length, the second capture oligonucleotide has a second capture oligonucleotide length, and the second oligonucleotide tag length is shorter than the second capture oligonucleotide length.

[0020] In one aspect, the sense probe comprises DNA. In one aspect, the sense binding portion of the sense probe comprises DNA. In one aspect, the first oligonucleotide tag of the sense probe comprises DNA. In one aspect, the sense binding portion of the sense probe and the oligonucleotide tag comprise DNA. In one aspect, the sense binding portion of the sense probe comprises DNA and the oligonucleotide tag of the sense probe comprises RNA.

[0021] In one embodiment, the antisense probe comprises DNA. In one embodiment, the antisense binding portion of the antisense probe comprises DNA. In one embodiment, the first oligonucleotide tag of the antisense probe comprises DNA. In one embodiment, the antisense binding portion and the oligonucleotide tag of the antisense probe comprise DNA. In one embodiment, the antisense binding portion of the antisense probe comprises DNA and the oligonucleotide tag of the antisense probe comprises RNA.

[0022] In one aspect, the sense probe comprises RNA. In one aspect, the sense binding portion of the sense probe comprises RNA. In one aspect, the first oligonucleotide tag of the sense probe comprises RNA. In one aspect, the sense binding portion of the sense probe and the oligonucleotide tag comprise RNA. In one aspect, the sense binding portion of the sense probe comprises RNA and the oligonucleotide tag of the sense probe comprises DNA.

[0023] In one embodiment, the antisense probe comprises RNA. In one embodiment, the antisense binding portion of the antisense probe comprises RNA. In one embodiment, the first oligonucleotide tag of the antisense probe comprises RNA. In one embodiment, the antisense binding portion and the oligonucleotide tag of the antisense probe comprise RNA. In one embodiment, the antisense binding portion of the antisense probe comprises RNA and the oligonucleotide tag of the antisense probe comprises DNA.

[0024] In one embodiment, the sense probe, the antisense probe, or both, comprise one or more modified nucleic acids. In one embodiment, the one or more modified nucleotides comprise a locked nucleic acid (LNA). In one embodiment, the one or more modified nucleotides are selected from phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoroamidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or a combination thereof.

[0025] In one embodiment, the single-strand specific nuclease comprises a single-strand specific DNase. In one embodiment, the single-strand specific DNase is S1 nuclease, P1 nuclease, or Mung Bean nuclease. In one embodiment, the single-strand specific nuclease comprises a single-strand specific RNase. In one embodiment, the single-strand specific RNase is RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, PNPase, RNase PH, RNase R, RNase D, RNase T, RNaseONE, oligoribonuclease, exoribonuclease I, or exoribonuclease II.

[0026] In one embodiment, (a) to (c) are performed simultaneously. In another embodiment, (a) to (c) are performed sequentially.

[0027] In one aspect, the hybridization conditions in (b) are: (i) incubating the probe with the sample at a first temperature to denature the sense and antisense strands of the oligonucleotide duplex; (ii) incubating the probe with the denatured sense strand and denatured antisense strand of the oligonucleotide duplex at a second temperature to allow the sense probe and antisense probe to hybridize to the sense strand and antisense strand.

[0028] In one embodiment, the hybridization further comprises incubating the sense and antisense complexes at a holding temperature of about 2°C to about 8°C.

[0029] In one aspect, the hybridization conditions in (b) are: (i) incubating the probe with the sample at a first temperature of about 60° C. to about 95° C. for about 1 minute to about 15 minutes; (ii) incubating the probe with the sample at a second temperature of about 10° C. to about 65° C. for about 30 seconds to about 5 minutes; (iii) incubating the probe with the sample at a holding temperature of about 2°C to about 8°C.

[0030] In one aspect, the hybridization conditions in (b) are: (i) incubating the probe at a first temperature of about 95° C. for about 2 minutes; (ii) incubating the probe with the sample at a second temperature of about 65° C. for about 1 minute; (iii) incubating the probe with the sample at a holding temperature of about 4°C.

[0031] In one aspect, the hybridization conditions include a first temperature transition rate between steps (i) and (ii) of about 1° C. / sec to about 2° C. / sec. In one aspect, the hybridization conditions include a first temperature transition rate between steps (i) and (ii) of about 1.8° C. / sec. In one aspect, the hybridization conditions include a second temperature transition rate between steps (ii) and (iii) of about 0.05° C. / sec to about 1° C. / sec. In one aspect, the hybridization conditions include a first temperature transition rate between steps (i) and (ii) of about 0.1° C. / sec.

[0032] In one embodiment, the probe is incubated with the sample in a buffer containing diluent 54 or N-PLEX Hybridization Buffer 1 or 2.

[0033] In one aspect, the sample comprises a plurality of oligonucleotide duplexes and the composition in (a) comprises a plurality of sets of probes, each set of probes hybridizing to a unique sense or antisense strand of a unique oligonucleotide duplex.

[0034] In one embodiment, (c) comprises incubating the support surface with the sense complex and the antisense complex at a temperature of about 20° C. to about 40° C. for about 15 minutes to about 12 hours. In one embodiment, (c) comprises incubating the support surface with the sense complex and the antisense complex at a temperature of about 20° C. to about 40° C. for about 1 hour to about 2 hours. In one embodiment, the support surface is incubated with the sense complex and the antisense complex while shaking. In one embodiment, (c) comprises incubating the support surface with the sense complex and the antisense complex at a temperature of about 37° C. for about 1 hour while shaking at about 705 rpm.

[0035] In one embodiment, the composition in (a) comprises about 20 pM to about 10 nM of the sense probe.In one embodiment, the composition in (a) comprises about 20 pM to about 10 nM of the antisense probe.

[0036] In one aspect, the sample comprises a biological sample. In one aspect, the sample comprises an unprocessed biological sample. In one aspect, the sample comprises a pre-processed biological sample. In one aspect, the sample comprises a purified sample. In one aspect, the sample is purified by precipitation, centrifugation, or column chromatography. In one aspect, the sample comprises an extracted sample. In one aspect, the sample comprises naturally occurring RNase. In one aspect, the method comprises mixing the sample with an RNase inhibitor prior to (a). In one aspect, the sample comprises cell-free DNA.

[0037] In one aspect, the biological sample comprises a fluid obtained from an organism. In one aspect, the biological sample comprises whole blood, plasma, serum, urine, feces, breast milk, saliva, or amniotic fluid. In one aspect, the sample comprises an environmental sample. In one aspect, the sample comprises a manufacturing process sample.

[0038] In one embodiment, the method has a limit of detection of less than about 200 pg / mL.

[0039] In one embodiment, the support surface comprises one or more electrodes. In one embodiment, the one or more electrodes comprise carbon electrodes. In one embodiment, the one or more electrodes comprise carbon ink electrodes. In one embodiment, the one or more electrodes are comprised in a multi-well plate. In one embodiment, each well of the multi-well plate comprises an electrode.

[0040] In one embodiment, the label comprises a member of a binding pair, hi one embodiment, the label comprises biotin.

[0041] In one aspect, the label comprises an electrochemiluminescent (ECL) label. In one aspect, the method comprises generating an assay signal by contacting an electrode with an electrochemiluminescent read buffer comprising an electrochemiluminescent coreactant and applying a potential to the electrode. In one aspect, the coreactant is selected from a tertiary amine, tripropylamine, N-butyldiethanolamine, and combinations thereof.

[0042] In one aspect, a composition is provided comprising a set of probes. In one aspect, the set of probes comprises: (a) a sense probe comprising a first single-stranded oligonucleotide tag that is complementary to at least a portion of a first capture oligonucleotide, a sense binding portion capable of hybridizing to a nucleotide sequence of a sense strand of an oligonucleotide duplex, and a first label; (b) an antisense probe comprising a second single-stranded oligonucleotide tag that is complementary to at least a portion of the second capture oligonucleotide, an antisense binding moiety capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label; The sense binding portion of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand, and the antisense binding portion of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand.

[0043] In one aspect, (a) an oligonucleotide duplex comprising a sense strand and an antisense strand; (b) (i) a sense probe comprising a first single-stranded oligonucleotide tag that is complementary to at least a portion of the first capture oligonucleotide, a sense binding portion capable of hybridizing to a nucleotide sequence of a sense strand of an oligonucleotide duplex, and a first label; (ii) an antisense probe comprising a second single-stranded oligonucleotide tag that is complementary to at least a portion of the second capture oligonucleotide, an antisense binding moiety capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label; The sense binding portion of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand, and the antisense binding portion of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand.

[0044] In one embodiment, a composition is provided comprising one or more hybridization complexes. In one embodiment, the hybridization complexes comprise: (a) a sense complex comprising a sense probe hybridized to a sense strand of an oligonucleotide duplex, the sense probe comprising a first single-stranded oligonucleotide tag that is complementary to at least a portion of a first capture oligonucleotide, a sense binding moiety capable of hybridizing to a nucleotide sequence of the sense strand of the oligonucleotide duplex, and a first label, wherein the sense binding moiety of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand; (b) an antisense complex comprising an antisense probe hybridized to an antisense strand of an oligonucleotide duplex, the antisense probe comprising a second single-stranded oligonucleotide tag that is complementary to at least a portion of the second capture oligonucleotide, an antisense binding moiety capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label, wherein the antisense binding moiety of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand; and combinations thereof.

[0045] In one embodiment, the sense and antisense strands of the oligonucleotide duplex in the composition each individually comprise from about 8 to about 50 nucleotides.In one embodiment, the sense and antisense strands of the oligonucleotide duplex in the composition each individually comprise from about 16 to about 30 nucleotides.

[0046] In one embodiment, the sense strand of the oligonucleotide duplex in the composition comprises DNA. In one embodiment, the sense strand of the oligonucleotide duplex in the composition comprises RNA. In one embodiment, the antisense strand of the oligonucleotide duplex in the composition comprises DNA. In one embodiment, the antisense strand of the oligonucleotide duplex in the composition comprises RNA. In one embodiment, the oligonucleotide duplex comprises a DNA / DNA duplex. In one embodiment, the oligonucleotide duplex comprises an RNA / RNA duplex. In one embodiment, the oligonucleotide duplex comprises a DNA / RNA heteroduplex.

[0047] In one embodiment, the sense strand, the antisense strand, or both the sense strand and the antisense strand of the oligonucleotide duplex in the composition each individually comprises one or more modified nucleic acids.In one embodiment, the sense strand, the antisense strand, or both the sense strand and the antisense strand of the oligonucleotide duplex in the composition each individually comprises a 5'-conjugate or a 3'-conjugate.In one embodiment, the conjugate comprises polyethylene glycol (PEG), N-acetylgalactosamine (GalNAc), cell penetrating peptide (CPP), α-tocopherol, an aptamer, an antibody, cholesterol, squalene, a fatty acid, or a nucleolipid.

[0048] In one embodiment, the sense binding length of the sense probe in the composition is at least one nucleotide shorter than the sense strand length of the sense strand. In one embodiment, the sense binding length of the sense probe in the composition is about 10 to about 16 nucleotides in length. In one embodiment, the sense binding portion of the sense probe in the composition has a 5' end that aligns with the 3' end of the sense strand of the oligonucleotide duplex.

[0049] In one embodiment, the antisense binding length of the antisense probe in the composition is at least one nucleotide shorter than the antisense strand length of the antisense strand. In one embodiment, the antisense binding length of the antisense probe in the composition is about 10 to about 16 nucleotides in length. In one embodiment, the antisense binding portion of the antisense probe in the composition has a 5' end that aligns with the 3' end of the antisense strand of the oligonucleotide duplex.

[0050] In one embodiment, the sense probe in the composition comprises DNA. In one embodiment, the sense binding portion of the sense probe comprises DNA. In one embodiment, the first oligonucleotide tag of the sense probe comprises DNA. In one embodiment, the sense binding portion of the sense probe and the oligonucleotide tag comprise DNA. In one embodiment, the sense binding portion of the sense probe comprises DNA and the oligonucleotide tag of the sense probe comprises RNA.

[0051] In one embodiment, the antisense probe in the composition comprises DNA. In one embodiment, the antisense binding portion of the antisense probe comprises DNA. In one embodiment, the first oligonucleotide tag of the antisense probe comprises DNA. In one embodiment, the antisense binding portion and the oligonucleotide tag of the antisense probe comprise DNA. In one embodiment, the antisense binding portion of the antisense probe comprises DNA and the oligonucleotide tag of the antisense probe comprises RNA.

[0052] In one embodiment, the sense probe in the composition comprises RNA. In one embodiment, the sense binding portion of the sense probe comprises RNA. In one embodiment, the first oligonucleotide tag of the sense probe comprises RNA. In one embodiment, the sense binding portion of the sense probe and the oligonucleotide tag comprise RNA. In one embodiment, the sense binding portion of the sense probe comprises RNA and the oligonucleotide tag of the sense probe comprises DNA.

[0053] In one embodiment, the antisense probe in the composition comprises RNA. In one embodiment, the antisense binding portion of the antisense probe comprises RNA. In one embodiment, the first oligonucleotide tag of the antisense probe comprises RNA. In one embodiment, the antisense binding portion and the oligonucleotide tag of the antisense probe comprise RNA. In one embodiment, the antisense binding portion of the antisense probe comprises RNA and the oligonucleotide tag of the antisense probe comprises DNA.

[0054] In one embodiment, the sense probe, the antisense probe, or both probes in the composition comprise one or more modified nucleic acids. In one embodiment, the one or more modified nucleotides comprise locked nucleic acids (LNA). In one embodiment, the one or more modified nucleotides are selected from phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or combinations thereof.

[0055] In one embodiment, the label in the composition comprises a member of a binding pair. In one embodiment, the label comprises biotin. In one embodiment, the label in the composition comprises an electrochemiluminescent label.

[0056] In one aspect, kits are provided for carrying out the methods described herein. [Brief description of the drawings]

[0057] [Figure 1A] FIG. 1 is a schematic diagram of an antisense binding complex in which an antisense binding probe described herein is hybridized to an antisense oligonucleotide sequence. [Figure 1B] FIG. 1 is a schematic diagram of a sense binding complex in which a sense binding probe described herein is hybridized to a sense oligonucleotide sequence. [Figure 2A]FIG. 2 is a schematic diagram of an antisense binding complex formed between the antisense binding strand of an oligonucleotide duplex and a "short" antisense binding probe. [Figure 2B] FIG. 2 is a schematic diagram of a sense binding complex formed between the sense binding strand of an oligonucleotide duplex and a "short" sense binding probe. [Figure 2C] FIG. 1 is a schematic diagram showing a "non-productive" binding complex formed between a "short" sense binding probe and a "short" antisense binding probe. [Figure 3A] FIG. 2 is a schematic diagram of an antisense binding complex formed between the antisense binding strand of an oligonucleotide duplex and a "full-length" antisense binding probe. [Figure 3B] FIG. 2 is a schematic diagram of a sense binding complex formed between the sense binding strand of an oligonucleotide duplex and a "full-length" sense binding probe. [Figure 3C] FIG. 1 is a schematic diagram showing a "non-productive" binding complex formed between a "full-length" sense binding probe and a "full-length" antisense binding probe. [Figure 4A] FIG. 1 is a schematic diagram of an oligonucleotide tag of a probe hybridized to a capture oligonucleotide immobilized on a support surface, the probe being part of an antisense or sense complex with a single-stranded overhang. [Figure 4B] FIG. 1 is a schematic diagram of an oligonucleotide tag of a probe hybridized to a capture oligonucleotide immobilized on a support surface, the probe being part of an antisense or sense complex that does not have a single-stranded overhang. [Figure 4C] FIG. 1 is a schematic diagram of an oligonucleotide tag of a probe hybridized to a capture oligonucleotide immobilized on a support surface, the probe not being part of an antisense or sense complex. [Figure 4D] FIG. 1 is a schematic diagram of an oligonucleotide tag of a probe hybridized to a capture oligonucleotide immobilized on a support surface, the probe being part of a probe-probe complex that includes a single-stranded overhang. [Figure 4E] FIG. 1 is a schematic diagram of an oligonucleotide tag of a probe hybridized to a capture oligonucleotide immobilized on a support surface, where the probe is part of a probe-probe complex that does not include a single-stranded overhang. [Figure 5A] 1 is a graph showing ECL signal curves for 1× and 4× concentrations of antisense (AS) probe hybridized in diluent 54. [Figure 5B] FIG. 1 is a graph showing ECL signal curves for 1× and 4× concentrations of antisense (AS) probe hybridized in hybridization buffer. [Figure 6A] 1 is a graph showing ECL signal curves for 1× and 4× concentrations of sense (SS) probe hybridized in diluent 54. [Figure 6B] FIG. 1 is a graph showing ECL signal curves at 1× and 4× concentrations of sense (SS) probe hybridized in hybridization buffer. [Figure 7A] 1 is a graph showing ECL signal curves for 16-mer (FL) and 12-mer antisense (AS) probes hybridized in diluent 54. [Figure 7B] FIG. 1 is a graph showing ECL signal curves for 16-mer (FL) and 12-mer antisense (AS) probes hybridized in hybridization buffer. [Figure 8A] Graph showing ECL signal curves with a 16-mer (FL) probe hybridized to the antisense (AS) strand alone or in a heteroduplex. [Figure 8B] FIG. 1 is a graph showing ECL signal curves with a 12-mer probe hybridized to the antisense (AS) strand alone or in a heteroduplex. [Figure 9A] Graph showing ECL signal curves with a 16-mer (FL) probe hybridized to the sense (SS) strand alone or in a heteroduplex. [Figure 9B]Graph showing ECL signal curves with a 12-mer probe hybridized to the sense (SS) strand alone or in a heteroduplex. [Figure 10A] FIG. 1 is a graph showing ECL signal curves with a 12-mer probe hybridized to the antisense (AS) strand alone or in a heteroduplex. [Figure 10B] Graph showing ECL signal curves with a 12-mer probe hybridized to the sense (SS) strand alone or in a heteroduplex. [Figure 11A] 1 is a graph showing ECL signal curves with a 16-mer (FL) probe hybridized to the antisense (AS) strand under long or short hybridization conditions. [Figure 11B] FIG. 1 is a graph showing ECL signal curves with a 12-mer probe hybridized to the antisense (AS) strand under long or short hybridization conditions. [Figure 12A] 1 is a graph showing ECL signal curves for individual sense (SS) strands in diluent 54 and in plasma. [Figure 12B] 1 is a graph showing ECL signal curves for the sense (SS) strand of the heteroduplex in diluent 54 and in plasma. [Figure 13A] 1 is a graph showing ECL signal curves for individual sense (SS) strands in diluent 54 or brain lysate. [Figure 13B] 1 is a graph showing ECL signal curves for the sense (SS) strand of the heteroduplex in diluent 54 or brain lysate. [Figure 14A] FIG. 1 is a graph showing ECL signal curves for individual antisense (AS) strands with or without LNA. [Figure 14B] FIG. 1 is a graph showing ECL signal curves for the antisense (AS) strand of a heteroduplex with or without LNA. [Figure 15A] 1 is a graph showing ECL signal curves with antisense (AS) LNA probes at various concentrations. [Figure 15B] 1 is a graph showing ECL signal curves with sense (SS) LNA probes at various concentrations. [Figure 16A] FIG. 13 is a graph showing ECL signal curves for multiplex detection of individual antisense (AS) strands using unmodified or LNA-modified 12-mer probes. [Figure 16B] FIG. 1 is a graph showing ECL signal curves for multiplex detection of antisense (AS) strands of heteroduplexes using unmodified or LNA-modified 12-mer probes. [Figure 17A] FIG. 13 is a graph showing ECL signal curves for multiplex detection of individual sense (SS) strands using unmodified or LNA-modified 12-mer probes. [Figure 17B] FIG. 1 is a graph showing ECL signal curves for multiplex detection of the sense (SS) strand of a heteroduplex using unmodified or LNA-modified 12-mer probes. [Figure 18A] 1 is a graph showing ECL signal curves for individual antisense (AS) strands detected using a FL probe, a 14-mer probe, a 13-mer probe, and a 12-mer probe. [Figure 18B] 1 is a graph showing ECL signal curves for individual sense (SS) strands detected using a FL probe, a 14-mer probe, a 13-mer probe, and a 12-mer probe. [Figure 19A] 1 is a graph showing ECL signal curves for the antisense (AS) strand of a heteroduplex detected using a FL probe, a 14-mer probe, a 13-mer probe, and a 12-mer probe. [Figure 19B] 1 is a graph showing ECL signal curves for the sense (SS) strand of a heteroduplex detected using a FL probe, a 14-mer probe, a 13-mer probe, and a 12-mer probe. [Figure 20A] 1 is a graph showing ECL signal curves for the antisense (AS) strand of a heteroduplex when detected using a combination of antisense (AS) and sense (SS) probe lengths. [Figure 20B]1 is a graph showing ECL signal curves for the sense (SS) strand of a heteroduplex when detected using a combination of antisense (AS) and sense (SS) probe lengths. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] A.Definition Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular, e.g., "a" or "an" includes the plural, e.g., "one or more" or "at least one," and the term "or" can mean "and / or," unless otherwise specified. The terms "including," "includes," and "included" are not limiting. Ranges provided herein of any type include all values ​​within the particular range described, as well as values ​​per endpoint in the particular range. As used herein, ranges expressed using the term "between" include the endpoints of the range. Thus, for example, a range of 50°C to 70°C includes 50°C to 70°C, i.e., it includes the endpoints of 50°C and 70°C.

[0059] As used herein, the term "about" is used to modify, for example, the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, and ranges of components in a composition used in describing the present invention. The term "about" refers to variations in the amount expressed as a number that may occur, for example, through typical measuring and handling procedures used to make a compound, composition, concentrate, or formulation, through inadvertent errors in these procedures, through differences in the manufacture, source, or purity of starting materials or components used to carry out the method, and other similar considerations. The term "about" also encompasses amounts that differ due to changes over time in a formulation having a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation having a particular initial concentration or mixture. When modified by the term "about," the claims appended hereto include such equivalents.

