Hybridization-based method for detection of therapeutic oligonucleotides

Abstract

The disclosure relates to compositions, methods, and kits for the detection of oligonucleotides, including RNA, DNA and mixed synthetic modified oligonucleotides, such as antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), microRNAs (miRNAs), and aptamers.

Description

[0001] Attorney Docket No.: ADV-001PC / 112625-5001

[0002] HYBRIDIZATION-BASED METHOD FOR DETECTION OF THERAPEUTIC

[0003] OLIGONUCLEOTIDES

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 703,317, filed on October 4, 2024, the entire contents of which are hereby incorporated by reference in their entirety.

[0006] FIELD

[0007] The present disclosure describes compositions, methods, and kits for the detection of oligonucleotides, including RNA, DNA and mixed synthetic modified oligonucleotides, such as antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), microRNAs (miRNAs), and aptamers.

[0008] DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0009] This application contains a Sequence Listing, which has been submitted electronically herewith in XML format via EFS-Web. The contents of the XML copy of the Sequence Listing named “ADV- 001PC_l 12625-5001_PCT Sequence Listing,” which was created on October 3, 2025, and is 37,506 bytes in size, are incorporated herein by reference in their entirety.

[0010] BACKGROUND

[0011] Oligonucleotides of known sequences are commonly used in a wide variety of chemical and biological applications, and have also gained high importance in the diagnosis and treatment of diseases. For instance, antisense oligonucleotides, short interfering RNA(siRNA) and aptamers are promising pharmacological tools and therapeutic agents.

[0012] Qualitative and quantitative detection of these oligonucleotides in samples like cells, tissue, blood or plasma is a prerequisite to assess their therapeutic use and to monitor their stability in vivo.

[0013] A variety of methods have been developed for detection of oligonucleotides in biological samples. The most commonly used in clinical research methods is mass spectrometry, which, while providing high sensitivity of detection, requires special and expensive equipment and is time consuming to develop.

[0014] DBl / 162941341.2 Attorney Docket No.: ADV-001PC / 112625-5001

[0015] As an alternative to mass-spectrometry a variety of biochemical methods have been developed, based on the hybridization properties of oligonucleotides. Many of the hybridization-based approaches for the detection of oligonucleotides are described in the literature and disclosed in published patent applications, e.g., WO / 2008 / 046645. Most established procedures are based on hybridization of target oligonucleotides with complementary oligonucleotides or their derivatives via Watson-Crick base pairing. For example, peptide nucleic acids (PNAs), oligonucleotide mimics in which the sugar-backbone is replaced by a pseudopeptide chain of N-aminoethylglycine monomers, are often used in probe-based oligonucleotide detection methods as they bind to complementary DNA or RNA sequences with high affinity, specificity and stability (U.S. Pat. No. 6,395,474). WO / 2008 / 046645 describes the use of PNA probes in a RT-PCR-based oligonucleotide detection assay. U.S. Pat. No. 6,045,995 describes the qualitative and quantitative detection of oligonucleotides by capillary gel electrophoresis. Rossi et al. describe the identification of PCR-amplified oligonucleotides by PNA probes in anion-exchange high performance liquid chromatography (HPLC) (J. Agric. Food Chem. 2007, 55, 2509-2516).

[0016] Detection of fully modified therapeutics siRNAs presents a special challenge due to the high stability of the siRNA duplexes, which need to be disrupted by the hybridization procedure. This requires particular attention to design and testing of several probes to achieve efficient detection, which makes assay development a challenging and time-consuming enterprise.

[0017] There remains a need for efficient compositions and methods to detect oligonucleotides.

[0018] SUMMARY

[0019] Accordingly, the present disclosure provides, inter alia, an effective and simple approach for ELISA-like qualitative and / or quantitative detection of oligonucleotides, based on a hybridization probe design.