[0060] Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein, and the techniques thereof, are those well known and commonly used in the art. Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted one-letter codes.

[0061] The term "nucleotide" refers to a monomeric unit that includes a nucleobase, a sugar, and one or more internucleotide bridges. As used herein, the term nucleotide includes naturally occurring and modified nucleotides. Naturally occurring nucleotides include guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U), as well as naturally occurring base analogs. In deoxyribonucleic acid (DNA), the sugar is deoxyribose. In ribonucleic acid (RNA), the sugar is ribose. The term "modified nucleotide" refers to a nucleotide that includes a modification in the nucleobase, sugar, or internucleotide linkage, where the modified nucleotide is still capable of base pairing with a complementary naturally occurring or modified nucleotide. The term "polynucleotide" refers to a polymer of two or more nucleotides covalently linked to each other by internucleoside linkages.

[0062] As used herein, the term "oligonucleotide" refers to a short polymer comprising two or more nucleotides, generally about 5 to about 100 nucleotides, covalently linked by an internucleoside bond. In one embodiment, an oligonucleotide is a polymer that is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 nucleotides in length up to about 30, 35, 40, 45, 50, or 100 nucleotides in length, or about 8 to about 50 nucleotides in length, about 10 to about 40 nucleotides in length, about 12 to about 30 nucleotides in length, about 18 to about 30 nucleotides in length. As used herein, the term oligonucleotide can refer to a single-stranded oligonucleotide or a double-stranded oligonucleotide, or the individual oligonucleotide strands of a double-stranded oligonucleotide. In one embodiment, the term "oligonucleotide" refers to a double-stranded oligonucleotide therapeutic agent.

[0063] An "oligonucleotide therapeutic" is an oligonucleotide that comprises at least one strand that is at least partially complementary to and can hybridize to a target nucleic acid. In one embodiment, the oligonucleotide therapeutic comprises a sense strand and an antisense strand. In one embodiment, the oligonucleotide therapeutic can hybridize to a target nucleic acid and modulate the expression or amount of the target nucleic acid. The term "modulate" can include increasing or decreasing the expression or amount of the target nucleic acid. The term "expression" refers to the process by which the information in a gene is used to produce a protein, including, but not limited to, transcription, splicing, post-transcriptional modification, and translation. In one embodiment, the oligonucleotide therapeutic increases the expression or amount of the target nucleic acid. In one embodiment, the oligonucleotide agent decreases the expression or amount of the target nucleic acid. In one embodiment, the oligonucleotide is chemically synthesized and purified or isolated. In one embodiment, the oligonucleotide is made by solid phase chemical synthesis. Methods for making oligonucleotides, including, for example, sense and antisense oligonucleotides, probes, tags, or capture oligonucleotides as described herein, are known.

[0064] As used herein, "base pairing" refers to specific hydrogen bonding between purines and pyrimidines that results in the formation of double-stranded oligonucleotides. In DNA, adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C). In RNA, adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C). Although not limited to any particular mechanism, the most common mechanism of base pairing involves hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding.

[0065] The term "chimeric" refers to a compound having at least two chemically distinct regions. In embodiments, each region has multiple subunits. As used herein, the term "chimeric probe" includes linked single-stranded DNA and / or RNA derived from two or more biological sources.

[0066] "Complementary" refers to nucleic acid molecules or oligonucleotides that interact by the formation of hydrogen bonds, for example, according to the Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bond base pairing models. Hybridization can occur between two complementary DNA molecules (DNA-DNA hybridization), between two RNA molecules (RNA-RNA hybridization), or between complementary DNA and RNA molecules (DNA-RNA hybridization). When used in relation to nucleotides, the term "complementary" refers to a pair of nucleotides, including, for example, purines and pyrimidines, that can base pair with each other. Complementary pairs of nucleotides can include pairs of naturally occurring nucleotides, pairs of modified nucleotides, or pairs that include naturally occurring and modified nucleotides. When used in relation to oligonucleotides, the term "complementary" means that a nucleotide or a portion thereof of one oligonucleotide can hydrogen bond with a nucleotide or a portion thereof of another oligonucleotide when the complementary nucleotides are aligned. Hybridization can occur between a short nucleotide sequence that is complementary to a portion of a longer nucleotide sequence. Although hybridization can occur between sequences that do not have 100% "sequence complementarity" (i.e., sequences in which less than 100% of the nucleotides align based on a base pairing model such as the Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bond base pairing model), sequences with lower sequence complementarity are less stable and less likely to hybridize than sequences with higher sequence complementarity. In one embodiment, the nucleotides of the complementary sequence have 100% sequence complementarity based on the Watson-Crick model (i.e., each nucleotide of one oligonucleotide strand or region can hydrogen bond with each nucleotide of a second oligonucleotide strand or region). In another embodiment, the nucleotides of the complementary sequence have at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity based on the Watson-Crick model.In one embodiment, "substantial complementarity" refers to sequences that are partially complementary and can hybridize under physiologically relevant conditions. In one embodiment, "substantial complementarity" refers to sequences that are partially complementary and can hybridize under stringent conditions. In one embodiment, complementarity refers to the complementarity between two oligonucleotides of a double-stranded oligonucleotide therapeutic. In one embodiment, complementarity refers to the complementarity between a single-stranded oligonucleotide therapeutic and a single-stranded oligonucleotide probe. In one embodiment, complementarity refers to the complementarity between a single-stranded oligonucleotide tag and a single-stranded capture oligonucleotide. In one embodiment, complementarity refers to the complementarity between a single-stranded oligonucleotide and a chimeric probe.

[0067] Whether two complementary sequences hybridize can depend on the stringency of the hybridization conditions, which can vary depending on conditions such as temperature, solvent, ionic strength, and other parameters. The stringency of the hybridization conditions can be selected to provide for the selective formation or maintenance of a desired hybridization product of two complementary nucleic acid sequences in the presence of other potentially cross-reacting or interfering sequences. Stringent conditions are sequence-dependent - typically, longer complementary sequences hybridize specifically at higher temperatures than shorter complementary sequences. In general, stringent hybridization conditions are determined by the thermal melting point (T) for a particular nucleotide sequence at a defined ionic strength, concentration of chemical denaturant, pH, and concentration of hybridization partner. m) (i.e., the temperature at which 50% of the sequence hybridizes to a substantially complementary sequence). In general, nucleotide sequences having a higher percentage of G and C bases will hybridize under more stringent conditions than nucleotide sequences having a lower percentage of G and C bases. In general, stringency can be increased by increasing the temperature, increasing the pH, decreasing the ionic strength, or increasing the concentration of chemical nucleic acid denaturants (e.g., formamide, dimethylformamide, dimethylsulfoxide, ethylene glycol, propylene glycol, and ethylene carbonate). Stringent hybridization conditions typically include salt concentrations of less than about 1 M, about 500 mM, or about 200 mM, hybridization temperatures of greater than about 20° C., about 30° C., about 40° C., about 60° C., or about 80° C., and chemical denaturing agent concentrations of greater than about 10%, about 20%, about 30%, about 40%, or about 50%. Because many factors can affect the stringency of hybridization, the combination of parameters may be more important than the absolute value of any parameter alone.

[0068] In one aspect, complementarity refers to the complementarity between the oligonucleotide and the target nucleic acid sequence. In one aspect, the target nucleic acid comprises DNA or RNA. In one aspect, the target RNA comprises mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA, mature microRNA, or promoter-directed RNA. In one aspect, the target nucleic acid is an mRNA transcribed from a cellular gene or a gene whose expression is associated with a particular disorder or disease. In one aspect, the target nucleic acid is a nucleic acid molecule from an infectious agent. In one aspect, the target nucleic acid is a viral or bacterial nucleic acid.

[0069] As used herein, "hybridize", "hybridizing" or "hybridization" refers to base pairing between two complementary oligonucleotides. In one embodiment, the single-stranded oligonucleotide strand of the double-stranded oligonucleotide therapeutic agent can hybridize to a target nucleic acid. In one embodiment, the two single-stranded oligonucleotide strands of the double-stranded oligonucleotide hybridize to each other. In one embodiment, the oligonucleotide probe hybridizes to the single-stranded oligonucleotide strand of the double-stranded oligonucleotide. In one embodiment, the single-stranded oligonucleotide tag hybridizes to the single-stranded capture oligonucleotide. "Specifically hybridize" refers to hybridization between two complementary oligonucleotides that occurs with higher affinity and without significant cross-hybridization with other oligonucleotides in the sample. In one embodiment, the complementary oligonucleotides specifically hybridize under physiologically relevant conditions, such as those found in the cytoplasm of a cell. In another embodiment, the complementary oligonucleotides specifically hybridize under stringent hybridization conditions.

[0070] As used herein, "oligonucleotide duplex" refers to a double-stranded oligonucleotide formed by hybridization of two single-stranded oligonucleotides that are at least partially complementary to each other and hybridize to each other through base pairing between complementary nucleobases. In one embodiment, at least one strand of the oligonucleotide duplex comprises DNA. In one embodiment, at least one strand of the oligonucleotide duplex comprises RNA. In one embodiment, both strands of the oligonucleotide duplex comprise DNA. In one embodiment, both strands of the oligonucleotide duplex comprise RNA. In one embodiment, the double-stranded oligonucleotide is a heteroduplex that comprises one strand that is DNA and one strand that is RNA. In one embodiment, the sugar-phosphate backbones of the two oligonucleotide strands of the oligonucleotide duplex are oriented in opposite directions (i.e., one strand extends from 5' to 3' and the other extends from 3' to 5'), which is referred to as "antiparallel". In one embodiment, the two oligonucleotide strands of the oligonucleotide duplex are the same length as each other, such that the oligonucleotide duplex is double-stranded over its entire length, i.e., the oligonucleotide duplex has blunt ends. In one embodiment, the two oligonucleotide strands of the oligonucleotide duplex are the same length as each other, but are aligned such that the oligonucleotide duplex is not double-stranded over its entire length, i.e., the oligonucleotide duplex has a single-stranded 3' overhang or a single-stranded 5' overhang at both ends of the duplex. In one embodiment, the two oligonucleotide strands of the oligonucleotide duplex are different lengths from each other, such that the oligonucleotide duplex is not double-stranded over its entire length, i.e., the oligonucleotide duplex has a single-stranded 3' overhang or a single-stranded 5' overhang at one or both ends of the oligonucleotide duplex. In one embodiment, the single-stranded overhang is about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2 nucleotides. In one embodiment, the oligonucleotide duplex has one blunt end and one end that includes a single-stranded overhang.The term "length," when used in reference to oligonucleotides, refers to the number of nucleotide residues in the polymer backbone of a single-stranded oligonucleotide.

[0071] As used herein, the term "overhang" refers to a double-stranded oligonucleotide in which at least one end of one strand is longer than the corresponding end of the other strand. In one embodiment, the single-stranded overhang is located at the 3'-end of one or both strands of the double-stranded oligonucleotide. In one embodiment, the single-stranded overhang is located at the 5'-end of one or both strands of the double-stranded oligonucleotide. In one embodiment, the single-stranded overhang comprises about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides. In one embodiment, one end of the double-stranded oligonucleotide is blunt and the other end comprises a 3' or 5' overhang. In one embodiment, both ends of the double-stranded oligonucleotide comprise single-stranded overhangs.

[0072] The term "antisense" refers to an oligonucleotide having a nucleic acid sequence that is inverted with respect to the orientation required for transcription of a target nucleic acid, so that the antisense oligonucleotide can hybridize to the target nucleic acid, for example, via Watson-Crick base pairing. The "sense strand" of an oligonucleotide duplex is complementary to the antisense strand, and is therefore "sense" to at least a portion of the target nucleic acid. In one embodiment, the antisense strand and the sense strand of an oligonucleotide duplex are at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% complementary to each other.

[0073] Single-stranded oligonucleotides have "direction" or "directionality" because adjacent nucleotides are joined by internucleoside linkages, such as phosphodiester linkages, between their 5' and 3' carbon atoms, such that the terminal 5' and 3' carbons are exposed at either end of the oligonucleotide, which may be referred to as the 5'-(phosphoryl) and 3'-(hydroxyl) ends of the molecule.

[0074] The term "identical" means that the oligonucleotide sequences contain identical nucleobases at the same positions over the comparison window. The term "sequence identity percentage" can be determined by comparing two aligned sequences over the comparison window, determining the number of positions where identical nucleobases are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The comparison window can include the full length sequence or can be a subportion of a larger sequence. Various methods and algorithms are known for determining the percent identity between two or sequences, including, but not limited to, MEGALIGN (DNASTAR, Inc. Madison, Wis.), FASTA, BLAST, or ENTREZ.

[0075] In one aspect, the nucleotides of the oligonucleotides described herein include structural analogs with non-naturally occurring chemical structures that can also participate in hybridization reactions. In one example, a nucleotide or nucleic acid can include chemical modifications that provide it with a reactive functional group that can link it to a label or link it to a label, for example, through the use of an amine- or thiol-modified nucleotide base, phosphate, or sugar. The term "reactive functional group" refers to an atom or a linkage of atoms that can undergo further chemical reaction to form, for example, a covalent bond with another functional group. Examples of reactive functional groups include, but are not limited to, amino, thiol, hydroxy, and carbonyl groups. In one aspect, a reactive functional group includes a thiol group. Labels that can be linked to a nucleotide or nucleic acid via these chemical modifications include, but are not limited to, detectable moieties such as biotin, haptens, fluorophores, and electrochemiluminescence (ECL) labels.

[0076] The term "modified oligonucleotide" refers to an oligonucleotide that contains at least one nucleoside modification, such as a sugar modification or a nucleobase modification, or an internucleoside linkage modification. In one aspect, the modified oligonucleotide contains one or more modifications, including, but not limited to, phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), phosphorothioate constrained ethyl (cEt), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or combinations thereof. "Locked nucleic acid nucleoside" or "LNA" refers to a nucleoside that contains a bicyclic sugar moiety with a 4'-CH2-O-2' bridge. "Phosphorothioate" refers to an internucleoside linkage in which one of the non-bridging oxygens is replaced by sulfur.

[0077] The term "conjugate" refers to an atom or group of atoms that is directly or indirectly attached to an oligonucleotide. In one aspect, the conjugate is connected to the oligonucleotide via a stable or cleavable linker. In one aspect, the conjugate modifies one or more properties of the oligonucleotide to which it is attached, including but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, or clearance properties. Examples of conjugates include, but are not limited to, polyethylene glycol (PEG), N-acetylgalactosamine (GalNAc), membrane-permeable peptides (CPPs), vitamin E (also known as α-tocopherol), aptamers, antibodies, cholesterol or cholesterol derivatives, squalene, fatty acids, nucleolipids, and globular nucleic acids.

[0078] The term "nuclease" refers to an enzyme, e.g., a hydrolase, that can cleave the backbone of an oligonucleotide polymer. In one aspect, a nuclease is a phosphodiesterase that cleaves phosphodiester bonds in the backbone of an oligonucleotide. "Ribonuclease" or "RNase" refers to an enzyme that preferentially cleaves ribonucleic acid (RNA). "Deoxyribonuclease" or "DNase" refers to an enzyme that preferentially cleaves deoxyribonucleic acid (DNA). In one aspect, a nuclease is a "single-strand specific nuclease" that preferentially cleaves the single-stranded region of a single-stranded oligonucleotide or a double-stranded oligonucleotide. A single-strand specific RNase is an enzyme that preferentially cleaves single-stranded RNA. A single-strand specific DNase is an enzyme that preferentially cleaves single-stranded DNA.

[0079] "Target nucleic acid" refers to a nucleic acid of interest with a known sequence to which an oligonucleotide is designed to hybridize. In one aspect, a target nucleic acid is a nucleic acid with a known sequence to which an oligonucleotide therapeutic is designed to hybridize. In one aspect, a target nucleic acid is a sequence found in DNA or RNA of a prokaryotic or eukaryotic organism. In one aspect, a target nucleic acid includes miRNA, therapeutic RNA, mRNA, RNA virus, or a combination thereof. In one aspect, hybridization of an oligonucleotide with a target nucleic acid in a cell changes the activity of a gene expressed by the cell. In one aspect, hybridization of an oligonucleotide with a target nucleic acid increases the activity of a gene. In one aspect, hybridization of an antisense oligonucleotide with a target nucleic acid decreases the activity of a gene.

[0080] In one embodiment, the target nucleic acid comprises 16S ribosomal DNA (16S ribosomal DNA, 16S rDNA) or 16 ribosomal RNA (16S rRNA, 16S rRNA). 16s rRNA is the ribosomal RNA component of the small subunit of the prokaryotic ribosome, which is involved in the essential process of converting genetic messages into functional cellular components through the translation of mRNA into proteins. The gene 16s rDNA encodes the 16s rRNA sequence. The 16S rRNA gene is conserved in bacteria and contains hypervariable regions that can provide species-specific signature sequences, and is widely used in bacterial identification and phylogenetic, identification, classification, and quantification studies.

[0081] The term "subject" or "patient" refers to an organism to which an oligonucleotide composition is administered for experimental, diagnostic, prophylactic, or therapeutic purposes, including, but not limited to, animals, e.g., mammals such as mice, rats, rabbits, non-human primates, and humans, insects, worms, and plants. In one aspect, a subject may be suffering from or susceptible to a disease or disorder.

[0082] As used herein, the term "human microbiome" refers to the collection of all microorganisms, such as bacteria, fungi, viruses, and their genes, that naturally live on and in the human body. In one aspect, the microorganisms of the human microbiome live on or in human organs, tissues, and bodily fluids, including the skin, mammary glands, nasal passages, semen, uterus, ovarian follicles, lungs, saliva, oral mucosa, conjunctiva, bile duct, and gastrointestinal tract. In one aspect, the human microbiome consists of commensal and symbiotic microorganisms that do no harm to humans. In one aspect, the human microbiome consists of microorganisms that are beneficial to the human body. In one aspect, the human microbiome consists of microorganisms that are harmful to the human body. In one aspect, the human microbiome consists of microbial communities that are both beneficial and harmful to the human body. In one aspect, the human microbiome consists of microorganisms that are mutualistic, where both the human body and the microflora benefit.

[0083] "Probe" refers to a reagent that comprises a single-stranded oligonucleotide sequence that can hybridize to one strand of an oligonucleotide duplex. In one aspect, a probe comprises a single-stranded oligonucleotide sequence that can hybridize to the sense or antisense strand of an oligonucleotide duplex. A "sense probe" is an oligonucleotide that comprises a single-stranded oligonucleotide sequence that can hybridize to the sense strand of an oligonucleotide duplex. An "antisense probe" is an oligonucleotide that comprises a single-stranded oligonucleotide sequence that can hybridize to the antisense strand of an oligonucleotide duplex. In one aspect, a probe comprises a single-stranded oligonucleotide sequence that is complementary or substantially complementary to the sense or antisense strand of an oligonucleotide duplex. In one aspect, a probe comprises an oligonucleotide tag (which can be referred to as a targeting sequence) that is complementary to the sequence of a capture oligonucleotide. A probe can comprise DNA or RNA, or a combination of DNA and RNA sequences, and can comprise one or more modified nucleotides or modified internucleotide linkages. A probe can be prepared by any suitable method known in the art, including, but not limited to, chemical or enzymatic synthesis.

[0084] "Linker" refers to one or more atoms that connect one chemical moiety to another. In one aspect, the linker attaches a reactive functional group or label to the oligonucleotide. The linker can be a nucleotide or non-nucleotide compound that contains one or more atoms, for example, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 atoms to about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 atoms, and can include atoms such as carbon, oxygen, sulfur, nitrogen, and phosphorus, and combinations thereof. Examples of linkers include low molecular weight groups such as amide groups, ester groups, carbonate groups, and ether groups, and high molecular weight linking groups such as polyethylene glycol (PEG) chains and alkyl chains. The linker can include one or more atoms, units, or molecules.

[0085] "Label" refers to a chemical group or moiety that has a detectable physical property or that can cause a chemical group or moiety to exhibit a detectable physical property, such as an enzyme that catalyzes the conversion of a substrate to a detectable product. Labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other methods. Examples of labels include, but are not limited to, radioisotopes, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, electrochemiluminescent moieties, magnetic particles, and bioluminescent moieties. In another aspect, the label is a compound that is a member of a binding pair, where a first member of the binding pair (which can be referred to as a "primary binding reagent") is attached to a substrate, e.g., an oligonucleotide, and the other member of the binding pair (which can be referred to as a "secondary binding reagent") has a detectable physical property or is attached to a moiety that has a detectable physical property. Non-limiting examples of binding pairs include biotin and streptavidin or avidin, complementary oligonucleotides, haptens and hapten binding partners, and antibody / antigen binding pairs.

[0086] As used herein, "concurrently" when used in connection with the method steps described herein refers to a method in which the steps are performed substantially simultaneously, i.e., the performance of at least a portion of one method step overlaps in time with the performance of a portion of another method step. "Concurrently" does not require precise simultaneous activity, i.e., it is not required that all method steps start or end at the same time. In one aspect, "concurrently" can mean that all reagents required for a method step are combined in the same reaction mixture, such that the reactions occur in the same reaction volume or during the same incubation period.

[0087] As used herein, "sequentially" when used in connection with the method steps described herein refers to a method in which the steps are performed at different times, e.g., separate events occur in the performance of the method. In one aspect, the sequential steps are performed during separate incubation periods. In one aspect, the sequential steps are performed in different reaction mixtures. In one aspect, the sequential method steps are performed at different times. In one aspect, the sequential method steps are performed at different times but in the same reaction cell or on the same surface.

[0088] "Capture oligonucleotide" refers to an oligonucleotide reagent that can be immobilized on a support surface and is designed to hybridize to a complementary oligonucleotide tag (and thus capture on the surface). In one aspect, the capture oligonucleotide is a single-stranded sequence that can selectively hybridize to a single-stranded oligonucleotide tag present on an oligonucleotide probe, for example, under stringent hybridization conditions. The capture oligonucleotide can be provided in solid form (e.g., lyophilized), in solution, or immobilized on a support surface, for example, on a particle (e.g., microparticle, bead) or array.

[0089] "Detection" can refer to detecting or quantifying the presence of a substance, such as an oligonucleotide, based on the presence or absence of a label. In one aspect, "detecting" refers to the process by which the presence or absence of a substance, such as an oligonucleotide, is determined. In one aspect, "quantifying" refers to the process by which the amount of a substance, such as an oligonucleotide, is determined.

[0090] "Corresponding" can be used to refer to the relationship between a capture oligonucleotide and an oligonucleotide tag, where the oligonucleotide tag is designed to specifically bind to a particular capture oligonucleotide sequence under stringent hybridization conditions. In one embodiment, an oligonucleotide tag specifically binds to its corresponding capture oligonucleotide under stringent conditions and does not bind or cross-react with other capture oligonucleotides. In one embodiment, an oligonucleotide tag specifically binds to its corresponding capture oligonucleotide under stringent conditions and does not bind or cross-react with other capture oligonucleotides in the array. In one embodiment, an oligonucleotide tag is a single-stranded oligonucleotide having a sequence that is complementary to at least a portion of the sequence of its "corresponding" capture oligonucleotide. In one embodiment, the nucleotides of the "corresponding" oligonucleotide tag sequence and the capture oligonucleotide sequence have 100% sequence complementarity based on the Watson-Crick model. In another embodiment, the nucleotides of the corresponding sequences have at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity based on the Watson-Crick model.

[0091] "Corresponding" can be used to refer to the relationship between the sense binding portion of a sense probe or the antisense binding portion of an antisense probe and the sense or antisense strand of an oligonucleotide duplex, respectively. In one embodiment, the sense binding portion of a sense probe specifically binds to its corresponding sense strand of an oligonucleotide duplex and does not bind or cross-react with the sense strand of other oligonucleotide duplexes in the sample, the antisense strand of an oligonucleotide duplex, or the oligonucleotide tag in the sample. In one embodiment, the antisense binding portion of an antisense probe specifically binds to its corresponding antisense strand of an oligonucleotide duplex and does not bind or cross-react with the antisense strand of other oligonucleotide duplexes in the sample, the sense strand of an oligonucleotide duplex, or the oligonucleotide in the sample. In one embodiment, the "corresponding" sense binding portion or antisense binding portion and the nucleotide of a sense oligonucleotide or antisense oligonucleotide, respectively, have 100% sequence complementarity based on the Watson-Crick model. In another embodiment, the nucleotides of the "corresponding" sense or antisense binding portion and the sense or antisense oligonucleotide, respectively, have at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity based on the Watson-Crick model.

[0092] "Cross-react" or "cross-reactivity" refers to the ability of an oligonucleotide sequence to hybridize to two or more other oligonucleotide sequences in a sample. In one aspect, the term "cross-react" refers to the ability of a first oligonucleotide sequence to hybridize to a second oligonucleotide sequence in a sample, where the second oligonucleotide sequence is not complementary or substantially not complementary to the first oligonucleotide sequence. In one aspect, the term "cross-react" or "cross-reactivity" refers to the ability of a capture oligonucleotide to hybridize to two or more oligonucleotide tags or two or more tagged target nucleotide sequences in a sample. In one aspect, a cross-reactive capture oligonucleotide hybridizes to one or more oligonucleotide tags in a sample under stringent capture hybridization conditions. "Non-cross-reactive" or "non-cross-reacting" refers to the ability of a first oligonucleotide sequence to hybridize only to a particular oligonucleotide sequence in a sample, e.g., a first oligonucleotide sequence to hybridize only to its corresponding complementary sequence in a sample. In one aspect, the term "non-cross-reactive" refers to the ability of a capture oligonucleotide to hybridize to only one oligonucleotide tag in a sample containing two or more oligonucleotide tags or two or more tagged target nucleotide sequences. In one aspect, a non-cross-reactive capture oligonucleotide hybridizes to only one oligonucleotide tag in a sample under stringent hybridization conditions. In one aspect, non-cross-reactive means that the proportion of a first oligonucleotide that binds to a sequence other than its complementary sequence in the sample is less than 0.05% under stringent hybridization conditions. In one aspect, stringent capture hybridization conditions include a temperature of 27°C to 47°C, a formamide concentration of 21% to 41%, a salt concentration of 300 mM to 500 mM, and a pH of 7.5 to 8.5. In one aspect, stringent capture hybridization conditions include a temperature of about 37°C, a formamide concentration of about 31%, a salt concentration of about 400 mM, and a pH of 8.0.

[0093] "Array" refers to one or more support surfaces having two or more spatially distinct (i.e., non-overlapping) addressable locations, referred to herein as binding domains or array elements. In one aspect, each addressable location contains an assay reagent, including, for example, a capture oligonucleotide.

[0094] "Support surface" refers to a surface material on which various substances, such as one or more capture oligonucleotides, can be immobilized. A "support surface" can be planar or non-planar. In one aspect, the support surface comprises a flat surface. In one aspect, the support surface is a plate with multiple wells, i.e., a "multiwell plate." A multiwell plate can contain any number of wells of any size or shape arranged in any pattern or configuration. In another aspect, the support surface has a curved surface. In one aspect, the support surface is provided by one or more particles, beads, or microspheres. The terms particles, beads, or microspheres can be used interchangeably unless otherwise indicated. In one aspect, the support surface comprises color-coded particles, beads, or microspheres. In one aspect, the support surface comprises an assay module, such as an assay plate, slide, cartridge, bead, or chip. In one aspect, the support surface comprises an assay flow cell or an assay fluid.

[0095] In one embodiment, the support surface comprises a plurality of addressable locations (which may be referred to as "spots"), for example, as is typical in "gene chip" devices. In another embodiment, the array comprises a plurality of support surfaces, each having one addressable location, as in the "bead array" approach, where each bead in a suspension of beads represents an addressable location (which may be addressed, for example, using flow cytometry or microscopic detection techniques). In another embodiment, the array comprises a plurality of support surfaces, each having one or more, or two or more addressable locations per surface. The addressable locations on the support surface may be arranged in uniform rows and columns, or may form other patterns. The number of addressable locations on the array may vary, for example, from less than about 10 to about 50, about 100, about 200, about 500, or more than about 1000. "Multiplexing" refers to the simultaneous analysis of two or more assay targets in a single assay.

[0096] In the context of an analyte measured in an assay, or a reagent used in an assay, the term "multiple" refers not only to two or more copies of an analyte or reagent (e.g., reagent A and another copy of reagent A), but also to two or more structurally or functionally different analytes or reagents (e.g., reagent A and reagent B). For example, the term "multiple detection reagents" refers to two or more structurally or functionally different detection reagents present in an assay, e.g., different detection reagents each specifically bind to a different target analyte, and does not describe a situation in which multiple copies of one reagent are present. However, the use of the term "multiple" in this context does not exclude the possibility that multiple copies of any of the multiple analytes or reagents are present. For example, multiple immobilized targeting reagent complements may refer to immobilized targeting reagent complements that include one or more copies of targeting reagent complement A and one or more copies of targeting reagent complement B. When referring to multiple analytes or reagents, the terms "first," "second," "third," etc., or "additional" can be used to distinguish between unique analytes or reagents. For example, a "first" detection reagent binds to a "first" target analyte and a "second" detection reagent binds to a "second" target analyte or a different portion of the target analyte.

[0097] "Unique" is a relative term that depends on other components present in a composition or mixture. For example, when used in relation to a nucleotide sequence, such as the nucleotide sequence of an analyte-binding portion of a probe, the term "unique" means that the nucleotide sequence of one analyte-binding portion is different from the nucleotide sequence of the analyte-binding portion of other probes in the composition or mixture. Similarly, when used in relation to a targeting reagent or oligonucleotide tag, the term "unique" means that the nucleotide sequence of the targeting reagent or oligonucleotide tag is different from the nucleotide sequence of other targeting reagents or tags in the composition or mixture. The term "unique" does not exclude the possibility that multiple copies of a "unique" analyte or reagent may be present in an assay or sample.

[0098] "Carbon-based" refers to materials that contain elemental carbon (C) as a major component. Examples of carbon-containing or carbon-based materials include, but are not limited to, carbon, carbon black, graphitic carbon, glassy carbon, carbon nanotubes, carbon fibrils, graphite, carbon fibers, and mixtures thereof. Carbon-based materials can include elemental carbon, including, for example, graphite, carbon black, or carbon nanotubes. In one aspect, carbon-based materials include conductive carbon-polymer composites, conductive polymers, or conductive particles dispersed in a matrix, such as, for example, carbon inks, carbon pastes, or metal inks. Conductive carbon particles can include, for example, carbon fibrils, carbon black, or graphitic carbon dispersed in a matrix, such as, for example, a polymer matrix, such as, for example, ethylene vinyl acetate (EVA), polystyrene, polyethylene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, or acrylonitrile butadiene styrene (ABS). Such polymer matrices can also include copolymers with two or more types of component monomers, which may include monomers selected from vinyl acetate, ethylene, vinyl alcohol, vinyl chloride, acrylonitrile, butadiene, styrene, or other monomers.

[0099] B. Overview Provided herein is a method for detecting or quantifying oligonucleotide duplexes in a sample.In one aspect, a method is provided for detecting or quantifying the first and second strands of an oligonucleotide duplex in a sample.In one aspect, a method is provided for detecting or quantifying the sense and antisense strands of an oligonucleotide duplex in a sample.In one aspect, the method is used to detect or quantify the sense and antisense strands of a double-stranded oligonucleotide therapeutic agent.

[0100] The methods described herein provide a robust and sensitive method for characterizing oligonucleotide therapeutics in a variety of complex biological samples, including biological fluids, including but not limited to plasma, serum, whole blood, urine, feces, breast milk, saliva, and amniotic fluid, as well as organs or organ homogenates, such as brain, liver, spleen, heart, lung, and kidney, or tissues or tissue homogenates, including but not limited to other tissues, such as muscle, skin, or bone marrow. In one aspect, the sample is an environmental sample. In one aspect, the sample is a manufacturing process sample.

[0101] In one aspect, the methods can be used to characterize the pharmacokinetics, biodistribution, and cellular uptake of therapeutic oligonucleotides, including, but not limited to, pharmacokinetics (PK), pharmacodynamics (PD), clearance, half-life, peak concentration, exposure-response relationship, biodistribution, tissue targeting, tissue accumulation, tissue bioavailability, or combinations thereof. In one aspect, the methods can be used to detect or quantitate both strands of an oligonucleotide duplex, e.g., to assess duplex stability, as well as the pharmacokinetics, biodistribution, and cellular uptake of the individual strands of an oligonucleotide duplex.

[0102] In one aspect, the method or kit is used to identify, detect, or quantify one or more nucleotide sequences or variants of a microorganism. In one aspect, the method or kit is used to identify, detect, or quantify one or more nucleotide sequences or variants of a bacterium, fungus, protozoan, or virus. In one aspect, the method or kit is used to identify, detect, or quantify one or more nucleotide sequences of a bacterium, fungus, protozoan, or virus that is a component of the human microbiome. In one aspect of the method or kit, it is used to identify, detect, or quantify one or more nucleotide sequences or variants of 16S rRNA or rDNA from bacteria.

[0103] In one aspect, the bacteria are Achromobacter, Acidaminococcus, Acinetobacter, Actinomycetales, Aerococcus, Anaerococcus, Aggregatibacter, Aeromonas, Alcaligenes, Anaerobiospirillum, Atopobium, Bacillus, Bacillota, Bacteroides, Bacterionema, Bartonella, Bifidobacterium, Bordetella, Borrelia, Brucella, Burkholderia, Buchnera, Butyriviberio, Campylobacter, Capnocytophaga, Cardiobacterium, Chlamydia, Chlamydophila, Collinsella, Citrobacter, Clostridium, Corynebacterium, Cutibacterium, Dialister, Demodex, Eggerthella, Eikenella, Enterococcus, Enterobacter, Escherichia, Eubacterium, Faecalibacterium, Finegoldia, Firmicutes, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gordonia, Haemophilus, Helicobacter, Kingella, Klebsiella, Lactobacillus, Legionella, Leptospira, Leptotrichia, Listeria, Megasphaera, Methanobrevibacter, Microbacterium, Micrococcus, Mobiluncus, Morganella, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Peptococcus, Peptoniphilus, Peptostreptococcus, Plesiomonas, Porphyromonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Pseudomonadota,Rickettsia, Roseburia, Rothia, Ruminococcus, Sarcina, Salmonella, Selenomonas, Shigella, Slackia, Sneathia, Spirochaeta, Staphylococcus, Stre ptobacillus, Streptococcus, Streptomyces, Tannerella, Treponema, Trichophyton, Ureaplasma, Veillonella, Vibrio, Wolinella, or Yersinia bacteria. ,

[0104] In one embodiment, a method is provided for detecting or quantifying the sense and antisense strands of an oligonucleotide duplex in a sample. In one embodiment, the method comprises contacting the sample with a composition comprising a set of probes comprising a sense probe and an antisense probe. In one embodiment, the sense probe comprises a single-stranded oligonucleotide tag that is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface, a sense binding moiety that can hybridize to a nucleotide sequence of the sense strand of the oligonucleotide duplex, and a label. In one embodiment, the antisense probe comprises a single-stranded oligonucleotide tag that is complementary to at least a portion of a second capture oligonucleotide immobilized on a support surface, an antisense binding moiety that can hybridize to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a label. In one embodiment, the sense binding moiety of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand. In one embodiment, the antisense binding moiety of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand.

[0105] In one aspect, the method further comprises incubating the probe with the sample to form a hybridization mixture. In one aspect, the hybridization mixture comprises "productive" or "desired" hybridization complexes, where the sense probe hybridizes to the sense strand of the oligonucleotide duplex to form a sense complex, and the antisense probe hybridizes to the antisense strand of the oligonucleotide duplex to form an antisense complex. In one aspect, the hybridization mixture comprises one or more "non-productive" or undesired hybridization complexes. An example of a non-productive hybridization complex is when the sense probe does not hybridize to the sense strand of the oligonucleotide, or when the antisense probe does not hybridize to the antisense strand of the oligonucleotide. Another example of a non-productive hybridization complex is when the sense probe and the antisense probe hybridize to each other to form a probe-probe complex.

[0106] Figure 1A is a schematic diagram of an antisense complex 10 comprising an antisense strand 11 and an antisense probe 12 of an oligonucleotide duplex, where the antisense probe 12 comprises an oligonucleotide tag 13, an antisense binding moiety 14, and a label 15. Figure 1B is a schematic diagram of a sense complex 20 comprising a sense strand 21 from an oligonucleotide duplex and a sense probe 22, where the sense probe 22 comprises an oligonucleotide tag 23, a sense binding moiety 24, and a label 25.

[0107] One difficulty that arises when attempting to detect the sense and antisense strands of an oligonucleotide duplex is the potential non-productive binding due to probe-probe hybridization. Possible hybridization complexes in a hybridization mixture containing the sense and antisense strands of an oligonucleotide duplex and the sense and antisense probes are shown in Figures 2A-2C (using probes with short binding moieties) and 3A-3C (using probes with full-length binding moieties).

[0108] 2A is a schematic diagram of an antisense complex 10 comprising an antisense strand 11 and an antisense probe 12 of an oligonucleotide duplex, where the antisense probe 12 comprises an oligonucleotide tag 13, a "short" antisense binding portion 14, and a label 15. FIG. 2B is a schematic diagram of a sense complex 20 comprising a sense strand 21 and a sense probe 22 from an oligonucleotide duplex, where the sense probe 22 comprises an oligonucleotide tag 23, a "short" sense binding portion 24, and a label 25. As used herein, the term "short" binding portion means that the binding portions of the antisense probe and the sense probe have a length shorter (i.e., containing at least one less nucleotide) than the antisense and sense strands of the oligonucleotide duplex, respectively, such that a single-stranded overhang 16 is present in the antisense complex 10 and a single-stranded overhang 26 is present in the sense complex 22.

[0109] In one embodiment, the terminal portion of the antisense 11 strand of the antisense complex 10 is single-stranded. In one embodiment, the 3' terminal portion of the antisense 11 strand of the antisense complex 10 is single-stranded. In one embodiment, the 5' terminal portion of the antisense 11 strand of the antisense complex 10 is single-stranded. In one embodiment, the terminal portion of the sense strand 21 of the sense complex 20 is single-stranded. In one embodiment, the 3' terminal portion of the sense strand 21 of the sense complex 20 is single-stranded. In one embodiment, the 5' terminal portion of the sense strand 21 of the sense complex 20 is single-stranded. In one embodiment, the single-stranded overhang is about 1 to about 10 nucleotides in length, about 1 to about 5 nucleotides in length, about 1 to about 3 nucleotides in length, or about 1 to about 2 nucleotides in length. In one embodiment, the single-stranded overhang is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.

[0110] 2C is a schematic diagram of a probe-probe binding complex 40 in which a "short" antisense binding portion 14 of antisense probe 12 hybridizes to a "short" sense binding portion 24 of sense probe 22. In this situation, when antisense probe 10 has a "short" antisense binding portion 14 and sense probe 20 has a "short" sense binding portion 24, there are exposed single-stranded overhangs 17 and 27 in probe-probe complex 40. In one embodiment, the 5' end of antisense binding portion 14 of antisense probe 12 in probe-probe complex 40 is single-stranded. In one embodiment, the 5' end of sense binding portion 24 of sense probe 22 in probe-probe complex 40 is single-stranded. In one embodiment, the 5' end of antisense binding portion 14 of antisense probe 12 and the 5' end of sense binding portion 24 of sense probe 22 in probe-probe complex 40 are each single-stranded. In one embodiment, the 3' end of the antisense binding portion 14 of the antisense probe 12 in the probe-probe complex 40 is single-stranded. In one embodiment, the 3' end of the sense binding portion 24 of the sense probe 22 in the probe-probe complex 40 is single-stranded. In one embodiment, the 3' end of the antisense binding portion 14 of the antisense probe 12 and the 3' end of the sense binding portion 24 of the sense probe 22 in the probe-probe complex 40 are each single-stranded. In one embodiment, the single-stranded overhang is about 1 to about 10 nucleotides in length, about 1 to about 5 nucleotides in length, about 1 to about 3 nucleotides in length, or about 1 to about 2 nucleotides in length. In one embodiment, the single-stranded overhang is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.

[0111] Figure 3A is a schematic diagram of an antisense complex 10' comprising an antisense strand 11 and an antisense probe 12' of an oligonucleotide duplex, where the antisense probe 12' comprises an oligonucleotide tag 13, a "full-length" antisense binding portion 14', and a label 15. Figure 3B is a schematic diagram of a sense complex 20' comprising a sense strand 21 and a sense probe 22' from an oligonucleotide duplex, where the sense probe 22' comprises an oligonucleotide tag 23, a "full-length" sense binding portion 24', and a label 25. As used herein, the term "full-length" binding portion means that the binding portions of the antisense probe and the sense probe are the same length (i.e., contain the same number of nucleotide bases) as the antisense and sense strands of the oligonucleotide duplex, respectively, such that there are no single-stranded overhangs in the antisense complex 10' or the sense complex 20'. FIG. 3C is a schematic diagram of a probe-probe binding complex 40' in which the "full length" antisense binding portion 14' of antisense probe 12' is hybridized to the "full length" sense portion 24' of sense probe 22' in which there is no single-stranded overhang.

[0112] In one embodiment, the support surface is contacted with a hybridization mixture under conditions in which the oligonucleotide tag of the sense probe or the antisense probe hybridizes to the capture oligonucleotide immobilized on the support surface. In one embodiment, the oligonucleotide tag of the antisense probe that is part of the antisense complex hybridizes to the capture oligonucleotide immobilized on the support surface. In one embodiment, the oligonucleotide tag of the sense probe that is part of the sense complex hybridizes to the capture oligonucleotide immobilized on the support surface. In one embodiment, the oligonucleotide tag of the sense probe that is part of the sense complex hybridizes to the capture oligonucleotide immobilized on the support surface. In FIG. 4A, the antisense binding portion of the antisense probe is "short" such that the antisense strand portion of the antisense complex includes and is a single-stranded overhang, and the sense binding portion of the sense probe is "short" such that the sense strand portion of the sense complex includes a single-stranded overhang. In FIG. 4B, the antisense binding portion of the antisense probe is "full length" such that the antisense strand portion of the antisense complex does not include a single-stranded overhang, and the sense binding portion of the sense probe is "full length" such that the sense strand portion of the sense complex does not include a single-stranded overhang. The situation in which the antisense or sense complex hybridizes to the support surface via the oligonucleotide tag of the antisense or sense probe, respectively, is referred to herein as "productive."

[0113] In one embodiment, the oligonucleotide tag of the antisense probe or the sense probe is not part of the antisense complex or the sense complex, respectively. The situation in which a hybridization complex that is not an antisense complex or a sense complex hybridizes to the support surface via the oligonucleotide tag of the antisense probe is referred to herein as "non-productive". In one embodiment, as shown in FIG. 4C, the oligonucleotide tag of the antisense probe 12 or the sense probe 22 only binds the antisense probe 12 or the sense probe 22 to the support surface 30. In this situation, the antisense binding portion 14 and the sense binding portion 24 of the antisense probe 12 or the sense probe 22, respectively, remain single-stranded.