[0020] The present methods, in aspects, involve a hybridization-ligation approach (FIG. 1 or FIG. 11), that includes binding and hybridization of the target oligonucleotide with a complimentary biotinylated hybridization probe to form a duplex, separation of the duplex from unbound oligonucleotides by the binding to streptavidin-coated matrix (e.g., 96-well plate), followed by ligation with a universal affinity-labeled detection probe. In embodiments, after washing the

[0021] DBl / 162941341.2 2 Attorney Docket No.: ADV-001PC / 112625-5001 unligated detection probe, the bound duplex is detected by the enzyme-ligated antibodies or by direct detection (e.g., fluorescent detection).

[0022] In aspects, the disclosure provides a hybridization probe (also referred to as a “capture probe”), which allows both for the efficient and specific binding to the target oligonucleotide and efficient ligation to a detection probe. In aspects, the hybridization probe / capture probe is described by the formula: 5 'N(6-IO)(<Y)N)(O-5))( N)(7-3O) [binding ligand] 3' where N is a nucleotide, Y is either ribose or deoxyribose, x is a 2' modification, optionally selected from 2'-O-methyl (2'0Me), 2'-fluoro (2'F), 2'-O-methoxyethyl (2'-M0E), 2'OH, 2'H, and BNA (2-4 bridged nucleic acid), including, by way of non-limitation, LNA and binding ligand is a binding ligand useful for the attachment to a matrix (e.g., biotin).

[0023] In aspects, the hybridization probe is described by the formula: 5'NNNNNNNNN (Y)N(Y)NxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxN- [binding ligand] 3', where N is a nucleotide, (YJN is a deoxynucleotide, and xN is 2'0Me nucleotide.

[0024] In aspects, the hybridization probe is described by the formula: 5' TAACTAGTG <Y)N(Y)N xNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxN- [binding ligand] 3', where N is a nucleotide, Y)N is a deoxynucleotide and xN is 2'0Me nucleotide.

[0025] In embodiments, all of the 2' modified nucleotides are 2'0Me nucleotides.

[0026] In embodiments, the hybridization probe is a biotinylated backbone-modified oligonucleotide complimentary to a target oligonucleotide and having an extension of a plurality of nucleotides, complimentary to a universal affinity-labeled detection probe. In embodiments, backbone modifications used in the design of the hybridization probe are 2'OMe, 2'F, 2'-M0E, 2'OH, 2'H, and BNA (2-4 bridged nucleic acid), including, by way of non-limitation, LNA, or a combination of them with several DNA backbones located next to the detection probe ligation site. In embodiments, the presence of these DNAs allows for efficient ligation of the detection probe.

[0027] In embodiments, the universal affinity-labeled detection probe is or comprises 5'P CACTAGTTA- [label] 3'.

[0028] In embodiments, the universal affinity-labeled detection probe is conjugated to the label. In embodiments, the universal affinity-labeled detection probe is conjugated to digoxigenin. In embodiments, the universal affinity-labeled detection probe is suitable for being bound by an

[0029] DBl / 162941341.2 3 Attorney Docket No.: ADV-001PC / 112625-5001 antibody, e.g. enzyme-conjugated, directed against the label or direct detection. In embodiments, the label is digoxigenin.

[0030] In aspects, the disclosure provides a composition of any of one those shown in FIG. 2 or substantially similar to any of one those shown in FIG. 2.

[0031] In embodiments, the present disclosure provides a method for detecting one or more oligonucleotides, comprising contacting one or more target oligonucleotides with the present hybridization probe and detecting the presence or absence of the one or more oligonucleotides.

[0032] In embodiments, the methods provide separation of a resultant duplex from unbound oligonucleotides by the binding to matrix (e.g., a streptavidin-coated matrix, e.g., in a 96-well plate format), followed by ligation with a universal affinity-labeled detection probe. In embodiments, the methods provide one or more washing steps. In embodiments, the bound duplex is detected by contacting it with one or more detection antibodies, e.g. , enzyme-conjugated antibodies directed against a label. In embodiments, the bound duplex is detected by direct detection (e.g. fluorescent detection).

[0033] In embodiments, the present disclosure provides a method for qualitatively and / or quantitatively detecting one or more target oligonucleotides, comprising (a) contacting a target oligonucleotide with the present hybridization probe (e.g., comprising biotin) to form a duplex; (b) separating the duplex from unbound oligonucleotides by the binding to matrix (e.g., a streptavidin-coated matrix, e.g., in a 96-well plate format); (c) ligating the duplex with a universal affinity-labeled detection probe to yield a labelled duplex; (d) contacting the labelled duplex with one or more detection antibodies (e.g., enzyme-conjugated antibodies); and (e) detecting the presence or absence of the one or more target oligonucleotides.