[0114] In one embodiment, the oligonucleotide tag of the antisense probe or the sense probe is part of the probe-probe complex, and the oligonucleotide tag of the antisense probe 12 or the sense probe 22 immobilizes the probe-probe complex 40' to the support surface 30. In one embodiment, as shown in Figure 4D, the antisense and sense binding portions of the probe are "short", such that there are single-stranded overhangs at the binding portions of the probe-probe complex 40'. In one embodiment, as shown in Figure 4E, the antisense and sense binding portions of the probe are "full length", such that there are no single-stranded overhangs at the binding portions of the probe-probe complex 40'.

[0115] In one aspect, the sample comprises a plurality of oligonucleotide duplexes and the composition comprises a plurality of sets of probes, each set of probes hybridizing to a unique sense or antisense strand of a unique oligonucleotide duplex.

[0116] In one aspect, the method includes step-down hybridization conditions, where the probes hybridize to their respective sense or antisense strands during a gradual decrease in annealing temperature. In one aspect, the hybridization conditions include a denaturation step in which the sample is incubated at a first temperature to denature the sense and antisense strands of the oligonucleotide duplex. In one aspect, the hybridization conditions include an annealing step in which the probes are incubated with the denatured sense and antisense strands of the oligonucleotide duplex at a second temperature to allow the sense and antisense probes to hybridize to the sense and antisense strands. In one aspect, the method includes incubating the sense and antisense complexes at a holding temperature of about 2°C to about 8°C.

[0117] In one embodiment, the denaturation step includes incubating the sample at a first temperature of about 60°C to about 95°C to denature the sense and antisense strands of the oligonucleotide duplex. In one embodiment, the denaturation step includes incubating the sample at a first temperature of at least about 60°C, about 65°C, about 70°C, about 75°C, or about 80°C, and up to about 85°C, about 90°C, about 95°C, or about 100°C. In one embodiment, the denaturation step includes incubating the sample at a first temperature of about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C. In one embodiment, the hybridization conditions include incubating the sample at a first temperature of about 80°C to about 95°C to denature the sense and antisense strands of the oligonucleotide duplex. In one embodiment, the hybridization conditions include incubating the sample at a first temperature of about 90° C. to about 95° C. to denature the sense and antisense strands of the oligonucleotide duplex. In one embodiment, the hybridization conditions include incubating the sample at a first temperature of about 95° C. to denature the sense and antisense strands of the oligonucleotide duplex. In one embodiment, the sample is incubated for about 1 minute to about 15 minutes. In one embodiment, the sample is incubated for at least about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes, and up to about 10 minutes or about 15 minutes. In one embodiment, the sample is incubated for about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, or about 15 minutes. In one embodiment, the sample is incubated for about 1 minute to about 5 minutes. In one embodiment, the sample is incubated for about 1 minute to about 2 minutes. In one embodiment, the sample is incubated for about 2 minutes. In one embodiment, the denaturing step comprises incubating the probe with the sample at a first temperature of about 60° C. to about 95° C. for about 1 minute to about 15 minutes to denature the sense and antisense strands of the oligonucleotide duplex. In one embodiment, the hybridization conditions comprise incubating the probe with the sample at a first temperature of about 80° C. to about 95° C. for about 1 minute to about 5 minutes to denature the sense and antisense strands of the oligonucleotide duplex.In one embodiment, the hybridization conditions include incubating the probe with the sample at a first temperature of about 95° C. for about 2 minutes to denature the sense and antisense strands of the oligonucleotide duplex.

[0118] In one embodiment, the annealing step includes incubating the probe with the sample at a second temperature of about 10° C. to about 65° C. to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one embodiment, the annealing step includes incubating the probe with the sample at a second temperature of at least about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., or about 40° C., and up to about 45° C., about 50° C., about 55° C., about 60° C., or about 65° C. to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one aspect, the annealing step comprises incubating the probe with the sample at a second temperature of about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, or about 65°C to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one aspect, the annealing step comprises incubating the probe with the sample at a second temperature from about 40°C and about 65°C to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one aspect, the hybridization conditions comprise incubating the probe with the sample at a second temperature of 60°C to about 65°C to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one aspect, the hybridization conditions include incubating the probe with the sample at a second temperature of about 65° C. to allow the sense and antisense probes to hybridize to the sense and antisense strands. In one aspect, the annealing step includes incubating the probe with the sample at the second temperature for about 30 seconds to about 5 minutes to allow the sense and antisense probes to hybridize to the sense and antisense strands. In one aspect, the annealing step includes incubating the probe with the sample at the second temperature for at least about 30 seconds, about 60 seconds, about 90 seconds, and up to about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes to allow the sense and antisense probes to hybridize to the sense and antisense strands.In one embodiment, the hybridization conditions include incubating the probe with the sample at a second temperature for about 1 minute to about 2 minutes to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one embodiment, the hybridization conditions include incubating the probe with the sample at a second temperature to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one embodiment, the annealing step includes incubating the probe with the sample at a second temperature of about 10°C to about 65°C for about 30 seconds to about 5 minutes to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one embodiment, the hybridization conditions include incubating the probe with the sample at a second temperature of about 40°C to about 65°C for about 1 minute to about 2 minutes to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand. In one aspect, the hybridization conditions include incubating the probe with the sample at a second temperature of about 65° C. for about 1 minute to hybridize the sense and antisense probes to the sense and antisense strands. In one aspect, the hybridization conditions include incubating the probe with the sample at a holding temperature of about 2° C. to about 8° C. In one aspect, the hybridization conditions include incubating the probe with the sample at a holding temperature of about 4° C.

[0119] In one embodiment, the temperature transition rate between the annealing step and the hold is about 0.05° C. / sec to about 0.5° C. / sec. In one embodiment, the temperature transition rate between the annealing step and the hold is about 0.1° C. / sec.

[0120] In one embodiment, the probe is incubated with the sample in a buffer containing diluent 54 or N-PLEX Hybridization Buffer 1 or 2.

[0121] In one embodiment, the hybridization mixture containing the hybridization complex is contacted with a single-strand specific nuclease.In one embodiment, the support surface is first contacted with the hybridization mixture under conditions that the first oligonucleotide tag and the second oligonucleotide tag of the sense probe and the antisense probe hybridize to the first capture oligonucleotide and the second capture oligonucleotide on the support surface, and the hybridization complex is immobilized on the support surface, and then the support surface is contacted with a single-strand specific nuclease.In one embodiment, the hybridization mixture containing the hybridization complex is contacted with a single-strand specific nuclease to form a reaction mixture, and then the support surface is contacted with a reaction mixture under conditions that the oligonucleotide tag of the sense probe and the antisense probe hybridize to the capture oligonucleotide immobilized on the support surface.

[0122] In one embodiment, the single-strand specific nuclease comprises a single-strand specific DNase. In one embodiment, the single-strand specific DNase is S1 nuclease, P1 nuclease, or mung bean nuclease. In one embodiment, the single-strand specific nuclease comprises a single-strand specific RNase. In one embodiment, the single-strand specific RNase is RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, PNPase, RNase PH, RNase R, RNase D, RNase T, RNaseONE, oligoribonuclease, exoribonuclease I, or exoribonuclease II.

[0123] In one embodiment, as shown in Figure 4C, a single-stranded nuclease cleaves unbound single-stranded probe in the hybridization complex and separates the label from the probe. Advantageously, this removes the label from the probe and reduces the background caused by immobilization of unbound probe to the support surface.

[0124] In one embodiment, as shown in FIG. 4D, a single-stranded nuclease cleaves single-stranded overhang probe-probe complexes formed using "short" probes, removing the label from the probe-probe complexes, thereby reducing background levels.

[0125] In one embodiment, the probe-probe complex is formed from the FL probe such that there are no single-stranded overhangs, as shown in Figure 4E, In this situation, the label remains immobilized on the support surface, causing high background levels.

[0126] In one embodiment, one or more of the method steps of contacting the sample with a composition comprising a set of probes, incubating the probes with the sample to form hybridization complexes comprising sense and antisense complexes, contacting the support surface with a hybridization mixture comprising hybridization complexes, and contacting the support surface with a single-strand specific nuclease are performed simultaneously. In one embodiment, the method steps of contacting the sample with a composition comprising a set of probes, incubating the probes with the sample to form hybridization complexes comprising sense and antisense complexes, contacting the support surface with a hybridization mixture comprising hybridization complexes, and contacting the hybridization complexes with a single-strand specific nuclease are all performed simultaneously.

[0127] In one embodiment, one or more of the method steps of contacting the sample with a composition comprising a set of probes, incubating the probes with the sample to form a hybridization mixture comprising hybridization complexes comprising sense and antisense complexes, contacting a support surface with the hybridization mixture, and contacting the hybridization complexes with a single-strand specific nuclease are performed sequentially. In one embodiment, the method steps of contacting the sample with a composition comprising a set of probes, incubating the probes with the sample to form a hybridization mixture comprising hybridization complexes comprising sense and antisense complexes, contacting a support surface with the hybridization mixture, and contacting the hybridization complexes with a single-strand specific nuclease are each performed sequentially.

[0128] In one embodiment, the support surface is incubated with the hybridization complex for about 15 minutes to about 12 hours. In one embodiment, the support surface is incubated with the hybridization complex for at least about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours, and up to about 6 hours or about 12 hours. In one embodiment, the support surface is incubated with the hybridization complex for about 30 minutes to about 3 hours. In one embodiment, the support surface is incubated with the hybridization complex for about 1 hour to about 2 hours.

[0129] In one embodiment, the support surface is incubated with the hybridization complex at a temperature of about 20°C to about 40°C. In one embodiment, the support surface is incubated with the hybridization complex at a temperature of at least about 20°C, about 25°C, or about 30°C, and up to about 25°C or about 40°C. In one embodiment, the support surface is incubated with the hybridization complex at a temperature of about 20°C to about 40°C. In one embodiment, the support surface is incubated with the hybridization complex at a temperature of about 30°C to about 40°C. In one embodiment, the support surface is incubated with the hybridization complex at a temperature of about 35°C to about 40°C. In one embodiment, the support surface is incubated with the hybridization complex at a temperature of about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In one embodiment, the support surface is incubated with the sense complex and the antisense complex at a temperature of about 37°C.

[0130] In one embodiment, the support surface is incubated with the hybridization complex while shaking at about 700 rpm to about 900 rpm. In one embodiment, the support surface is incubated with the hybridization complex while shaking at about 700 rpm, 705 rpm, 710 rpm, 725 rpm, 750 rpm, and up to about 800 rpm, about 850 rpm, or about 900 rpm. In one embodiment, the support surface is incubated with the hybridization complex while shaking at about 705 rpm.

[0131] In one embodiment, the support surface is incubated with the hybridization complex at a temperature of about 20° C. to about 40° C. for about 15 minutes to about 12 hours. In one embodiment, the support surface is incubated with the hybridization complex at a temperature of about 20° C. to about 40° C. for about 1 hour to about 2 hours. In one embodiment, the support surface is incubated with the sense complex and the antisense complex while shaking. In one embodiment, the support surface is incubated with the sense complex and the antisense complex at a temperature of about 37° C. for about 1 hour while shaking at about 705 rpm.

[0132] In one embodiment, the sample comprises a plurality of oligonucleotide duplexes, and the probe composition comprises a plurality of sets of probes, each set of probes hybridizing to a unique sense or antisense strand of a unique oligonucleotide duplex. In one embodiment, the probe composition comprises about 20 pM to about 10 nM of sense probe. In one embodiment, the probe composition comprises about 20 pM, about 50 pM, about 100 pM, about 150 pM, about 200 pM, or about 250 pM, up to about 0.5 nM, about 1 nM, about 5 nM, or about 10 nM of sense probe. In one embodiment, the probe composition comprises about 100 pM to about 500 pM of sense probe. In one embodiment, the probe composition comprises about 20 pM to about 200 pM of sense probe. In one embodiment, the probe composition comprises about 20 pM to about 100 pM of sense probe. In one embodiment, the probe composition comprises about 20 pM, about 50 pM, about 100 pM, about 150 pM, about 200 pM, or about 250 pM, up to about 0.5 nM, about 1 nM, about 5 nM, or about 10 nM of antisense probe. In one embodiment, the probe composition comprises about 100 pM to about 500 pM of antisense probe. In one embodiment, the probe composition comprises about 20 pM to about 200 pM of antisense probe.

[0133] In one embodiment, the method comprises detecting or quantifying the sense and antisense strands of an oligonucleotide duplex based on the presence of a label immobilized on a support surface. In one embodiment, the method has a lower limit of detection (LLOD) of less than about 200 pg / mL.

[0134] C. Oligonucleotide duplex In one aspect, the method described herein is used to detect an oligonucleotide duplex in a sample. In one aspect, the method described herein is used to detect the first strand and the second strand of the oligonucleotide duplex. In one aspect, the oligonucleotide duplex is a double-stranded oligonucleotide therapeutic. In one aspect, the oligonucleotide therapeutic comprises a sense and an antisense strand.

[0135] In one embodiment, both strands of the oligonucleotide duplex comprise DNA. In one embodiment, the oligonucleotide duplex is a DNA / DNA duplex. In one embodiment, both strands of the oligonucleotide duplex comprise RNA. In one embodiment, the oligonucleotide duplex is an RNA / RNA duplex. In one embodiment, one strand of the oligonucleotide duplex comprises DNA and one strand of the oligonucleotide duplex comprises RNA. In one embodiment, the oligonucleotide duplex is a DNA / RNA heteroduplex. In one embodiment, the sense strand of the oligonucleotide duplex comprises DNA. In one embodiment, the sense strand of the oligonucleotide duplex comprises RNA. In one embodiment, the antisense strand of the oligonucleotide duplex comprises DNA. In one embodiment, the antisense strand of the oligonucleotide duplex comprises RNA.

[0136] In one embodiment, one or both strands of the oligonucleotide duplex comprise one or more modified nucleotides. In one embodiment, one of the strands of the oligonucleotide duplex comprises DNA and one or more modified nucleotides. In one embodiment, one of the strands of the oligonucleotide duplex comprises RNA and one or more modified nucleotides. In one embodiment, the antisense strand of the oligonucleotide duplex comprises DNA and one or more modified nucleotides. In one embodiment, the antisense strand of the oligonucleotide duplex comprises RNA and one or more modified nucleotides. In one embodiment, the sense strand of the oligonucleotide duplex comprises DNA and one or more modified nucleotides. In one embodiment, the sense strand of the oligonucleotide duplex comprises RNA and one or more modified nucleotides. In one embodiment, the modified nucleotide comprises a modification in the nucleobase, sugar, or internucleotide linkage. In one embodiment, the sense strand, the antisense strand, or both the sense strand and the antisense strand of the oligonucleotide duplex individually comprise one or more modified nucleic acids. In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands of the oligonucleotide duplex individually comprise a 5'- or 3'-bioconjugate. In one embodiment, the bioconjugate comprises polyethylene glycol (PEG), N-acetylgalactosamine (GalNAc), a cell penetrating peptide (CPP), α-tocopherol, an aptamer, an antibody, cholesterol, squalene, a fatty acid, or a nucleolipid.

[0137] In one embodiment, each strand of the oligonucleotide duplex independently comprises about 5 to about 100 nucleotides. In one embodiment, each strand of the oligonucleotide duplex independently comprises about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, or about 30 nucleotides, up to about 30, about 35, about 40, about 45, about 50, or about 100 nucleotides in length. In one embodiment, each strand of the oligonucleotide duplex independently comprises about 8 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 12 to about 30 nucleotides, about 16 to about 30, or about 18 to about 30 nucleotides.

[0138] D. Oligonucleotide Probes In one aspect, a sense probe is provided that can hybridize to the sense strand of an oligonucleotide duplex. In one aspect, an antisense probe is provided that can hybridize to the antisense strand of an oligonucleotide duplex. In one aspect, an antisense probe is provided that comprises an oligonucleotide tag, an antisense binding moiety, and a label. In one aspect, a sense probe is provided that comprises an oligonucleotide tag, a sense binding moiety, and a label.

[0139] 1. Oligonucleotide tags In one aspect, the sense and antisense probes comprise an oligonucleotide tag having a nucleic acid sequence that hybridizes to an oligonucleotide sequence of a capture oligonucleotide. In one aspect, the oligonucleotide tag comprises a single-stranded oligonucleotide that is complementary to at least a portion of the nucleotide sequence of the single-stranded capture oligonucleotide. In one aspect, the oligonucleotide tag hybridizes to its corresponding capture oligonucleotide.

[0140] In one aspect, an oligonucleotide tag does not cross-react or hybridize with a capture oligonucleotide that is not its corresponding capture oligonucleotide. In one aspect, an oligonucleotide tag does not cross-react or hybridize with a capture oligonucleotide that is not its corresponding capture oligonucleotide under stringent hybridization conditions. In one aspect, an oligonucleotide tag does not hybridize to a sense or antisense strand of an oligonucleotide duplex. In one aspect, an oligonucleotide tag of a sense probe does not hybridize to an antisense binding portion of an antisense probe, or vice versa. In one aspect, an oligonucleotide tag of a sense probe does not hybridize to an antisense binding portion of an antisense probe under physiologically relevant or stringent conditions. In one aspect, an oligonucleotide tag of an antisense probe does not hybridize to a sense binding portion of a sense probe under physiologically relevant or stringent conditions. In one aspect, an oligonucleotide tag does not hybridize to a sense or antisense strand of an oligonucleotide duplex under physiologically relevant or stringent conditions.

[0141] In one embodiment, the oligonucleotide tag and its corresponding capture oligonucleotide have 100% "sequence complementarity" based on the Watson-Crick model. In one embodiment, the oligonucleotide tag and the capture oligonucleotide have sequences that have at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity based on the Watson-Crick model.

[0142] In one embodiment, the oligonucleotide tag is recombinantly produced. In one embodiment, the oligonucleotide tag is chemically synthesized. In one embodiment, the oligonucleotide tag is not a naturally occurring sequence. In one embodiment, the oligonucleotide tag comprises a single-stranded DNA sequence. In one embodiment, the oligonucleotide tag comprises a single-stranded RNA sequence. In one embodiment, the oligonucleotide tag of the sense probe comprises RNA. In one embodiment, the oligonucleotide tag of the antisense probe comprises RNA. In one embodiment, the oligonucleotide tag of the sense probe comprises DNA. In one embodiment, the oligonucleotide tag of the antisense probe comprises DNA.

[0143] In one embodiment, the oligonucleotide tag of the antisense probe comprises one or more modified nucleic acids. In one embodiment, the oligonucleotide tag of the antisense probe comprises one or more modified nucleotides selected from phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or combinations thereof. In one embodiment, the oligonucleotide tag of the antisense probe comprises locked nucleic acid (LNA).

[0144] In one embodiment, the oligonucleotide tag of the sense probe comprises one or more modified nucleic acids. In one embodiment, the oligonucleotide tag of the sense probe comprises one or more modified nucleotides selected from phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or combinations thereof. In one embodiment, the oligonucleotide tag of the sense probe comprises locked nucleic acid (LNA).

[0145] In one embodiment, the oligonucleotide tag comprises one or more modified nucleotides.

[0146] In one embodiment, the oligonucleotide tag is attached to the 5' end of the antisense probe. In one embodiment, the oligonucleotide tag is attached to the 3' end of the antisense probe. In one embodiment, the oligonucleotide tag is attached to the 5' end of the sense probe. In another embodiment, the oligonucleotide tag is attached to the 3' end of the sense probe. In one embodiment, the oligonucleotide tag is not complementary to and does not hybridize with the sense or antisense strand of the oligonucleotide duplex.

[0147] In one aspect, the oligonucleotide tag has a nucleotide sequence that is at least about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25, and up to about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40, or about 15 and about 40, or about 20 and about 30 nucleotides in length. In one embodiment, the oligonucleotide tag comprises a nucleotide sequence that is at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10, and up to about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20, or about 1 to about 20, about 10 to about 15, or about 12 to about 13 nucleotides shorter than the complementary capture oligonucleotide sequence. In one embodiment, the tag has a nucleotide sequence that is at least about 24, about 30, or about 36 nucleotides in length. In one embodiment, the oligonucleotide tag has a length that is the same as the length of the corresponding capture oligonucleotide. In one embodiment, the oligonucleotide tag has a length that is shorter than the length of the corresponding capture oligonucleotide.

[0148] 2.Joining part In one embodiment, the antisense probe comprises an antisense binding portion. In one embodiment, the antisense binding portion of the antisense probe has a nucleic acid sequence that is complementary to the nucleic acid sequence of the antisense strand of the oligonucleotide duplex. In one embodiment, the antisense binding portion of the antisense probe can hybridize to the antisense strand of the oligonucleotide duplex. In one embodiment, the antisense binding portion and the antisense strand of the oligonucleotide duplex have 100% "sequence complementarity" based on the Watson-Crick model. In one embodiment, the antisense binding portion and the antisense strand of the oligonucleotide duplex have a sequence that has at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity based on the Watson-Crick model.

[0149] In one embodiment, the sense probe comprises a sense binding portion. In one embodiment, the sense binding portion of the sense probe has a nucleic acid sequence that is complementary to the nucleic acid sequence of the sense strand of the oligonucleotide duplex. In one embodiment, the sense binding portion of the sense probe can hybridize to the sense strand of the oligonucleotide duplex. In one embodiment, the sense binding portion and the sense strand of the oligonucleotide duplex have 100% "sequence complementarity" based on the Watson-Crick model. In one embodiment, the sense binding portion and the sense strand of the oligonucleotide duplex have a sequence that has at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity based on the Watson-Crick model.

[0150] In one embodiment, the antisense binding length of the antisense probe is at least one nucleotide shorter than the antisense strand length of the antisense strand. In one embodiment, the antisense binding portion has a length of about 1 to about 10 nucleotides shorter than the antisense strand of the oligonucleotide duplex. In one embodiment, the antisense binding portion has a length of about 1 to about 5 nucleotides shorter than the antisense strand of the oligonucleotide duplex. In one embodiment, the antisense binding portion has a length of about 10 to about 25 nucleotides, or about 10 to about 20 nucleotides, or about 10 to about 16 nucleotides. In one embodiment, the antisense binding portion of the antisense probe has a length that is about 50% to about 99% of the length of the antisense strand of the oligonucleotide duplex. In one embodiment, the antisense binding portion of the antisense probe has a length that is about 75% to about 95% of the length of the antisense strand of the oligonucleotide duplex.

[0151] In one embodiment, the sense binding length of the sense probe is at least one nucleotide shorter than the sense strand length of the sense strand. In one embodiment, the sense binding portion has a length of about 1 to about 10 nucleotides shorter than the sense strand of the oligonucleotide duplex. In one embodiment, the sense binding portion has a length of about 1 to about 5 nucleotides shorter than the sense strand of the oligonucleotide duplex. In one embodiment, the sense binding portion has a length of about 10 to about 25 nucleotides, or about 10 to about 20 nucleotides, or about 10 to about 16 nucleotides. In one embodiment, the sense binding portion of the sense probe has a length that is about 50% to about 99% of the length of the sense strand of the oligonucleotide duplex. In one embodiment, the sense binding portion of the sense probe has a length that is about 75% to about 95% of the length of the sense strand of the oligonucleotide duplex.

[0152] In one embodiment, the sense binding portion of the sense probe has a 5' end that aligns with the 3' end of the sense strand of the oligonucleotide duplex. In one embodiment, the sense binding portion of the sense probe has a 3' end that aligns with the 5' end of the sense strand of the oligonucleotide duplex. In one embodiment, the antisense binding portion of the antisense probe has a 5' end that aligns with the 3' end of the antisense strand of the oligonucleotide duplex. In one embodiment, the antisense binding portion of the antisense probe has a 3' end that aligns with the 5' end of the antisense strand of the oligonucleotide duplex.

[0153] In one embodiment, the antisense binding portion of the antisense probe comprises DNA. In one embodiment, the antisense binding portion of the antisense probe comprises RNA. In one embodiment, the sense binding portion of the sense probe comprises DNA. In one embodiment, the sense binding portion of the sense probe comprises RNA.

[0154] In one embodiment, the antisense binding portion of the antisense probe and the oligonucleotide tag comprise DNA. In one embodiment, the antisense binding portion of the antisense probe and the oligonucleotide tag comprise RNA. In one embodiment, the antisense binding portion of the antisense probe comprises DNA and the oligonucleotide tag of the antisense probe comprises RNA. In one embodiment, the antisense binding portion of the antisense probe comprises RNA and the oligonucleotide tag of the antisense probe comprises DNA.

[0155] In one aspect, the sense binding portion of the sense probe and the oligonucleotide tag comprise DNA. In one aspect, the sense binding portion of the sense probe and the oligonucleotide tag comprise RNA. In one aspect, the sense binding portion of the sense probe comprises DNA and the oligonucleotide tag of the sense probe comprises RNA. In one aspect, the sense binding portion of the sense probe comprises RNA and the oligonucleotide tag of the sense probe comprises DNA.

[0156] In one embodiment, the antisense probe is a chimeric probe comprising an antisense binding portion comprising DNA and an oligonucleotide tag comprising RNA, the antisense binding portion antisense probe having an antisense binding length at least one nucleotide shorter than the antisense strand length of the antisense strand of the oligonucleotide duplex. In one embodiment, the antisense probe is a chimeric probe comprising an antisense binding portion comprising RNA and an oligonucleotide tag comprising DNA, the antisense binding portion antisense probe having an antisense binding length at least one nucleotide shorter than the antisense strand length of the antisense strand of the oligonucleotide duplex. In one embodiment, the sense probe is a chimeric probe comprising a sense binding portion comprising DNA and an oligonucleotide tag comprising RNA, the sense binding portion of the sense probe having a sense binding length at least one nucleotide shorter than the sense strand length of the sense strand of the oligonucleotide duplex. In one embodiment, the sense probe is a chimeric probe comprising a sense binding portion comprising RNA and an oligonucleotide tag comprising DNA, the sense binding portion of the sense probe having a sense binding length at least one nucleotide shorter than the sense strand length of the sense strand of the oligonucleotide duplex.

[0157] In one embodiment, the binding portion of the antisense probe comprises one or more modified nucleic acids. In one embodiment, the binding portion of the antisense probe comprises one or more modified nucleotides selected from phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or combinations thereof. In one embodiment, the binding portion of the antisense probe comprises locked nucleic acid (LNA).

[0158] In one embodiment, the binding portion of the sense probe comprises one or more modified nucleic acids. In one embodiment, the binding portion of the sense probe comprises one or more modified nucleotides selected from phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or combinations thereof. In one embodiment, the binding portion of the sense probe comprises locked nucleic acid (LNA).

[0159] 3.Signs In one embodiment, the probe comprises a label. In one embodiment, the label is directly attached to the probe. In another embodiment, the label is attached to the probe via a linker. In one embodiment, the label is attached to the 5' end of the probe. In one embodiment, the label is attached to the 3' end of the probe. In one embodiment, the label is a primary label. In one embodiment, the primary label has a detectable physical property. Examples of primary labels include, but are not limited to, radioisotopes, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, electrochemiluminescent (ECL) moieties, magnetic particles, and bioluminescent moieties. In one embodiment, the primary label is an electrochemiluminescent (ECL) label. In one embodiment, the ECL label is an organometallic complex that includes a transition metal, such as ruthenium. In one embodiment, the primary label is an MSD SULFO-TAG label (Meso Scale Diagnostics, LLC, Rockville, MD, USA).

[0160] In one embodiment, the label is a compound that is a member of a binding pair, where a first member of the binding pair (which can be referred to as a "primary binding reagent") is bound to a substrate, e.g., an oligonucleotide, and the other member of the binding pair (which can be referred to as a "secondary binding reagent") has a detectable physical property or is bound to a moiety that has a detectable physical property. Non-limiting examples of binding pairs include biotin and streptavidin, or avidin, complementary oligonucleotides, haptens and hapten binding partners, and antibody-antigen binding pairs. In one embodiment, the label is a primary binding agent that includes biotin. In one embodiment, the secondary binding reagent includes streptavidin. In one embodiment, the secondary binding reagent includes MSD SULFO-TAG label (Meso Scale Diagnostics, LLC, Rockville, MD, USA).

[0161] E. Sample In one aspect, the oligonucleotide duplex is in a sample. In one aspect, the sample is a biological sample obtained or derived from a source of interest. In one aspect, the sample is an organism or is obtained from an organism. In one aspect, the sample is a plant or is obtained from a plant. In one aspect, the sample is an animal or is obtained from an animal. In one aspect, the sample is obtained from a mammal. In one aspect, the sample is obtained from a human. In one aspect, the source of interest comprises a bioreactor. In one aspect, the sample is a manufacturing process sample. In one aspect, the sample is an environmental sample. In one aspect, the environmental sample comprises a water sample, including, for example, an aquifer sample, a groundwater sample, or a wastewater sample. In one aspect, the environmental sample comprises a soil sample. In one aspect, the environmental sample comprises a soil microbial sample. In one aspect, the sample comprises cell-free DNA.

[0162] In one embodiment, the sample comprises a biological sample. In one embodiment, the sample comprises an untreated biological sample. In one embodiment, the sample comprises a pretreated biological sample. In one embodiment, the sample is pretreated, for example, to remove one or more components or to add one or more agents. In one embodiment, the sample comprises a purified sample. Methods for purifying oligonucleotides from biological samples are known, and include, for example, precipitation, centrifugation, and column chromatography. In one embodiment, column chromatography includes high performance liquid chromatography (HPLC), for example, reverse phase high performance liquid chromatography (RP-HPLC), anion exchange high pressure liquid chromatography (AEX HPLC), or polyacrylamide gel electrophoresis (PAGE). In one embodiment, the sample is an extracted sample. In one embodiment, the sample is filtered, for example, using a semipermeable membrane. In one embodiment, the sample comprises oligonucleotides extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of mRNA.

[0163] In one embodiment, the sample comprises one or more target oligonucleotide sequences. In one embodiment, the sample comprises one or more amplified target oligonucleotide sequences. In one embodiment, the sample comprises one or more amplified target oligonucleotide sequences obtained by methods including, but not limited to, polymerase chain reaction (PCR) or rolling circle amplification (RCA).

[0164] In one aspect, the biological sample includes biological tissues or biological fluids, including, for example, bodily fluids, secretions, excretions, cells, tissues or organs, or homogenates thereof. In one aspect, biological fluids include plasma, serum, whole blood, lymph, urine, feces, breast milk, sputum, saliva, peritoneal fluid, cerebrospinal fluid, peritoneal fluid, pleural fluid, and amniotic fluid. In one aspect, the biological tissue includes tissues or tissue homogenates, including, but not limited to, organs or organ homogenates, such as brain, liver, spleen, heart, lung, and kidney, or other tissues, such as, for example, muscle, skin, or bone marrow. In one aspect, the biological sample includes tissues or fine needle biopsy samples, cell-containing bodily fluids, suspended nucleic acids, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings, or lavages, such as ductal or bronchoalveolar lavages, aspirates, scrapings, surgical specimens.

[0165] In one aspect, the biological sample includes a sample isolated from a part of the human body. In one aspect, the biological sample includes a sample isolated from the nasal cavity, oral cavity, skin, ear, mucous membrane, gastrointestinal tract, genitourinary tract, respiratory tract, eye, or a combination thereof. In one aspect, the biological sample includes a sample obtained from a part of the human body using methods known in the art, including, but not limited to, swabbing, puncture sampling, and serum sampling.

[0166] In one embodiment, the sample contains a naturally occurring RNase. In samples containing naturally occurring RNase, it may be desirable to contact the sample with an RNase inhibitor prior to contacting the sample with the sense and antisense probes.

[0167] F. Capture Oligonucleotides In one embodiment, the method or kit includes one or more capture oligonucleotides that are immobilized or can be immobilized to a distinct binding domain on a support surface. In one embodiment, the capture oligonucleotide is not a naturally occurring sequence. In another embodiment, the capture oligonucleotide is recombinantly produced. In one embodiment, the capture oligonucleotide is chemically synthesized.

[0168] In one aspect, the capture oligonucleotide is a single stranded capture oligonucleotide having a nucleotide sequence that is complementary to the nucleotide sequence of the single stranded oligonucleotide tag, hi one aspect, the oligonucleotide tag is attached to a sense or antisense probe.

[0169] In one embodiment, the method or kit includes a unique capture oligonucleotide sense strand and an antisense strand of an oligonucleotide duplex of interest. In one embodiment, the capture oligonucleotide is immobilized on a support surface. In one embodiment, the capture oligonucleotide is immobilized on an array. In one embodiment, the array includes two or more capture oligonucleotides. In one embodiment, the array includes about 2 to about 150 or more capture oligonucleotides.

[0170] In one embodiment, one or more capture oligonucleotides comprise single-stranded nucleic acid sequences, including, for example, nucleic acid sequences that contain deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or structural analogs that contain non-natural chemical structures that can also participate in hybridization reactions.

[0171] In one embodiment, the capture oligonucleotides used in a particular array have similar binding energies or melting temperatures (Tm), e.g., within at least about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, or about 5°C of each other, and the melting temperatures (T m ) refers to the temperature at which 50% of the oligonucleotides are hybridized to their complements and 50% are free in solution. m can be determined using known methods, for example, by measuring the change in absorbance of an oligonucleotide having its complement as a function of temperature. In one embodiment, the capture oligonucleotide has a melting temperature (T) in 50 mM NaCl of about 50° C. to about 70° C., about 55° C. to about 65° C., or at least about 50° C., about 55° C., or about 60° C., up to about 60° C., about 65° C., or about 70° C. m In one embodiment, the capture oligonucleotide has a GC content of about 40% to about 60%, or about 40% to about 50%.

[0172] In one embodiment, the capture oligonucleotide is about 20 to about 100, about 30 to about 50, or about 35 to about 40 nucleotides in length, for example, at least about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36, and up to about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 75, or about 100 nucleotides in length. In one embodiment, the capture oligonucleotide comprises at least 20, about 24, about 30, or about 36 nucleotides. In one embodiment, one or more capture oligonucleotides in the array are not identical in length to the nucleic acid sequence of their complementary oligonucleotide tags. In one embodiment, a capture oligonucleotide has a sequence that is, for example, up to about 5, about 10, about 15, about 20, or about 25 bases longer than the sequence of its complementary single-stranded oligonucleotide tag.

[0173] In one embodiment, one or more capture oligonucleotides are immobilized on the support surface by covalent or non-covalent bonds. In one embodiment, one or more capture oligonucleotides are immobilized on one or more binding domains on the support surface by covalent or non-covalent bonds. In one embodiment, the capture oligonucleotides are adsorbed on the support surface via electrostatic interactions, e.g., between negatively charged phosphate groups on the oligonucleotides and positive charges on the support surface. In one embodiment, one or more capture oligonucleotides are immobilized on the support surface via binding between a first binding partner bound to the capture oligonucleotide (directly or via a linker moiety) and a second binding partner immobilized on the surface. In one embodiment, one or more capture oligonucleotides are immobilized on the support surface by covalent bonds. In one embodiment, one or more capture oligonucleotides are immobilized directly on the support surface. In another embodiment, the capture oligonucleotides are immobilized on the support surface via a linker.

[0174] Capture oligonucleotides are disclosed in International Application Publication No. WO2020 / 227016, entitled "KITS FOR DETECTING ONE OR MORE TARGET NUCLEIC ACID ANALYTES IN A SAMPLE AND METHOD OF MAKING AND USING THE SAME" (Meso Scale Technologies, LLC., Rockville, MD, USA), the disclosure of which is incorporated by reference in its entirety.

[0175] G.Support surface In one embodiment, one or more capture oligonucleotides are immobilized on a support surface. The capture oligonucleotides can be immobilized on a variety of support surfaces, including support surfaces used in traditional binding assays. In one embodiment, the support surface has a flat surface. In another embodiment, the support surface has a curved surface. In one embodiment, the support surface comprises an assay module, such as an assay plate, slide, cartridge, bead, or chip. In one embodiment, the support surface comprises color-coded microspheres. See, for example, Yang et al. (2001) BADGE, Beads Array for the Detection of Gene Expression, a High-Throughput Diagnostic Bioassay. Genome Res. 11(11): 1888-1898. In one embodiment, the support surface comprises one or more beads on which one or more capture oligonucleotides are immobilized.

[0176] The support surface can be made from a variety of suitable materials, including polymers such as polystyrene and polypropylene, ceramics, glasses, composite materials, including carbon-polymer composites, such as carbon-based inks, etc. In one aspect, the support surface is a carbon-based support surface.

[0177] In one embodiment, the support surface is provided by one or more particles or "beads". In one embodiment, the beads can have a diameter of up to about 1 cm (or about 10,000 μm), about 5000 μm, about 1,000 μm, about 500 μm, or about 100 μm. In one embodiment, the beads have a diameter of about 10 nm and about 100 μm, about 100 nm and about 10 μm, or about 0.5 μm and about 5 μm. In one embodiment, the beads are paramagnetic, providing the ability to capture the beads through the use of a magnetic field. In one embodiment, the support surface is provided by streptavidin-coated magnetic particles or avidin-coated magnetic beads, and biotin-labeled capture oligonucleotides are immobilized on the beads.

[0178] In one aspect, the support surface is a plate having multiple wells, i.e., a "multiwell plate." A multiwell plate can contain any number of wells of any size or shape arranged in any pattern or configuration. In one aspect, the multiwell plate contains about 1 to about 10,000 wells. In one aspect, the multiwell assay plate uses an industry standard format for the number, size, shape, and configuration of plates and wells. Examples of standard formats include 96-well plates, 384-well plates, 1536-well plates, and 9600-well plates, where the wells are arranged in a two-dimensional array. Other multiwell formats include single wells, 2-wells, 6-wells, and 24-wells, as well as 6144-well plates. In one aspect, the support surface comprises a 96-well plate.

[0179] In one embodiment, the support surface comprises a two-dimensional patterned array in which capture oligonucleotides are printed at known locations called binding domains. In one embodiment, the support surface comprises a patterned array of distinct, non-overlapping, addressable binding domains to which the capture oligonucleotides are immobilized, and the sequence of the capture oligonucleotides in each binding domain is known and can be correlated with the appropriate target analyte. In one embodiment, all capture oligonucleotides in a particular binding domain have the same sequence, and the capture oligonucleotides in one binding domain have a different sequence than the capture oligonucleotides in other binding domains. In one embodiment, the multiple binding domains are arranged in regular rows and columns on the support surface, and the exact location and sequence of each binding domain is recorded in a computer database. In one embodiment, the array is arranged in a symmetric grid pattern. In other embodiments, the array is arranged in another pattern, including but not limited to radially distributed lines, spiral lines, or regular clusters. In another embodiment, each binding domain is arranged on the surface of one or more microparticles or beads, and the microparticles or beads are coded to allow discrimination between different binding domains.

[0180] In one embodiment, the support surface is a multi-well plate that contains one or more separate addressable binding domains in each well that correspond to one or more capture oligonucleotides. In one embodiment, the support surface contains at least one binding domain for detecting wild-type nucleotide sequences and another binding domain for detecting mutant nucleotide sequences. In one embodiment, each well contains at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 binding domains. In one embodiment, each well contains at least about 7, about 10, about 16, or about 25 binding domains.

[0181] In one embodiment, the support surface is a multi-well plate comprising at least 24, 96, or 384 wells, each well comprising an array of up to 10 binding domains, with a different capture oligonucleotide immobilized to each distinct binding domain. In a more particular embodiment, the support surface is a 96-well plate, each well comprising an array with up to 10 binding domains. In one embodiment, each well of the 96-well plate comprises up to 10 binding domains, with up to 10 distinct capture oligonucleotides immobilized thereon. In one embodiment, each well comprises the same patterned array with the same capture oligonucleotide. In another embodiment, different wells may comprise different patterned arrays of capture oligonucleotides.

[0182] H. Nuclease protection assay (NPA) In one aspect, a method is provided for detecting or quantifying the sense and antisense strands of an oligonucleotide duplex in a sample using a nuclease protection assay. In one aspect, the sample is contacted with a set of probes, the set of probes including a sense probe and an antisense probe. In one aspect, the sense probe includes a single-stranded oligonucleotide tag that is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface, a sense binding moiety that can hybridize to a nucleotide sequence of the sense strand of the oligonucleotide duplex, and a label. In one aspect, the antisense probe includes a single-stranded oligonucleotide tag that is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface, an antisense binding moiety that can hybridize to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a label. In one aspect, the method includes incubating the probe with the sample to form a hybridization mixture containing a hybridization complex. In one aspect, the method includes contacting the support surface with the hybridization mixture under conditions in which the oligonucleotide tag of the hybridization complex hybridizes to the capture oligonucleotide immobilized on the support surface. In one aspect, the method comprises detecting or quantifying the sense and antisense strands of an oligonucleotide duplex based on the presence of a label immobilized on a support surface.In one aspect, the method comprises detecting or quantifying the sense and antisense strands of an oligonucleotide duplex based on the presence of labeled sense and antisense hybridization complexes immobilized on a support surface.

[0183] In one embodiment, the sense binding portion of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand.In one embodiment, the sense binding portion of the sense probe has a sense binding length that is at least one nucleotide shorter than the sense strand length of the sense strand.In one embodiment, the antisense binding portion of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand.In one embodiment, the antisense binding portion of the antisense strand has an antisense binding length that is at least one nucleotide shorter than the antisense strand length of the antisense strand.

[0184] In one aspect, the sense probe comprises DNA. In one aspect, the sense binding portion of the sense probe comprises DNA. In one aspect, the oligonucleotide tag of the sense probe comprises DNA. In one aspect, the sense binding portion of the sense probe and the oligonucleotide tag comprise DNA.

[0185] In one aspect, the sense probe comprises RNA. In one aspect, the sense binding portion of the sense probe comprises RNA. In one aspect, the oligonucleotide tag of the sense probe comprises RNA. In one aspect, the sense binding portion of the sense probe and the oligonucleotide tag comprise RNA.

[0186] In one embodiment, the sense binding portion of the sense probe comprises DNA and the oligonucleotide tag of the sense probe comprises RNA.In one embodiment, the sense binding portion of the sense probe comprises RNA and the oligonucleotide tag of the sense probe comprises DNA.

[0187] In one embodiment, the antisense probe comprises DNA. In one embodiment, the antisense binding portion of the antisense probe comprises DNA. In one embodiment, the oligonucleotide tag of the antisense probe comprises DNA. In one embodiment, the antisense binding portion and the oligonucleotide tag of the antisense probe comprise DNA.

[0188] In one embodiment, the antisense probe comprises RNA. In one embodiment, the antisense binding portion of the antisense probe comprises RNA. In one embodiment, the oligonucleotide tag of the antisense probe comprises RNA. In one embodiment, the antisense binding portion and the oligonucleotide tag of the antisense probe comprise RNA.

[0189] In one embodiment, the antisense binding portion of the antisense probe comprises DNA and the oligonucleotide tag of the antisense probe comprises RNA.In one embodiment, the antisense binding portion of the antisense probe comprises RNA and the oligonucleotide tag of the antisense probe comprises DNA.