[0034] In embodiments, the present disclosure provides a method for qualitatively and / or quantitatively detecting one or more target oligonucleotides, comprising (a) contacting a target oligonucleotide with the present hybridization probe (e.g., comprising biotin) to form a duplex; (b) separating the duplex from unbound oligonucleotides by the binding to matrix (e.g., a streptavidin-coated matrix, e.g., in a 96-well plate format); (c) ligating the duplex with a universal affinity -labeled detection probe to yield a labelled duplex; (d) directly detecting the label; and (e) detecting the presence or absence of the one or more target oligonucleotides.

[0035] DBl / 162941341.2 4 Attorney Docket No.: ADV-001PC / 112625-5001

[0036] In embodiments, the present hybridization probe comprises biotin and is bound to a streptavidin- coated matrix. In embodiments, the universal affinity-labeled detection probe comprises a label, such as digoxigenin, DNP, or FITC and is detected using an enzyme-conjugated antibody, e.g., directed to the label, such as digoxigenin, B-galactosidase, urease, alkaline phosphatase or peroxidase. In embodiments, the enzyme-conjugated antibody comprises an enzyme that is suitable for detection, e.g., alkaline phosphatase, 13-galactosidase, urease, or peroxidase. In embodiments, the label is fluorescent and allows direct detection (e.g., without the use of an antibody), optionally wherein the label is Cy3.

[0037] In embodiments, the methods use the composition of any of one those shown in FIG. 2 or substantially similar to any of one those shown in FIG. 2.

[0038] In embodiments, the methods comprise the steps of FIG. 1 or FIG. 11 or substantially similar to the steps of FIG. 1 or FIG. 11.

[0039] In embodiments, the methods comprise the use of an oligonucleotide of any one of SEQ ID NOs: 7-21, or a variant thereof.

[0040] In embodiments, there is provided a composition comprising an oligonucleotide of any one of SEQ ID NOs: 7-21, or a variant thereof.

[0041] In embodiments, the one or more target oligonucleotides are in a biological sample. In embodiments, the biological sample is selected from cells, tissue, blood, plasma, saliva, sweat, urine, and tears. In embodiments, the tissue is from an organ, such as a liver, spleen, kidney, or lungs. In embodiments, the one or more target oligonucleotides are in a liver.

[0042] In embodiments, the methods described herein are used for detecting or measuring the biodistribution of one or more target oligonucleotides in an organism, such as a human, a mouse, or a non-human primate.

[0043] In aspects, there is provided a kit for qualitatively and / or quantitatively detecting one or more target oligonucleotides comprising the present hybridization probe, one or more of the universal affinity-labeled detection probes, and / or the one or more detection antibodies.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 shows a non-limiting scheme of the present methods.

[0046] DBl / 162941341.2 5 Attorney Docket No.: ADV-001PC / 112625-5001

[0047] FIG. 2 shows a non-limiting scheme of the present hybridization (i.e., binding) probe.

[0048] FIG. 3 shows the efficiency of HTT siRNA detection using different ligases (HTT). To test the efficiency of ligation of siRNA with detection probe dose curves for HTT siRNA were obtained under identical hybridization conditions using different ligases.

[0049] FIG. 4 shows the effect of chemical modifications of the probe on the efficiency of siRNA detection. To measure the efficiency of siRNA detection an illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2, occasionally referred to as “Illustrative #1 herein) was hybridized with probes having the same sequence but different backbone modifications.

[0050] FIG. 5 shows the detection of HTT siRNA in different buffers. To evaluate the effect of hybridization conditions the dose curves were obtained by spiking HTT siRNA in different buffers.

[0051] FIG. 6 shows the detection of an illustrative fully modified siRNA (SEQ ID NO: 5 and / or 6, occasionally referred to as “Illustrative #2 herein) in different buffers. To evaluate the effect of hybridization conditions the dose curves were obtained by spiking HTT siRNA in different buffers.