[0190] In one embodiment, the antisense probe is a chimeric probe comprising an antisense binding portion comprising DNA and an oligonucleotide tag comprising RNA, the antisense binding portion antisense probe having an antisense binding length at least one nucleotide shorter than the antisense strand length of the antisense strand of the oligonucleotide duplex. In one embodiment, the antisense probe is a chimeric probe comprising an antisense binding portion comprising RNA and an oligonucleotide tag comprising DNA, the antisense binding portion antisense probe having an antisense binding length at least one nucleotide shorter than the antisense strand length of the antisense strand of the oligonucleotide duplex. In one embodiment, the sense probe is a chimeric probe comprising a sense binding portion comprising DNA and an oligonucleotide tag comprising RNA, the sense binding portion of the sense probe having a sense binding length at least one nucleotide shorter than the sense strand length of the sense strand of the oligonucleotide duplex. In one embodiment, the sense probe is a chimeric probe comprising a sense binding portion comprising RNA and an oligonucleotide tag comprising DNA, the sense binding portion of the sense probe having a sense binding length at least one nucleotide shorter than the sense strand length of the sense strand of the oligonucleotide duplex.

[0191] In one aspect, the method includes incubating a set of probes with a sample to form a hybridization mixture. In one aspect, the hybridization mixture contains hybridization complexes including a sense complex and an antisense complex. In one aspect, the hybridization mixture includes a sense complex including a sense probe hybridized to a sense strand of an oligonucleotide duplex, and an antisense complex including an antisense probe hybridized to an antisense strand of an oligonucleotide duplex.

[0192] In one embodiment, the hybridization mixture further contains one or more of the following non-productive hybridization complexes: a sense probe that does not hybridize to the sense strand of the oligonucleotide duplex, an antisense probe that does not hybridize to the antisense strand of the oligonucleotide duplex, or a probe-probe complex in which a sense probe and an antisense probe hybridize to each other. In one embodiment, the non-productive hybridization complex comprises a single-stranded overhang. In one embodiment, the non-productive hybridization complex comprises a single-stranded oligonucleotide sequence. In one embodiment, the non-productive hybridization complex comprises a single-stranded RNA sequence. In one embodiment, the non-productive hybridization complex comprises a single-stranded DNA sequence.

[0193] In one embodiment, the single-stranded overhang or single-stranded oligonucleotide sequence in the non-productive hybridization complex is digested by a single-strand specific nuclease. In one embodiment, the single-stranded overhang or single-stranded oligonucleotide sequence is a DNA sequence that is digested with a single-strand specific DNase. In one embodiment, the single-stranded overhang or single-stranded oligonucleotide sequence is an RNA sequence that is digested with a single-strand specific RNase.

[0194] In one aspect, digestion of the single-stranded overhangs or single-stranded oligonucleotide sequences in the non-productive hybridization complexes is performed while the hybridization complexes are in solution (i.e., before the hybridization complexes are immobilized to the support surface via the oligonucleotide tag of the sense or antisense probe). In one aspect, digestion of the single-stranded overhangs or single-stranded oligonucleotide sequences in the non-productive hybridization complexes is performed after the hybridization complexes are immobilized to the support surface via the oligonucleotide tag of the sense or antisense probe. In one aspect, the method includes a washing step after the single-stranded overhangs or single-stranded oligonucleotide sequences are digested with single-strand specific RNase. In one aspect, the method includes a washing step after the single-stranded overhangs or single-stranded oligonucleotide sequences are digested with single-strand specific RNase and immobilized to the support surface. In one aspect, the sense and antisense strands of the oligonucleotide duplex are detected or quantified based on the presence of a label immobilized to the support surface. In one embodiment, the sense and antisense strands of an oligonucleotide duplex are detected or quantified based on the presence of a labeled sense or antisense complex immobilized on a support surface.

[0195] I. Electrode In one embodiment, the sense or antisense strand of an oligonucleotide duplex is detected or quantified using electrochemiluminescence (ECL). Multiplexed measurement of analytes using electrochemiluminescence is described in U.S. Patent Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entireties.

[0196] In one embodiment, the support surface comprises one or more electrodes. In one embodiment, the support surface comprises one or more working electrodes and one or more counter electrodes. In one embodiment, the support surface comprises one or more binding domains formed on the one or more electrodes for use in an electrochemical or electrochemiluminescent assay.

[0197] In one embodiment, the binding domains are formed by collecting beads coated with capture oligonucleotides on an electrode surface, hi one embodiment, the beads are paramagnetic and the beads are collected on the electrode through the use of a magnetic field.

[0198] In one embodiment, the electrodes are provided in an assay module that provides an assay receptacle, an assay flow cell, an assay fluidics, or other components useful for performing an assay. Examples of assay modules for performing electrochemiluminescence assays include, for example, multi-array cases, assay plate cases, cartridge cases, and the like. In one embodiment, the electrodes are provided in an assay module that provides an assay receptacle, an assay flow cell, an assay fluidics, or other components useful for performing an assay. Examples of assay modules for performing electrochemiluminescence assays can be found in U.S. Patent Nos. 6,673,533, 7,842,246, 9,731,297, and 8,298,834. In one embodiment, the support surface is a multi-well plate that includes at least one electrode. In one embodiment, each well of the multi-well assay plate includes at least one electrode. In one embodiment, at least one well of the multi-well assay plate includes a working electrode. In another embodiment, at least one well of the multi-well assay plate includes a working electrode and a counter electrode. In another aspect, each well of the multi-well assay plate comprises a working electrode and a counter electrode, hi one aspect, the working electrode is adjacent to, but not in electrical contact with, the counter electrode.

[0199] In one aspect, the electrodes are comprised of conductive materials including metals such as, for example, gold, silver, platinum, nickel, steel, iridium, copper, aluminum, conductive alloys, or combinations thereof. In another aspect, the electrodes include semiconductor materials such as silicon and germanium, or semiconductor thin films such as indium tin oxide (ITO) and antimony tin oxide (ATO). In another aspect, the electrodes include oxide-coated metals such as aluminum oxide-coated aluminum. In one aspect, the electrodes include carbon-based materials. In one aspect, the electrodes include conductive composites, inks, pastes, polymer blends, and mixtures of materials including metal / non-metal composites including, for example, mixtures of conductive or semiconductive materials and non-conductive materials. In one aspect, the electrodes include carbon-based materials such as carbon, glassy carbon, carbon black, graphitic carbon, carbon nanotubes, carbon fibrils, graphite, carbon fibers, and mixtures thereof. In one aspect, the electrode comprises a conductive carbon-polymer composite, a conductive polymer, or conductive particles dispersed in a matrix, e.g., carbon ink, carbon paste, or metal ink. In one aspect, the working electrode is made from a carbon-polymer composite, e.g., comprising conductive carbon particles, e.g., carbon fibrils, carbon black, or graphitic carbon, dispersed in a matrix, e.g., a polymer matrix, e.g., ethylene vinyl acetate (EVA), polystyrene, polyethylene, polyvinyl acetate, polyvinyl chloride, polyvinyl alcohol, acrylonitrile butadiene styrene (ABS), or a copolymer of one or more of these polymers.

[0200] In one embodiment, the working electrode is made from a continuous conductive sheet or thin film of one or more conductive materials, which may be extruded, compressed, or molded. In another embodiment, the working electrode is made from conductive materials deposited or patterned on a substrate, for example, by printing, painting, coating, spin coating, evaporation, chemical vapor deposition, electrolytic deposition, electroless deposition, photolithography, or other electronic microfabrication techniques. In one embodiment, the working electrode comprises a conductive carbon ink printed on a polymeric support, for example, by inkjet printing, laser printing, or screen printing. Carbon inks are known and include materials manufactured by Acheson Colloids Co. (e.g., Acheson 440B, 423ss, PF407A, PF407C, PM-003A, 30D071, 435A, Electrodag 505SS, and Aquadag™), EI Du Pont de Nemours and Co. (e.g., Dupont 7105, 7101, 7102, 7103, 7144, 7082, 7861D, and CB050), Conductive Compounds Inc. (e.g., C-100), and Ercon Inc. (e.g., G-451).

[0201] In one embodiment, the working electrode is a continuous thin film. In another embodiment, the working electrode includes one or more discrete regions or a pattern of discrete regions. Alternatively, the working electrode may include multiple connected regions. One or more regions of the electrode surface exposed to the working electrode may be defined by a patterned insulating layer covering the working electrode, for example, by screen printing a patterned layer of insulating ink onto the working electrode or by adhering a die-cut insulating thin film. The exposed regions may define array elements of an array of reagents printed on the working electrode, resulting in array shapes and patterns as described above. In one embodiment, the insulating layer defines a series of annular regions (or "spots") of the exposed working electrode surface.

[0202] The counter electrode may have one or more of the properties generally described above for the working electrode. In one embodiment, the working electrode and the counter electrode are composed of the same material. In another embodiment, the working electrode and the counter electrode are not composed of the same material, for example, the working electrode may be a carbon electrode and the counter electrode may be a metal electrode.

[0203] In one embodiment, one or more capture oligonucleotides are immobilized to one or more electrodes by passive adsorption. In another embodiment, one or more capture oligonucleotides are immobilized to the electrode by covalent bonding. In one embodiment, the electrode is derivatized or modified, for example, to immobilize a reagent, such as a capture oligonucleotide, on the surface of the electrode. In one embodiment, the electrode is modified by chemical or mechanical treatment to improve the immobilization of the reagent, for example, to introduce a functional group for the immobilization of the reagent or to enhance its adsorption properties. Examples of functional groups that can be introduced include carboxylic acid (COOH), hydroxy (OH), amino (NH2), activated carboxyl (e.g., N-hydroxysuccinimide (NHS)-ester), poly-(ethylene glycol), thiol, alkyl ((CH2) n) groups, or combinations thereof). In one aspect, one or more reagents, e.g., capture oligonucleotides, are immobilized to a carbon-containing electrode, e.g., carbon black, fibrils, or carbon dispersed in another material, by either covalent or non-covalent means. It has been found that capture oligonucleotides bearing thiol groups can be covalently attached to a carbon-containing electrode, e.g., a screen-printed carbon ink electrode, without the need to first deposit an additional thiol-reactive layer, such as a protein layer or a chemical cross-linking layer. In one aspect, a method is provided for the direct attachment of capture oligonucleotides bearing thiol groups, such as thiol-modified oligonucleotides, to an electrode, which provides a simple, robust, efficient, and reproducible process for forming capture surfaces and arrays on the electrode. In one aspect, one or more capture oligonucleotides bearing thiol groups are directly immobilized to a carbon-containing electrode, such as a screen-printed carbon ink electrode, via reaction of the thiol with the electrode, without first adding a thiol-reactive layer to the electrode.

[0204] In one embodiment, the electrode is treated with a plasma, e.g., a low-temperature plasma such as a glow discharge plasma, to change the physical properties, chemical composition, or surface chemical properties of the electrode, for example, to aid in the immobilization of reagents such as capture oligonucleotides, or to reduce contaminants, improve adhesion to other materials, change the wettability of the surface, promote material deposition, create patterns, or improve uniformity. Examples of useful plasmas include oxygen, nitrogen, argon, ammonia, hydrogen, fluorocarbons, water, and combinations thereof. In one embodiment, an oxygen plasma is used to treat an electrode having carbon particles in a carbon-polymer composite. In another embodiment, oxygen is used to introduce carboxylic acid or other oxidized carbon functional groups to carbon or organic materials (e.g., activated esters or acyl chlorides) to facilitate coupling of reagents. In another embodiment, an ammonia-containing plasma can be used to introduce amino groups for use in coupling assay reagents. In one embodiment, the electrode is not pretreated to aid in the immobilization of one or more capture oligonucleotides.

[0205] In one embodiment, the support surface comprises an assay module such as a multiwell plate having one or more working or counter electrodes in each well. In one embodiment, the multiwell plate comprises multiple working or counter electrodes in each well. In one embodiment, the working or counter electrodes of the multiwell plate comprise a screen-printed layer of carbon, e.g., carbon ink. In one embodiment, one or more capture oligonucleotides are immobilized in the screen-printed carbon ink via thiol moieties on the capture oligonucleotides. In one embodiment, the working electrode is used to induce an ECL signal from the ECL label. In one embodiment, the ECL signal is released from ruthenium-tris-bipyridine in the presence of a coreactant such as a tertiary alkylamine, e.g., tripropylamine or butyldiethanolamine.

[0206] In one embodiment, the electrodes include the binding domains described above defined by a dielectric ink (i.e., an electrically insulating ink). The electrodes are working electrodes having a dielectric printed thereon in a pattern that defines the binding domains described above. In one embodiment, the binding domains are approximately annular regions (or "spots") of exposed working electrodes. The electrodes are in a 96-well plate formed by adhering an injection molded 96-well plate top to a Mylar sheet that defines the bottom of the wells. The top surface of the Mylar sheet has screen-printed carbon ink electrodes printed thereon, such that each well contains a carbon ink working electrode approximately in the center of the well and two carbon ink counter electrodes approximately toward the two edges of the well. Electrodes printed on the bottom of the Mylar sheet and connected to the top of the sheet via conductive through-holes provide contacts for applying voltages to the working and counter electrodes.

[0207] J. Detection In one embodiment, the presence of one or more oligonucleotide sequences is detected or quantified based on the detection of a label immobilized on the support surface. In one embodiment, the presence of one or more oligonucleotide sequences is detected or quantified based on the detection of a label on the hybridization complex immobilized on the support surface. In one embodiment, the presence of one or more target oligonucleotide sequences is detected or quantified based on the detection of a label on the sense complex immobilized on the support surface. In one embodiment, the presence of one or more target oligonucleotide sequences is detected or quantified based on the detection of a label on the antisense complex immobilized on the support surface. In one embodiment, the oligonucleotide sequences are detected or quantified in an array.

[0208] In one aspect, the presence of a hybridization complex, e.g., an antisense or sense complex, is detected by monitoring emission from the label, including, but not limited to, fluorescence, time-resolved fluorescence, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), luminescence, chemiluminescence, bioluminescence, phosphorescence, light scattering, or electrode-induced luminescence. In another aspect, the label comprises an enzyme or other chemically reactive species that has chemical activity that results in a measurable signal, such as light scattering, absorbance, or fluorescence. Examples of enzymatic labels include, but are not limited to, horseradish peroxidase or alkaline phosphatase. In one aspect, the label is a detectable hapten, including, but not limited to, biotin, fluorescein, or digoxigenin. In one aspect, the label comprises biotin.

[0209] In one embodiment, the hybridization complex, for example, antisense or sense complex, is immobilized on one or more binding domains located on a support surface. In one embodiment, the one or more binding domains are located on one or more electrodes, and detecting or quantifying comprises applying a voltage waveform to one or more electrodes to stimulate the label with the captured reaction product and generate an electrochemical or luminescent signal. In one embodiment, detecting or quantifying comprises measuring an ECL signal and correlating the signal with the presence or amount of sense or antisense oligonucleotide in the sample. In one embodiment, the intensity of the emitted light is proportional to the amount of sense or antisense oligonucleotide in the sample, such that the emitted light can provide a quantitative determination of the amount of sense or antisense oligonucleotide in the sample.

[0210] In one embodiment, the support surface is contacted with the detection mixture after the hybridization complexes are immobilized thereon. In one embodiment, the support surface is contacted with the detection mixture after the sense or antisense complexes are immobilized thereon. In one embodiment, the support surface is contacted with the detection mixture after the single-stranded overhangs in any non-productive hybridization complexes are digested with a single-stranded nuclease. In one embodiment, the digestion of the single-stranded overhangs in the non-productive hybridization complexes is performed while the hybridization complexes are in solution (i.e., before the hybridization complexes are immobilized on the support surface via the oligonucleotide tag of the sense probe or the antisense probe). In one embodiment, the digestion of the single-stranded overhangs in the non-productive hybridization complexes is performed after the hybridization complexes are immobilized on the support surface via the oligonucleotide tag of the sense probe or the antisense probe. In one embodiment, the detection mixture comprises an ECL label. Examples of ECL labels include i) organometallic compounds in which the metal is from the VIII group of noble metals, including, for example, Ru-containing organometallic compounds such as tris-bipyridyl-ruthenium (RuBpy) moieties and Os-containing organometallic compounds, and ii) luminol and related compounds. In one embodiment, the detection mixture also includes one or more electrochemiluminescence coreactants and one or more additional components such as pH buffers, surfactants, preservatives, antifoaming agents, salts, metal ions, or metal chelators. The term "electrochemiluminescence coreactant" refers to the species involved in the electrochemiluminescence label, including, but not limited to, tertiary amines such as tripropylamine (TPA), oxalate ions, ascorbic acid and persulfate for RuBpy, and hydrogen peroxide for luminol. Methods for measuring electrochemiluminescence are known, and instruments for performing the measurements are commercially available.For example, multiplexed measurements of analytes using electrochemiluminescence are used in Meso Scale Diagnostics, LLC, MULTI-ARRAY®, and SECTOR® Imager lines or products (see, e.g., U.S. Pat. Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated by reference in their entireties herein).

[0211] In one embodiment, biotin is covalently bound to the hybridization complex, and the detection mixture comprises a streptavidin-conjugated label that binds to the immobilized hybridization complex via an avidin moiety. In one embodiment, the streptavidin-conjugated label is an electrochemiluminescence (ECL) label. In one embodiment, the electrochemiluminescence label is an n-hydroxysuccinimide ester, such as Sulfo-TAG NHS-Ester (Meso Scale Diagnostics, Rockville, MD, USA).

[0212] K.Kit In one aspect, a kit is provided for carrying out the methods described herein. A "kit" refers to a set of components provided or assembled to be used together, for example, to make a composition, manufacture a device, or carry out a method. A kit can include one or more components. The components of the kit can be provided in one package or multiple packages, each of which can include one or more components. The listed components of the kit can also be provided as a single physical part, or as multiple parts that are combined for kit use. For example, the instrument components of the kit can be provided fully assembled, or as multiple instrument parts that are assembled before use. Similarly, the liquid reagent components of the kit can be provided as a complete liquid formulation in a container, as one or more dry reagents and one or more liquid diluents that are combined to provide a complete liquid formulation, or as two or more liquid solutions that are combined to provide a complete liquid formulation. As is known in the art, kit components for assays are often shipped and stored separately because they have different storage needs, for example, storage temperatures of 4°C vs. -70°C.

[0213] In one embodiment, the kit comprises a support surface. In one embodiment, the kit comprises a capture oligonucleotide that can be immobilized on the support surface. In one embodiment, the kit comprises a capture oligonucleotide immobilized on the support surface. In one embodiment, the kit comprises a capture oligonucleotide immobilized on the support surface in an array. In one embodiment, the kit comprises one or more capture oligonucleotides immobilized on one or more distinct binding domains having known positions in the array. In one embodiment, the kit comprises two or more capture oligonucleotides immobilized on a bead array.

[0214] In one embodiment, the kit comprises a carbon-based support surface. In one embodiment, the support surface comprises at least one electrode. In one embodiment, the electrode is a carbon-based electrode. In one embodiment, the support surface comprises one or more carbon ink electrodes. In one embodiment, the support surface comprises at least one working electrode and at least one counter electrode.

[0215] In one embodiment, the kit comprises a support surface comprising a multi-well assay plate. In one embodiment, one or more wells of the multi-well plate comprise one or more electrodes. In one embodiment, the support surface comprises a multi-well plate, and one or more wells comprise one or more working electrodes and one or more counter electrodes. In one embodiment, the support surface comprises one or more reference electrodes.

[0216] In one aspect, the kit includes multi-well plates in standard formats known in the art, including but not limited to 24, 96, and 384 well plates. In one aspect, the kit includes one or more 96 well plates. In one aspect, the kit includes one multi-well plate. In another aspect, the kit includes 10 multi-well plates. In another aspect, the kit includes 10-100 multi-well plates.

[0217] In one embodiment, the kit comprises a support surface having one or more electrodes onto which one or more arrays of capture oligonucleotides are printed. In one embodiment, the kit comprises one or more multiwell plates onto which one or more arrays of capture oligonucleotides are printed. In another embodiment, the kit comprises one or more multiwell plates and one or more vials containing one or more capture oligonucleotides, where the capture oligonucleotides can be printed onto the multiwell plates.

[0218] In one embodiment, the kit comprises one or more capture oligonucleotides immobilized to one or more binding domains on a support surface. In one embodiment, the kit comprises one or more capture oligonucleotides immobilized to one or more binding domains in wells of a multi-well plate. In one embodiment, the kit comprises one or more capture oligonucleotides immobilized to one or more binding domains on an electrode. In one embodiment, the kit comprises one or more capture oligonucleotides immobilized to one or more binding domains on an electrode in one or more wells of a multi-well plate.

[0219] In another embodiment, the kit comprises one or more oligonucleotide tags. In one embodiment, the kit comprises one or more oligonucleotide tags provided in containers, the oligonucleotide tags in the containers have the same sequence, and each container contains an oligonucleotide tag with a sequence that is different (and not complementary) to the sequence of the oligonucleotide tag in the other container. In one embodiment, the kit comprises at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25, and up to about 64 unique oligonucleotide tags in separate containers. In one embodiment, the kit comprises a set of up to 10 unique oligonucleotide tags.

[0220] In one embodiment, the kit comprises one or more multiwell plates in which up to 10 capture oligonucleotides are immobilized to one or more binding domains within wells of the multiwell plate, each binding domain comprising a capture oligonucleotide having a sequence that is different from the sequences of the capture oligonucleotides in other binding domains within the well. In one embodiment, the kit comprises a support surface having at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, or about 25 distinct capture oligonucleotides immobilized to at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, or about 25 unique binding domains. In one aspect, the kit comprises a multiwell plate having at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, or about 25 distinct capture oligonucleotides immobilized to at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, or about 25 unique binding domains in one or more wells. In one aspect, the kit comprises one or more multiwell plates, each well comprising up to about 10 capture oligonucleotides immobilized in an array. In one aspect, the multiwell plate can be configured to generate from about 1 to about 10 detection assays within each well of the multiwell plate.

[0221] In one embodiment, the kit comprises a single-stranded nuclease. In one embodiment, the kit comprises a single-stranded nuclease tag provided in a container. In one embodiment, the single-stranded specific nuclease comprises a single-stranded specific DNase. In one embodiment, the single-stranded specific DNase is S1 nuclease, P1 nuclease, or mung bean nuclease. In one embodiment, the single-stranded specific nuclease comprises a single-stranded specific RNase. In one embodiment, the single-stranded specific RNase is RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, PNPase, RNase PH, RNase R, RNase D, RNase T, RNaseONE, oligoribonuclease, exoribonuclease I, or exoribonuclease II.

[0222] In one aspect, a kit is provided for performing a luminescence assay, such as an electrochemiluminescence assay, to detect or quantitate one or more target nucleotide sequences in a sample. In one aspect, the kit includes one or more assay components useful for performing an electrochemiluminescence assay.

[0223] In one embodiment, the kit comprises a hybridization buffer that can be used to provide appropriate conditions (e.g., stringent conditions) for hybridization of the oligonucleotide tags with their corresponding complementary capture oligonucleotide sequences. In one embodiment, the hybridization buffer comprises Diluent 54 (Meso Scale Diagnostics, LLC, Rockville, MD, USA). In one embodiment, the hybridization buffer comprises Hybridization Buffer 1 or Hybridization Buffer 2 (Meso Scale Diagnostics, LLC, Rockville, MD, USA).

[0224] In one embodiment, the kit comprises one or more containers comprising a label. In one embodiment, the label is selected from radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light absorption, light scattering, electrochemical, magnetic, and enzymatic labels. In one embodiment, the label comprises an electrochemiluminescent label. In one embodiment, the label comprises an organometallic complex comprising a transition metal. In one embodiment, the transition metal comprises ruthenium. In one embodiment, the label is an MSD SULFO-TAG™ label (Meso Scale Diagnostics, LLC, Rockville, MD, USA).

[0225] In one embodiment, the label comprises a primary binding reagent that is a binding partner of a secondary binding reagent. In one embodiment, the secondary binding reagent comprises biotin, streptavidin, avidin, or an antibody. In one embodiment, the secondary binding reagent comprises avidin, streptavidin, or an antibody. In one embodiment, the label comprises a hapten selected from biotin, fluorescein, and digoxigenin. In one embodiment, the label is a primary binding agent that comprises a first oligonucleotide sequence and the secondary binding reagent comprises a second oligonucleotide sequence that is complementary to the first oligonucleotide sequence of the primary binding agent.

[0226] In one embodiment, the kit comprises one or more containers containing an electrochemiluminescent label. In a more particular embodiment, the kit comprises one or more containers containing a Ru-containing organometallic compound, such as tris-bipyridyl-ruthenium (RuBpy), or an Os-containing organometallic compound. In one embodiment, the label comprises an organometallic complex containing a transition metal. In one embodiment, the transition metal comprises ruthenium. In one embodiment, the label comprises an MSD SULFO-TAG™ label (Meso Scale Diagnostics, LLC, Rockville, MD, USA). In another embodiment, the kit comprises one or more containers containing luminol or other related compounds.

[0227] In one embodiment, the kit includes one or more containers having one or more electrochemiluminescent coreactants. In one embodiment, the one or more electrochemiluminescent coreactants are covalently or non-covalently immobilized on a support surface. In one embodiment, the one or more electrochemiluminescent coreactants are immobilized on one or more working electrodes of the support surface.

[0228] In one aspect, the label included in the kit comprises a primary binding reagent and a secondary binding reagent, hi one aspect, the secondary binding reagent comprises biotin, streptavidin, avidin, or an antibody.

[0229] In one embodiment, the kit includes one or more of the following assay components: one or more capture oligonucleotides, and one or more buffers, such as a wash buffer, hybridization buffer, binding buffer, or read buffer.

[0230] In one embodiment, the kit includes one or more assay components, such as a label. In one embodiment, the label is a luminescent label, such as an electrochemiluminescent label. In one embodiment, the kit includes at least one electrochemiluminescent coreactant. In one embodiment, the electrochemiluminescent coreactant includes a tertiary amine, tripropylamine, or N-butyldiethanolamine.

[0231] In one embodiment, the kit includes one or more other assay components. In one embodiment, the kit includes one or more assays, including but not limited to diluents, blocking agents, stabilizers, detergents, salts, pH buffers, and preservatives. In one embodiment, the kit includes containers for one or more such components. In another embodiment, one or more reagents are included on an assay support surface provided with the kit.

[0232] L. Incorporation by Reference All references cited herein, including patents, patent applications, articles, textbooks, and the like, and the references cited therein, unless already cited, are hereby incorporated by reference in their entirety for all purposes.

[0233] Working Example Example 1. RNase Protection Assay (RPA) Lower Limit of Detection (LLOD) Using Full-Length Probes The detection limits of the sense (SS) and antisense (AS) strands of a model 16-mer heteroduplex antisense oligonucleotide (ASO) (one DNA strand and one RNA strand, both modified) were determined in the absence of the opposing strand using an RNase protection assay (RPA).

[0234] A chimeric full-length antisense probe was generated comprising an RNA strand having a sequence complementary to a nucleic acid-based therapeutic molecule and a DNA strand having a sequence complementary to a capture oligonucleotide, and contained a single-stranded oligonucleotide tag, a full-length (16-mer) antisense binding portion, and a biotin label. A chimeric full-length sense probe was generated comprising an RNA strand having a sequence complementary to a nucleic acid-based therapeutic molecule and a DNA strand having a sequence complementary to a capture oligonucleotide, and contained a single-stranded oligonucleotide tag, a full-length (16-mer) sense binding portion, and a biotin label.

[0235] Briefly, individual antisense (AS) or sense (SS) strands (10 mg / mL) were diluted to 200 μg / mL and 10-point calibration curves were generated for each individual antisense or sense strand by spiking ASO at the highest calibrator concentration of 40,000 pg / mL in diluent 54 as shown in Table 1 and using 4-fold serial dilutions (+2 blank wells).

[0236] [Table 1]

[0237] Two concentrations of full-length probe were used to assess the effect on positive signal and background: 50 pM or 200 pM (4x). Chimeric probes were hybridized to the antisense or sense strand in Diluent 54 (Meso Scale Diagnostics, Rockville, MD, USA) using the hybridization protocol shown in Table 2.

[0238] [Table 2]

[0239] 96-well N-PLEX® plates (Meso Scale Discovery, Rockville, MD, USA ("MSD")) with immobilized single-stranded capture oligonucleotides (having sequences complementary to the oligonucleotide tag sequences of the chimeric sense and antisense probes) were blocked with N-PLEX™ Blocking Buffer (MSD) and washed 3× with a minimum of 150 μL / well of Dulbecco's phosphate-buffered saline (DPBS). After the probes were hybridized to their target strands, the probe / analyte complexes were diluted in buffer, added to the plate, and hybridized for 1 hour at 37° C. with shaking. Two different buffers were used to determine their effect on hybridization with the plate: Diluent 54, or a 2:3 ratio blend of NPLEX™ Hybridization Buffers 1 and 2 (MSD).

[0240] Plates were washed again (3x with a minimum of 150 μL / well DPBS) and RNase cocktail (RNase A, RNase I, and RNase T1) in diluent 54 was added to the plates and incubated at 37°C for 30 minutes with shaking.

[0241] The plate was washed again and SULFO-TAG™ Streptavidin (MSD) was added to the plate in Diluent 54 + 1% Blocker A (MSD) and incubated for 30 minutes at room temperature with shaking. The plate was washed and MSD GOLD™ Read Buffer A (MSD) was added and the plate was read using a SECTOR® S6000 or MESO® SECTOR® S600 imager (MSD).

[0242] The ECL signals for the antisense strand and probe are shown in Table 3 and for the sense strand and probe in Table 4. The probe against the antisense (DNA) strand had a higher ECL reading than the probe against the sense (RNA) strand in Diluent 54 and Hybridization Buffer blend. Increasing the amount of probe increased the positive and negative signals, with signal saturation at approximately 10,000 pg / mL (10 ng / mL).

[0243] 5A and 5B show ECL signal curves for antisense (AS) strand diluent 54 plate hybridization (FIG. 5A) and mixed hybridization buffer plate hybridization (FIG. 5B), and sense (SS) strand diluent 54 plate hybridization (FIG. 6A) and mixed hybridization buffer plate hybridization (FIG. 6B).

[0244] Tables 5 and 6 show the lower limit of detection (LLOD) for the antisense (AS) and sense (SS) probes and strands, respectively.

[0245] The results demonstrate that the antisense probe has a higher ECL signal and a lower LLOD than the sense probe, and although the background tends to be higher with the sense probe, both assays give good results. Results for the blend of Diluent 54 and Hybridization Buffer were similar.

[0246] [Table 3]

[0247] [Table 4]

[0248] [Table 5]

[0249] [Table 6]

[0250] Example 2. Hybridization of truncated probes The ability of truncated probes (12-mer, 11-mer, and 10-mer) to hybridize, in the absence of the opposing strand, to the sense (SS) and antisense (AS) strands of a model 16-nucleotide heteroduplex antisense oligonucleotide (ASO) from Example 1 was assessed in an RNase protection assay (RPA). The probes were truncated at the 3' end.

[0251] Briefly, a 10-point calibration curve (+2 blank wells) was generated using individual antisense (AS) or sense (SS) strands from the ASO with 4-fold serial dilutions and calibrators up to 40,000 pg / mL.

[0252] Four different probe lengths were evaluated for detection according to the methods described in Example 1 above: a full-length (FL) probe, a 12-mer probe, an 11-mer probe, and a 10-mer probe using a probe concentration of 200 pM. Probes were hybridized to the antisense or sense strand in Diluent 54 and hybridized to the plate in either Diluent 54 or Hybridization Buffer 1. Calibration curves were split between Diluent 54 or Hybridization Buffer 1 in series rather than in duplicate wells.

[0253] Four chimeric antisense (AS) probes were generated that contained a single-stranded oligonucleotide tag, an antisense binding portion (full-length 16-mer (FL), 12-mer, 11-mer, or 10-mer), and a biotin label.

[0254] Four chimeric sense (SS) probes were generated that contained a single-stranded oligonucleotide tag, a sense binding portion (full-length 16-mer (FL), 12-mer, 11-mer, or 10-mer), and a biotin label.

[0255] Tables 7 and 8 show the ECL signals for the antisense probe and strand hybridized in diluent 54 or hybridization buffer, respectively. Tables 9 and 10 show the ECL signals for the sense probe and strand hybridized in diluent 54 or hybridization buffer, respectively. The data from Tables 7-10 are shown graphically in Figures 7A-7B.

[0256] Table 11 shows the lower limit of detection (LLOD) for the antisense (AS) probe and strand for the 16-mer (FL) probe and the 12-mer probe in Diluent 54 and hybridization buffer. Tables 12 and 13 show the LLOD for the sense (SS) probe and strand for the 16-mer (FL) probe, the 12-mer probe, the 11-mer probe, and the 10-mer probe in Diluent 54 and hybridization buffer, respectively.

[0257] The data show that hybridization with hybridization buffer results in a loss of signal with the 12-mer probe on the antisense (AS) strand compared to diluent 54. For the antisense strand, shortening the probe reduces the ECL signal and LLOD. est For the sense strand, shortening the probe has a negative effect on the ECL signal and LLOD. est The type of hybridization buffer had no effect on the assay.

[0258] [Table 7]

[0259] [Table 8]

[0260] [Table 9]

[0261] [Table 10]

[0262] [Table 11]

[0263] [Table 12]

[0264] [Table 13]

[0265] Example 3. Heteroduplex detection of antisense and sense strands The detection limits of the antisense (AS) and sense (SS) strands of the model ASO from Example 1 were determined for RNase protection assays (RPA) using the full-length (16-mer) chimeric probe or the 12-mer truncated chimeric probe from Example 2.

[0266] Ten-point calibration curves were generated using individual AS strands, SS strands, or heteroduplexes with a top calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (plus two blank wells).

[0267] Two different probe lengths were evaluated: full length 16-mer (FL) and 12-mer at a concentration of 200 pM.

[0268] Probes were hybridized to either the antisense or sense strand in diluent 54 essentially as described in Example 2 and added in diluent 54 in duplicate wells of a 96-well plate.

[0269] The ECL signals for antisense strand detection and sense strand detection are shown in Tables 14 and 15, respectively. Data from Table 14 is shown graphically in Figures 8A-8B, and data from Table 15 is shown graphically in Figures 9A-9B.

[0270] Table 16 shows the lower limit of detection (LLOD) for the antisense (AS) strand alone or in a heteroduplex using 16-mer (FL) and 12-mer probes. Table 17 shows the LLOD for the sense (SS) strand alone or in a heteroduplex using 16-mer (FL) and 12-mer probes.

[0271] The results show that the 12-mer probe reduced the ECL signal in the antisense strand but not in the sense strand. The full-length probe had similar ECL signals for both strands, whether from individual strands or in heteroduplexes. The 12-mer probe reduced the ECL signal for both the antisense and sense strands in the heteroduplex. The heteroduplex did not show any significant ECL signal or LLOD when using full-length probes for both the antisense and sense strands. est The 12-mer probe reduced the ECL signal for both the antisense and sense strands in the heteroduplex and did not affect the LLOD. est Increased.

[0272] [Table 14]

[0273] [Table 15]

[0274] [Table 16]

[0275] [Table 17]

[0276] Example 4. Lower limit of detection (LLOD) using full-length (16-mer) probes The detection limits for individual antisense (AS) or sense (SS) strands or the complete model heteroduplex ASO from Example 1 were determined for the RNase protection assay (RPA) from Example 1 using the 16-mer full-length (FL) probe.

[0277] Briefly, 10-point calibration curves were generated from individual antisense strands, sense strands, or heteroduplexes using 4-fold serial dilutions (+2 blank wells) with a highest calibrator concentration of 40,000 pg / mL. For individual strands, 2 wells were used for each calibrator or individual strand, and 4 wells were used for each calibrator for heteroduplexes along with 24 individual blank wells for total LLOD determination. Two probe lengths were evaluated: full length 16-mer (FL) and 12-mer at a concentration of 200 pM. Probes were hybridized to the antisense (AS) or sense (SS) strands and hybridized to the plate in diluent 54.

[0278] As shown in Table 18, the ECL signals are similar between the individual antisense or sense strands and the heteroduplex. The results shown in Table 19 show that the LLOD of the singleplex assay on the antisense strand was less than 0.5 pg / mL for both the individual strands and the heteroduplex, and the LLOD of the singleplex assay on the sense strand was 25 pg / mL and 14 pg / mL for the individual strands and the heteroduplex, respectively. The coefficients of variation (CV) in Cal-3 were all less than 15%, and the CV on the antisense strand was much lower than that of the sense strand.

[0279] [Table 18]

[0280] [Table 19]

[0281] Example 5. Multiplex detection of heteroduplexes This experiment was designed to determine whether the antisense (AS) and sense (SS) strands of the model heteroduplex antisense oligonucleotide (ASO) from Example 1 could be detected using the RNase Protection Assay (RPA) from Example 1 in a multiplex format.

[0282] Briefly, 10-point calibration curves were generated from individual antisense (AS) strands, sense (SS) strands, or heteroduplexes using 4-fold serial dilutions (+2 blank wells) with a top calibrator concentration of 40,000 pg / mL. Wells with probe only were included to determine whether probe-probe interactions occurred with antisense and sense probes and whether probe-probe interactions could be eliminated with a shortened 12-mer probe.

[0283] Two probe lengths were evaluated: a full length 16-mer (FL) and a 12-mer (probe sequences are shown in Example 2) at a concentration of 200 pM.

[0284] Probes were hybridized to either the antisense or sense strand and were hybridized in diluent 54 in duplicate wells of a 96-well plate.

[0285] The ECL signals for antisense strand detection using 16-mer (FL) and 12-mer probes are shown in Tables 20 and 21, respectively. The ECL signals for sense strand detection using 16-mer (FL) and 12-mer probes are shown in Tables 22 and 23, respectively. The ECL data are shown graphically in Figures 10A and 10B. Table 24 shows the lower limit of detection (LLOD) for multiplex detection using 12-mer probes for antisense (AS) and sense (SS) strands alone or in heteroduplexes.

[0286] The results show that when full-length probes were used in multiplex assays, antisense and sense probes cross-reacted, causing greatly elevated background signals. At high concentrations, there was an additive effect with the antisense strand (individually or heteroduplexes), while there was an inverse correlation with the sense strand in the antisense spot signal and vice versa (likely due to loss of probe-probe interaction upon target binding to the probe).

[0287] Use of the 12-mer probe in either singleplex (Example 2) or multiplex (This Example) resulted in similar ECL levels, indicating no detrimental effects of multiplexing the probes.

[0288] [Table 20]

[0289] [Table 21]

[0290] [Table 22]

[0291] [Table 23]

[0292] [Table 24]

[0293] Example 6. Effect of hybridization time on heteroduplex detection The effect of shortened hybridization times on the detection limits of the antisense (AS) and sense (SS) strands of the model ASO from Example 1 was determined.

[0294] Briefly, a 10-point calibration curve was generated using a 4-fold serial dilution (+2 blank wells) using the heteroduplex with a highest calibrator concentration of 40,000 pg / mL. Two probe lengths were evaluated: full length 16-mer (FL) and 12-mer at a concentration of 200 pM. Probes were hybridized to the antisense or sense strand in Diluent 54 using the "long hybridization" protocol (Table 25) and the "short hybridization" protocol (Table 26). Probes were hybridized in Diluent 54 in duplicate wells of a 96-well plate.

[0295] The full length (FL) probe had better signals than the 12-mer probe in detecting both the antisense (AS) and sense (SS) strands in the heteroduplex. The results are shown graphically in Figures 11A and 11B. Varying the hybridization time did not have a significant effect on ECL signal generation.

[0296] The detection sensitivity of the ASO or SO strand of a heteroduplex is not affected by decreasing the amount of time for the probe to hybridize to its corresponding strand. To shorten the assay time, it is recommended to perform the probe / strand hybridization under shortened hybridization conditions.

[0297] [Table 25]

[0298] [Table 26]

[0299] Example 7. Detection of antisense and sense strands in plasma The detection limits for the antisense (AS) or sense (SS) strands of the model ASO from Example 1 were determined by spiking into mouse plasma.

[0300] Briefly, 10-point calibration curves were generated using individual antisense strands, sense strands, or heteroduplexes with a top calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (+2 blank wells). Both mouse plasma and diluent calibration curves were pretreated with RNAsecure™ RNase inactivation reagent for 10 min at 60°C and held at 4°C.

[0301] RNAsecure™ reagent was added to Diluent 54 in a 1:1 ratio (20 μL calibrator or blank: 20 μL RNAsecure™ reagent). Full length 16-mer (FL) probes were used at a final concentration of 200 pM. 5× probes were added in 10 μL volumes prepared in Diluent 54 for a total volume of 50 μL. Probes were hybridized to the antisense or sense strands in Diluent 54 or in plasma in duplicate wells using the shortened hybridization protocol shown in Table 26.

[0302] Data (not shown) show that the ECL signal generated from antisense strand detection does not change significantly with either individual strands or portions of the heteroduplex using plasma as a matrix. As shown in Figures 12A and 12B, the ECL signal was lost when individual sense strands (RNA) were added to mouse plasma, and decreased when the sense strand of the heteroduplex was added to mouse plasma. This is likely due to endogenous RNases in plasma that degrade ssRNA. Table 27 shows the lower limit of detection (LLOD) for the antisense strand in diluent 54 or in plasma. Table 28 shows the lower limit of detection (LLOD) for the sense strand in diluent 54 or in plasma.

[0303] The results demonstrate that the ability to sensitively detect the antisense strand (DNA) was not altered when measured in plasma. For the antisense strand, both the individual strands of the heteroduplex and the antisense strand had similar LLODs in diluent and plasma. est However, the ability to detect the sense strand (RNA) was altered when measured in plasma: individual sense strands were undetectable, and the sense strand of the heteroduplex was detectable, but with reduced sensitivity and a reduced dynamic range.

[0304] [Table 27]

[0305] [Table 28]

[0306] Example 8. Singleplex detection of antisense and sense strands in mouse plasma The limit of detection for the antisense and sense strands from Example 1 or the full model heteroduplex ASO was determined in mouse plasma using a full-length (16-mer) probe and an RNase protection assay (RPA) essentially as described in Example 1 (with the modifications provided below).

[0307] Briefly, a 10-point calibration curve was generated using heteroduplexes with a highest calibrator concentration of 40,000 pg / mL and 4-fold serial dilutions (+2 blank wells) in mouse plasma. For total lower limit of detection (LLOD) determination, 4 wells were used for each calibrator along with 24 blank wells. Calibrator samples and blanks were pretreated with RNAsecure™ as described in Example 7.

[0308] Probes were hybridized to either the antisense or sense strands using the shortened hybridization protocol described in Example 6, with one modification: the denaturation temperature was lowered to 80° C., since heating plasma to 95° C. makes it too viscous for easy pipetting. The revised protocol is shown in Table 29.

[0309] [Table 29]

[0310] Results (not shown) show that the ECL signals are consistent with the plasma experiments in Example 7. The top of curve (TOC) signal for the antisense strand (DNA) is significantly higher than that for the sense strand (RNA). The LLOD for the antisense strand (DNA) of the heteroduplex was about 1 pg / mL, and the LLOD for the sense strand (RNA) was about 31 pg / mL in the singleplex assay. The CVs for Cal-3 and blank were all less than 10%, indicating low signal variability.