[0052] FIG. 7 shows the detection of an illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in different buffers. To evaluate the effect of hybridization conditions the dose curves were obtained by spiking the illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in different buffers.

[0053] FIG. 8 shows the detection of an illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in human plasma. To evaluate the effect of biological matrix the dose curves were obtained by spiking the illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in dilutions of plasma in dilution buffer.

[0054] FIG. 9 shows the detection of an illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in liver extract. To evaluate the effect of biological matrix the dose curves were obtained by spiking the illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in dilutions of mouse liver extract in dilution buffer.

[0055] FIG. 10 shows discrimination between full lengths and N-l, N-2 and N-3 illustrative fully modified siRNAs (SEQ ID NO: 1 and / or 2). To evaluate whether the assay can detect siRNA degradation products, illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) detection probe was hybridized with siRNAs containing full-length, and 3' N-l, N-2 and N-3 antisense strands (see SEQ ID NO: 1 and / or 2 and 12-14) .

[0056] DBl / 162941341.2 6 Attorney Docket No.: ADV-001PC / 112625-5001

[0057] FIG. 11 shows a non-limiting illustration of a scheme of the assay with direct fluorescent detection.

[0058] FIG. 12 shows detection of an illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) using Cy3-labeled detection probe.

[0059] FIGs. 13A-13C show detection of (i) a Huntingtin (HTT) siRNA conjugated to docosanoic acid (DCA), (ii) an Illustrative siRNA #1 conjugated to N-acetylgalactosamine (NAc), and (iii) an Illustrative siRNA #2 conjugated to cholesterol (Choi), respectively, in mouse liver biopsies 7 days post administration of these siRNA at different doses using compositions and / or methods described herein. FIGs. 13D-13F show the relative expression levels of HTT mRNA, Illustrative siRNA #1 target mRNA, and Illustrative siRNA #2 target mRNA, respectively, in the corresponding liver biopsies of mice administered with HTT (DCA) siRNA, Illustrative siRNA #1, and Illustrative siRNA #2, respectively, 7 days post administration of these siRNA at different doses.

[0060] DETAILED DESCRIPTION

[0061] Compositions

[0062] In aspects, there is provided a hybridization probe defined by the formula:

[0063] 5' N(6-i0)((Y)N)(0-5))(xN)(7-30) [binding ligand] 3' wherein: N is a nucleotide, Y is either ribose or deoxyribose, x is a 2' modification, and binding ligand is a ligand suitable for the attachment to a matrix.

[0064] In embodiments, the hybridization probe is defined by the formula:

[0065] 5' NNNNNNNNN (Y)N(Y)N xNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxN- [binding ligand] 3', wherein N is a nucleotide, <Y)N is a deoxynucleotide, and xN is 2'0Me nucleotide.

[0066] In embodiments, the hybridization probe is defined by the formula:

[0067] 5 ' TAACT AGTG(Y)N(Y)NXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXN- [binding ligand] 3', wherein N is a nucleotide, (YJN is a deoxynucleotide and xN is 2'0Me nucleotide.

[0068] In embodiments, the Y is ribose.

[0069] DBl / 162941341.2 7 Attorney Docket No.: ADV-001PC / 112625-5001

[0070] In embodiments, the Y is deoxyribose.

[0071] In embodiments, the number of Y)N is about 1 to about 4, or about 2 nucleotides.

[0072] In embodiments, the number of N is about 8 to about 10, or about 9 nucleotides.

[0073] In embodiments, the number of xN is about 15 to about 25, or about 18 to about 22, or about 20, or about 21, or about 22 nucleotides.

[0074] In embodiments, the 2' modification is selected from 2'-O-methyl (2'0Me), 2'-fluoro (2'F), 2'-O- methoxyethyl (2'-M0E), 2'OH, 2'H, and BNA (2-4 bridged nucleic acid), including, by way of non-limitation, LNA, or combinations thereof.

[0075] In embodiments, the 2' modifications are solely 2'OMe. In embodiments, the 2' modifications are solely 2'0Me and the 2' modified nucleotides are immediately 3' to two deoxyribonucleotides.