[0311] Example 9. Effect of Blocker S1 on Background The effect of adding blocker S1 to the N-PLEX™ blocking buffer was evaluated for its impact on the background associated with antisense (AS) and sense (SS) singleplex assays.

[0312] Briefly, a 10-point calibration curve was generated from the heteroduplex using 4-fold serial dilutions (+2 blank wells) with a top calibrator concentration of 40,000 pg / mL. Full-length (16-mer) probes were used at a concentration of 200 pM, essentially following the procedure for simplex antisense and sense assays described in Example 8. Blocker S1 (MSD) was added to the N-PLEX™ blocker (MSD). Probes were hybridized to the sense or antisense strands using Diluent 54 (MSD).

[0313] The results show that including blocker S1 in the N-PLEX™ blockers had no effect on positive or background signal generation for either the AS or SS strands in singleplexes.

[0314] Example 10. Effect of double RNase digestion on background The effect of performing two RNase digestion steps was evaluated for its impact on the background associated with antisense (AS) and sense (SS) singleplex assays described in Example 1.

[0315] Briefly, a 10-point calibration curve was generated from the heteroduplex using 4-fold serial dilutions (+2 blank wells) with a top calibrator concentration of 40,000 pg / mL. Full-length (16-mer) probes were used at a concentration of 200 pM essentially following the method for simplex antisense and sense assays described in Example 8. RNase digestion steps were performed once or twice. Wells that had undergone one RNase digestion step were incubated with Diluent 54 during the second RNase digestion step. Probes were hybridized to the antisense or sense strands using Diluent 54.

[0316] Data (not shown) show that adding an additional RNase digestion step significantly reduced the positive or background ECL signal or LLOD est It shows that it had little effect on the measurements.

[0317] Example 11. Detection of antisense and sense strands in brain lysates The limit of detection for the antisense and sense strands from Example 1 or the full model heteroduplex ASO was determined using full-length (16-mer) probes in brain lysates using an RNase protection assay (RPA) essentially as described in Example 8 (with the modifications provided below).

[0318] Briefly, a 10-point calibration curve was generated using the antisense strand, sense strand, and heteroduplex spiked into diluted brain lysate using 4-fold serial dilutions (+2 blank wells) with a top calibrator concentration of 40,000 pg / mL. The full-length 16-mer (FL) probe was used at a concentration of 200 pM.

[0319] Probes were hybridized to the antisense or sense strand according to the protocol described in Example 8. Brains (BioIVT, Westbury, NY, USA) were homogenized and lysed in Diluent 54. The homogenates were centrifuged and the lysates were collected and diluted 1:4 in Diluent 54.

[0320] Data (not shown) show that ECL signal generation from AS strands (individually or as part of a heteroduplex) is very similar between diluent 54 or 1:4 diluted brain lysate. As shown in Figures 13A and 13B, ECL signal was much lower from sense (SS) strands (individually or as part of a heteroduplex) in brain lysate compared to diluent 54. In contrast to the results of Example 8 showing results in plasma, individual sense (SS) strands generate higher ECL signal than the sense (SS) strand of the heteroduplex in brain lysate. This may be due to the release of RNase H upon lysis of brain cells.

[0321] The LLODs of the antisense (AS) and sense (SS) strands alone or in heteroduplexes in diluent 54 or in brain lysates are shown in Tables 30 and 31. These results show that the antisense (DNA) strand can be easily measured in diluted brain lysates.

[0322] [Table 30]

[0323] [Table 31]

[0324] Example 12. Addition of LNA to the probe The effect of including a locked nucleic acid (LNA) in a 12-mer RNase protection probe on the detection of either the antisense or sense strand of the model ASO from Example 1 was evaluated and determined in a singleplex assay. The method was performed essentially as described in Example 8.

[0325] Briefly, 10-point calibration curves were generated using spiked antisense, sense, and heteroduplexes with a top calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (+2 blank wells). Full-length 16-mer (FL) or 12-mer probes with LNA were used at a concentration of 200 pM. Probes were hybridized to the antisense or sense strands of model ASOs using diluent 54.

[0326] Figures 14A and 14B show the effect of LNA on 16mer (FL) or 12mer RNase probes for detecting the antisense (AS) strand alone or in a heteroduplex. Table 32 shows the effect of LNA on 16mer (FL) or 12mer RNase probes for detecting the sense (SS) strand alone or in a heteroduplex. Table 33 shows the LLOD on FL or 12mer antisense probes with and without LNA. Table 34 shows the LLOD on FL or 12mer sense probes with and without LNA.

[0327] The results show that the full-length 16-mer (FL) probe produced higher positive signals and lower background signals for both AS and SS singleplex assays. The higher background observed with the LNA probe is likely due to the resistance of the LNA oligo to nuclease degradation. Although the target of degradation in the probe is the RNA residue, the LNA helped to protect the entire oligo from RNases.

[0328] [Table 32]

[0329] [Table 33]

[0330] [Table 34]

[0331] Example 13. Concentration test of LNA probes The effect of decreasing concentrations of LNA probes on background levels and sensitivity of singleplex AS and SS assays was evaluated for the model ASO from Example 1.

[0332] Briefly, a 10-point calibration curve was generated using a heteroduplex with a highest calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (+2 blank wells). AS and SS assays were performed in singleplex, and a 12-mer probe with LNA was used in both AS and SS assays at 4 concentrations: 200 pM (0.2 nM), 100 pM (0.1 nM), 50 pM (0.05 nM), and 20 pM (0.02 nM). The probe was hybridized to the antisense (AS) strand or the sense strand (SS) using diluent 54.

[0333] Figures 15A and 15B show that decreasing the concentration of the LNA probe to either the AS or SS strand of the heteroduplex reduces both the positive and background ECL signals at probe concentrations of 0.02 mM and 0.1 nM, respectively, providing improved positive and background signals.

[0334] Tables 35 and 36 show the LLOD at various LNA probe concentrations for detecting the antisense or sense strand, respectively, in a heteroduplex.

[0335] [Table 35]

[0336] [Table 36]

[0337] Example 14. LNA probes for multiplex detection of antisense and sense strands Multiplex detection of the AS and SS strands of the model heteroduplex from Example 1 was evaluated using LNA probes.

[0338] Briefly, 10-point calibration curves were generated using individual antisense (AS) strands, sense (SS) strands, or heteroduplexes with a top calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (+2 blank wells). The assay included wells with probe only to determine whether probe-probe interactions occurred between the antisense and sense probes.

[0339] Two different probe types were evaluated: unmodified 12-mer probe or LNA 12-mer probe. Probe concentration of 200 pM for unmodified 12-mer probe. 12-mer AS probe was used at a concentration of 20 pM and 12-mer SS probe was used at a concentration of 100 pM for AS. SS LNA probe was also used at a concentration of 100 pM. The concentration of LNA probe was varied to account for higher background. Probes were hybridized to the antisense or sense strand using diluent 54.

[0340] Figures 16A and 16B show that there was no off-target signal from either the unmodified 12-mer probe or the LNA 12-mer probe, and no probe-probe interactions occurred in either case, but the LNA probe gave a lower positive ECL signal and similar background for the AS strand than the unmodified 12-mer probe.

[0341] Figures 17A and 17B show that there was no off-target signal from either the unmodified 12-mer probe or the LNA 12-mer probe, indicating that no probe-probe interactions occurred in either case with the SS probe.

[0342] Table 37 shows the LLODs for multiplex detection of individual antisense (AS) strands or antisense strands in heteroduplexes using unmodified and LNA-modified 12-mer probes. Table 38 shows the LLODs for multiplex detection of individual sense (SS) strands or sense strands in heteroduplexes using unmodified and LNA-modified 12-mer probes.

[0343] The LNA probe was able to detect both the AS and SS strands, either as individual strands or as part of a heteroduplex. There was no advantage to using the 12-mer LNA probe over the full-length probe in a singleplex assay. The LNA probe had a higher background for both assays and had to be diluted to bring the background to an acceptable level. The specific signal was also sacrificed. The unmodified 12-mer probe outperformed the 12-mer LNA probe in multiplex detection of the AS and SS strands.

[0344] [Table 37]

[0345] [Table 38]

[0346] Example 15. Singleplex detection of antisense and sense strands using 13-mer and 14-mer probes Detection of the individual antisense (AS) and sense (SS) strands of the model heteroduplex from Example 1 was assessed using 13-mer and 14-mer probes according to the methods described in Example 8.

[0347] Briefly, a 10-point calibration curve was generated using a heteroduplex with a highest calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (+2 blank wells). Four different probe types were evaluated: unmodified full-length (FL) probe, 14-mer probe, 13-mer probe, and 12-mer probe at a concentration of 200 pM. Probes were hybridized to the antisense or sense strand using diluent 54.

[0348] Figures 18A and 18B show that the ECL signal for the 14-mer probe was similar to the FL probe in antisense and sense strand detection, but slight signal loss was observed with the 13-mer probe. A more significant loss was observed with the 12-mer probe compared to the FL probe. The background was increased with the 12-mer and 13-mer probes.

[0349] Tables 39 and 40 show the LLODs for detection of the antisense (AS) strand or the sense (SS) strand using a full length (FL) probe, a 14-mer probe, a 13-mer probe, or a 12-mer probe.

[0350] [Table 39]

[0351] [Table 40]

[0352] Example 16. Heteroduplex detection of antisense and sense strands using 13-mer and 14-mer probes Detection of the antisense (AS) and sense (SS) strands of the model heteroduplex from Example 1 was assessed using 13-mer and 14-mer probes essentially as described in Example 8.

[0353] Briefly, a 10-point calibration curve was generated using a heteroduplex with a highest calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (+2 blank wells). Four different probe types were evaluated: unmodified full-length (FL) probe, 14-mer probe, 13-mer probe, and 12-mer probe at a concentration of 200 pM. Probes were hybridized to the antisense or sense strand using diluent 54.

[0354] As shown in Figures 19A and 19B, the EL and 14-mer probes gave similar signals when measuring the AS strand of the heteroduplex. When measuring the AS strand of the heteroduplex, the 13-mer probe showed a significant loss of signal, and the 12-mer probe was unable to generate sufficient signal. The background was increased with the 13-mer probe. For SS probe and strand detection, the 14-mer and 13-mer probes were similar to the FL probe, while there was some signal loss with the 12-mer probe. The background was increased with the 12-mer probe.

[0355] Tables 41 and 42 show the LLODs for heteroduplex detection of the antisense (AS) or sense (SS) strands using full length (FL), 14-mer, 13-mer, or 12-mer probes.

[0356] [Table 41]

[0357] [Table 42]

[0358] Example 17. Multiplexed detection of antisense and sense strands using 13-mer and 14-mer probes This experiment was designed to determine whether a 13-mer probe or a 14-mer probe would allow for multiplex detection of the antisense (AS) and sense (SS) strands or the model heteroduplex from Example 1.

[0359] Briefly, a 10-point calibration curve was generated using the heteroduplex with a highest calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (+2 blank wells). Probe-only wells were included to assess probe-probe interactions.

[0360] Two different probe types were evaluated: a 14-mer probe and a 13-mer probe at a concentration of 200 pM. The probes were hybridized in diluent 54 to either the antisense or sense strand.

[0361] As shown in Table 43 and Table 44, the 14-mer probe allowed detection of both individual AS strands and heteroduplex AS strands, but the 14-mer probe showed a slight decrease in signal when measuring heteroduplexes compared to individual strands. Also, the 13-mer probe allowed detection of individual AS strands and heteroduplex AS strands, but signal reduction was observed. Importantly, both the 14-mer probe and the 13-mer probe were suitable for multiplex detection of heteroduplex AS strands and did not cause probe-probe interaction.

[0362] As shown in Tables 45 and 46, both the 14-mer probe and the 13-mer probe allowed detection of the SS strand of the heteroduplex, but the 14-mer probe gave a slightly higher signal than the 13-mer probe. Importantly, both the 14-mer probe and the 13-mer probe were suitable for multiplex detection of the SS strand of the heteroduplex and did not cause probe-probe interactions.

[0363] Tables 47 and 48 show the LLODs for detection of the antisense (AS) or sense (SS) strands alone or as part of a heteroduplex using 14-mer and 13-mer probes.

[0364] Therefore, the 14-mer probe is the probe of choice for the antisense strand, while either the 13-mer or 14-mer probe can be used to detect the sense strand.

[0365] [Table 43]

[0366] [Table 44]

[0367] [Table 45]

[0368] [Table 46]

[0369] [Table 47]

[0370] [Table 48]

[0371] Example 18. Probe cross-check for multiplex detection This experiment was designed to evaluate different combinations of AS and SS probes in the multiplex detection of both strands of a heteroduplex.

[0372] Briefly, a 10-point calibration curve was generated using a heteroduplex with a highest calibrator concentration of 40,000 pg / mL using 4-fold serial dilutions (+2 blank wells). Three different probe types were evaluated: a 14-mer probe, a 13-mer probe, and a 12-mer probe at a concentration of 200 pM. Probes were hybridized to the antisense or sense strand using diluent 54.

[0373] Each probe was crossed with the other two probes as follows.

[0374] 14mer AS vs 14mer SS 14-mer AS vs 13-mer SS 14-mer AS vs 12-mer SS 13-mer AS vs 14-mer SS 13-mer AS vs 13-mer SS 13-mer AS vs 12-mer SS 12mer AS vs 14mer SS 12mer AS vs 13mer SS 12mer AS vs. 12mer SS was not evaluated.

[0375] As shown in Figure 20A, the ECL signal was consistent for a given AS probe length, regardless of the length of the SS probe. The 14-mer AS probe showed less signal loss than the 13-mer and 12-mer AS probes. As shown in Figure 20B, the change in the length of the SS probe had a less dramatic effect on the ECL signal compared to the change in the AS probe length. The SS signal decreased as the AS probe decreased, especially for the 12-mer AS probe. Importantly, either the 14-mer SS probe or the 13-mer SS probe performed well for multiplex detection.

[0376] Table 49 provides the LLOD and Hill slope measurements for the probe combinations. These data demonstrate that by using truncated probes it is possible to sensitively measure both the sense and antisense strands of a heteroduplex in the same well.

[0377] [Table 49]

Claims

1. 1. A method for detecting or quantifying sense and antisense strands of an oligonucleotide duplex in a sample, the method comprising: (a) contacting the sample with a composition comprising a set of probes, wherein the set of probes comprises: (i) a sense probe comprising a first single-stranded oligonucleotide tag that is complementary to at least a portion of a first capture oligonucleotide immobilized on a support surface, a sense binding moiety capable of hybridizing to a nucleotide sequence of the sense strand of the oligonucleotide duplex, and a first label; and (ii) an antisense probe comprising a second single-stranded oligonucleotide tag that is complementary to at least a portion of the second capture oligonucleotide immobilized on the support surface, an antisense binding moiety capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label; the sense binding portion of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand; contacting, wherein the antisense binding portion of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand; (b) incubating the probe with the sample; (i) a sense complex comprising the sense probe hybridized to the sense strand of the oligonucleotide duplex; and (ii) forming a hybridization mixture comprising a hybridization complex, the hybridization complex comprising an antisense complex comprising the antisense probe hybridized to the antisense strand of the oligonucleotide duplex; (c) contacting the support surface with the hybridization mixture in which the first and second oligonucleotide tags of the sense and antisense probes hybridize to the first and second capture oligonucleotides immobilized on the support surface, and contacting the hybridization complexes in the hybridization mixture with a single-strand-specific nuclease; (d) detecting or quantifying the sense and antisense strands of the oligonucleotide duplex based on the presence of the label on the support surface.

2. (c) is (i) contacting the support surface with the hybridization mixture under conditions such that the first and second oligonucleotide tags of the hybridization complexes hybridize to the first and second capture oligonucleotides on the support surface, thereby immobilizing the hybridization complexes on the support surface; (ii) contacting the immobilized hybridization complex with a single-strand-specific nuclease; or (i) contacting the hybridization mixture with a single-strand-specific nuclease to form a reaction mixture; 2. The method of claim 1, comprising: (ii) contacting the support surface with the reaction mixture of (i) under conditions in which the first and second oligonucleotide tags of the hybridization complex hybridize to the first and second capture oligonucleotides immobilized on the support surface.

3. 2. The method of claim 1, wherein the sense strand and the antisense strand of the oligonucleotide duplex each individually comprise from about 8 to about 50 nucleotides.

4. 2. The method of claim 1, wherein the sense binding length of the sense probe is at least one nucleotide shorter than the sense strand length of the sense strand, or the sense binding portion of the sense probe has a 5' end that aligns with the 3' end of the sense strand of the oligonucleotide duplex.

5. 2. The method of claim 1, wherein the antisense binding length of the antisense probe is at least one nucleotide shorter than the antisense strand length of the antisense strand, or has a 5' end that aligns with the 3' end of the antisense strand of the oligonucleotide duplex.

6. 2. The method of claim 1, wherein the first oligonucleotide tag has a first oligonucleotide tag length, the first capture oligonucleotide has a first capture oligonucleotide length, and the first oligonucleotide tag length is the same as or shorter than the first capture oligonucleotide length.

7. 2. The method of claim 1, wherein the second oligonucleotide tag has a second oligonucleotide tag length, the second capture oligonucleotide has a second capture oligonucleotide length, and the second oligonucleotide tag length is the same as or shorter than the second capture oligonucleotide length.

8. The method of claim 1 , wherein steps (a) to (c) are carried out simultaneously.

9. The method of claim 1 , wherein steps (a) to (c) are performed sequentially.

10. (b) the hybridization conditions are (i) incubating the probe with the sample at a first temperature to denature the sense and antisense strands of the oligonucleotide duplex; 2. The method of claim 1, comprising: (ii) incubating the probe with the denatured sense strand and denatured antisense strand of the oligonucleotide duplex at a second temperature to hybridize the sense probe and the antisense probe to the sense strand and the antisense strand.

11. The hybridization conditions in (b) are: (i) incubating the probe with the sample at a first temperature of about 60°C to about 95°C for about 1 minute to about 15 minutes; (ii) incubating the probe with the sample at a second temperature of about 10°C to about 65°C for about 30 seconds to about 5 minutes; (iii) incubating the probe with the sample at a holding temperature of about 2°C to about 8°C.

12. 2. The method of claim 1, wherein the sample comprises a plurality of oligonucleotide duplexes and the composition in (a) comprises a plurality of probe sets, each probe set hybridizing to a unique sense or antisense strand of a unique oligonucleotide duplex.

13. The method of claim 1, wherein the composition in (a) comprises about 20 pM to about 10 nM of sense probe and / or about 20 pM to about 10 nM of antisense probe.

14. 10. The method of claim 1, wherein the sample comprises a biological sample, an environmental sample, a manufacturing process sample, or a combination thereof.

15. 10. The method of claim 1, wherein the method has a limit of detection of less than about 200 pg / mL.

16. The method of claim 1 , wherein the support surface comprises one or more electrodes.

17. 1. A composition comprising a set of probes, said set of probes comprising: (a) a sense probe comprising a first single-stranded oligonucleotide tag complementary to at least a portion of the first capture oligonucleotide, a sense binding portion capable of hybridizing to a nucleotide sequence of the sense strand of the oligonucleotide duplex, and a first label; (b) an antisense probe comprising a second single-stranded oligonucleotide tag complementary to at least a portion of the second capture oligonucleotide, an antisense binding moiety capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label; the sense binding portion of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand; The composition, wherein the antisense binding portion of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand.

18. 1. A composition comprising: (a) an oligonucleotide duplex comprising a sense strand and an antisense strand; (b) a set of probes, (i) a sense probe comprising a first single-stranded oligonucleotide tag complementary to at least a portion of the first capture oligonucleotide, a sense binding portion capable of hybridizing to a nucleotide sequence of the sense strand of the oligonucleotide duplex, and a first label; (ii) an antisense probe comprising a second single-stranded oligonucleotide tag that is complementary to at least a portion of the second capture oligonucleotide, an antisense binding moiety that can hybridize to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label; the sense binding portion of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand; The composition, wherein the antisense binding portion of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand.

19. 1. A composition comprising one or more hybridization complexes, said hybridization complexes comprising: (a) a sense complex comprising a sense probe hybridized to a sense strand of an oligonucleotide duplex, the sense probe comprising a first single-stranded oligonucleotide tag complementary to at least a portion of a first capture oligonucleotide, a sense binding portion capable of hybridizing to a nucleotide sequence of the sense strand of the oligonucleotide duplex, and a first label, wherein the sense binding portion of the sense probe has a sense binding length that is shorter than the sense strand length of the sense strand; (b) an antisense complex comprising an antisense probe hybridized to the antisense strand of the oligonucleotide duplex, wherein the antisense probe comprises a second single-stranded oligonucleotide tag complementary to at least a portion of a second capture oligonucleotide, an antisense binding moiety capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label, wherein the antisense binding moiety of the antisense strand has an antisense binding length that is shorter than the antisense strand length of the antisense strand; and combinations thereof.

20. (i) one or more capture oligonucleotides; (ii) at least a portion of a first capture oligonucleotide immobilized on a support surface; a sense probe comprising a first single-stranded oligonucleotide tag complementary to, a sense binding moiety capable of hybridizing to a nucleotide sequence of the sense strand of the oligonucleotide duplex, and a first label; (iii) an antisense probe comprising a second single-stranded oligonucleotide tag that is complementary to at least a portion of the second capture oligonucleotide immobilized on the support surface, an antisense binding moiety capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label; and A kit for carrying out the method according to any one of claims 1 to 16, comprising (iv) a single-stranded nuclease.

Images