[0076] In embodiments, the binding ligand is biotin.

[0077] In embodiments, the hybridization probe is suitable for specific binding to a target oligonucleotide and ligation to a detection probe.

[0078] In embodiments, the hybridization probe is suitable for binding a matrix via interaction of the binding ligand with a matrix. In embodiments, the matrix is a solid substrate. In embodiments, the matrix is a well plate. In embodiments, the matrix is a 96-well plate. In embodiments, the matrix is a bead, such as a nanobead or a microbead. In embodiments, the matrix is coated with a binding partner described herein (e.g. avidin, streptavidin and the like). In embodiments the matrix is coated, e.g. streptavidin- or PEG-coated. In embodiments, the matrix is a bead or a particle, e.g. a magnetic nano- or microbead, optionally coated, which allows the bead or particle to be held and / or manipulated by magnets. Examples of magnetic beads or particles include DYNABEADs (THERMO FISHER), MACS beads (MILTENYI BIOTEC), TURBOBEADS (TURBOBEADS), ABSOLUTE MAG STREPTAVIDIN MAGNETIC PARTICLES (CREATIVE DIAGNOSTICS), and GOLD NANOPARTICLES (SIGMA ALDRICH).

[0079] In embodiments, the hybridization probe is suitable for attaching to a 96-well plate via biotin / avidin binding.

[0080] In aspects, there is provided a universal affinity-labeled detection probe defined by the formula:

[0081] DBl / 162941341.2 8 Attorney Docket No.: ADV-001PC / 112625-5001

[0082] 5'P CACTAGTTA-[label] 3', wherein the label is detectable by specific binding by a binding partner or detectable directly (e.g., using fluorescence).

[0083] In embodiments, the label is selected from digoxigenin (DIG), biotin, fluorescein isothiocyanate (FITC), and dinitrophenol (DNP).

[0084] In embodiments, the universal affinity-labeled detection probe is a branched oligonucleotide probe. In embodiments, the universal affinity-labeled detection probe is a branched DNA probe.

[0085] In embodiments, the binding partner is an antibody or other protein-based binding partner. In embodiments, the binding partner is selected from avidin (e.g., streptavidin), anti-FITC antibody, anti-DNP antibody, and anti-DIG antibody. In embodiments, the label comprises biotin and the binding partner comprises avidin (e.g., streptavidin); the label comprises biotin and the binding partner comprises streptavidin; the label comprises fluorescein isothiocyanate (FITC) and the binding partner comprises anti-FITC antibody; the label comprises dinitrophenol (DNP) and the binding partner comprises anti-DNP antibody; or the label comprises digoxigenin (DIG) and the binding partner comprises anti-DIG antibody. In embodiments, the label is digoxigenin. In embodiments, the label comprises digoxigenin (DIG) and the binding partner comprises anti-DIG antibody.

[0086] In embodiments, the binding partner is an enzyme-conjugated antibody. In embodiments, the enzyme-conjugated antibody comprises an enzyme that is suitable for qualitative and / or quantitative detection, e.g., alkaline phosphatase, B-galactosidase, urease, or peroxidase.

[0087] In embodiments, the label is fluorescent and allows direct qualitative and / or quantitative detection (e.g., without the use of an antibody), optionally wherein the label is Cy3.

[0088] In embodiments, the one or more target oligonucleotides are selected from DNA and RNA. In embodiments the RNA is selected from antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), microRNAs (miRNAs), and aptamers.

[0089] In embodiments, the hybridization probe described herein and / or universal affinity-labeled detection probe described herein is as shown in FIG. 2 or substantially similar to any of one those shown in FIG. 2.

[0090] In aspects, there is provided a composition comprising the hybridization probe described herein, the universal affinity-labeled detection probe described herein, and / or a suitable buffer.

[0091] DBl / 162941341.2 9 Attorney Docket No.: ADV-001PC / 112625-5001

[0092] In aspects, there is provided a nucleic acid complex comprising the hybridization probe described herein and / or universal affinity-labeled detection probe described herein. In embodiments, the nucleic acid complex further comprises one or more target oligonucleotides. In embodiments, the one or more target oligonucleotides is hybridized to the hybridization probe. In embodiments, the universal affinity -labeled detection probe is hybridized to the hybridization probe. In embodiments, the universal affinity-labeled detection probe comprises a label. In embodiments, the nucleic acid complex further comprises an antibody bound to the label of the universal affinity- labeled detection probe.

[0093] Methods

[0094] In aspects, there is provided a method for qualitatively and / or quantitatively detecting one or more oligonucleotides, comprising contacting one or more target oligonucleotides with the hybridization probe described herein to yield a duplex and detecting the presence or absence of the one or more oligonucleotides.

[0095] In embodiments, the method further comprises separating the duplex from unbound oligonucleotide by the binding to matrix. In embodiments, the matrix is a solid substrate. In embodiments, the matrix is a well plate. In embodiments, the matrix is a 96-well plate. In embodiments, the matrix is a bead, such as a nanobead or a microbead. In embodiments, the matrix is coated with a binding partner described herein (e.g. avidin, streptavidin and the like). In embodiments the matrix is coated, e.g. streptavidin- or PEG-coated. In embodiments, the matrix is a bead or a particle, e.g. a magnetic nano- or microbead, optionally coated, which allows the bead or particle to be held and / or manipulated by magnets. Examples of magnetic beads or particles include DYNABEADs (THERMO FISHER), MACS beads (MILTENYI BIOTEC), TURBOBEADS (TURBOBEADS), ABSOLUTE MAG STREPTAVIDIN MAGNETIC PARTICLES (CREATIVE DIAGNOSTICS), and GOLD NANOPARTICLES (SIGMA ALDRICH).

[0096] In embodiments, the hybridization probe is suitable for adhering to a 96-well plate viabiotin / avidin binding.

[0097] In embodiments, the method further comprises ligating the matrix-bound duplex with a universal affinity-labeled detection probe comprising a label to yield a labeled duplex.

[0098] DBl / 162941341.2 10 Attorney Docket No.: ADV-001PC / 112625-5001

[0099] In embodiments, the method further comprises one or more washing steps.

[0100] In embodiments, the method further comprises contacting the labeled duplex with one or more binding partners.

[0101] In embodiments, the one or more binding partners are detection antibodies. In embodiments, the detection antibodies are enzyme-conjugated antibodies directed against the label.

[0102] In embodiments, the binding partner is selected from avidin (e.g. streptavidin), anti-FITC antibody, anti-DNP antibody, and anti-DIG antibody. In embodiments, the label comprises biotin and the binding partner comprises avidin (e.g. streptavidin); the label comprises biotin and the binding partner comprises streptavidin; the label comprises fluorescein isothiocyanate (FITC) and the binding partner comprises anti-FITC antibody; the label comprises dinitrophenol (DNP) and the binding partner comprises anti-DNP antibody; or the label comprises digoxigenin (DIG) and the binding partner comprises anti-DIG antibody.

[0103] In embodiments, the enzyme-conjugated antibody comprises an enzyme that is suitable for detection, e.g., alkaline phosphatase, B-galactosidase, urease, or peroxidase.

[0104] In embodiments, the method further comprises detecting bound duplex by measuring enzyme activity. In embodiments, the detecting of bound duplex provides detection of the one or more target oligonucleotides.

[0105] In embodiments, the label is fluorescent and allows direct detection (e.g., without the use of an antibody), optionally wherein the label is Cy3.

[0106] In aspects, there is provided a method for qualitatively and / or quantitatively detecting one or more target oligonucleotides, comprising (a) contacting one or more target oligonucleotides with the present hybridization probe (e.g., comprising biotin) to form a duplex; (b) separating the duplex from unbound oligonucleotides by the binding to matrix (e.g., a streptavidin-coated matrix, e.g., in a 96-well plate format); (c) ligating the duplex with a universal affinity-labeled detection probe to yield a labelled duplex; (d) contacting the labelled duplex with one or more detection antibodies (e.g., enzyme-conjugated antibodies) or directly detecting (e.g. via fluorescence); and (e) detecting the presence or absence of the one or more target oligonucleotides.

[0107] In embodiments, the hybridization probe comprises biotin and is bound to a streptavidin-coated matrix.

[0108] DBl / 162941341.2 11 Attorney Docket No.: ADV-001PC / 112625-5001

[0109] In embodiments, the universal affinity-labeled detection probe comprises a label, such as digoxigenin, DNP, or FITC.

[0110] In embodiments, the universal affinity-labeled detection probe comprises a label, such as fluorescent label which allows direct detection (e.g., without the use of an antibody). In embodiments, the label is Cy3.

[0111] In embodiments, the universal affinity-labeled detection probe is a branched oligonucleotide probe. In embodiments, the universal affinity-labeled detection probe is a branched DNA probe.

[0112] In embodiments, the methods use one or more ligases.

[0113] In embodiments, the ligase is chlorellavirus DNA ligase.

[0114] In embodiments, the ligase is T4 RNA ligase 2.

[0115] In embodiments, the ligase is T4 DNA ligase.

[0116] In embodiments, the method uses the composition of any one of those shown in FIG. 2 or substantially similar to any one of those shown in FIG. 2.

[0117] In embodiments, the method comprises the steps of FIG. 1 or FIG. 11 or substantially similar to the steps of FIG. 1 or FIG. 11.

[0118] In embodiments, the methods comprise the use of an oligonucleotide of any one of SEQ ID NOs: 7-21, or a variant thereof.

[0119] In embodiments, the method provides qualitative and / or quantitative detection in about 1 to about 1000 finol range. In embodiments, the method is adaptable to qualitative and / or quantitative detection of any target short oligonucleotide sequence.

[0120] In embodiments, the one or more target oligonucleotides are selected from DNA and RNA. In embodiment the RNA is selected from antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), microRNAs (miRNAs), and aptamers.

[0121] In embodiments, there is provided a composition comprising an oligonucleotide of any one of SEQ ID NOs: 7-21, or a variant thereof.

[0122] In embodiments, the one or more target oligonucleotides are in a biological sample. In embodiments, the biological sample is selected from cells, tissue, blood, plasma, saliva, sweat,

[0123] DBl / 162941341.2 12 Attorney Docket No.: ADV-001PC / 112625-5001 urine, and tears. In embodiments, the tissue is from a liver. In embodiments, the tissue is from an organ selected from adrenal glands, anus, appendix, arteries, bladder, blood cells, bone marrow, bones, brain, bronchi, bulbourethral glands, capillaries, cerebellum, diaphragm, ears, esophagus, eyes, fallopian tubes, gallbladder, genitals, hair, heart, human skeleton, hypothalamus, joints, kidneys, large intestine, larynx, ligaments, liver, lungs, lymph nodes, lymphatic vessels, mammary glands, mesentery, mouth, nails, nasal cavity, nerves, nose, olfactory epithelium, ovaries, pancreas, parathyroid glands, parathyroid glands, penis, pharynx, pineal gland, pituitary gland, placenta, prostate, rectum, salivary glands, scrotum, seminal vesicles, skeletal muscles, skin, small intestine, spinal cord, spleen, stomach, subcutaneous tissue, teeth, tendons, testes, the vestibular system of the ear, thoracic ducts, thymus gland, thyroid, tongue, tonsils, trachea, ureters, urethra, uterus, vagina, vas deferens, and veins.

[0124] In embodiments, the methods described herein are used for detecting or measuring the biodistribution of one or more target oligonucleotides in an organism. In embodiments, the organism is selected from a human, a mouse, or a non-human primate. In embodiments, the methods are used for confirming target delivery of the one or more target oligonucleotides. In embodiments, the methods are used for identifying off-target delivery of the one or more target oligonucleotides. In embodiments, the methods are used for refining target delivery of the one or more target oligonucleotides.

[0125] Kits

[0126] In aspects, there is provided a kit for qualitatively and / or quantitatively detecting one or more target oligonucleotides comprising the present hybridization probe, one or more of the universal affinity-labeled detection probes, and / or the one or more detection antibodies. In embodiments, the kit further comprises a multi-well plate for use.

[0127] In aspects, there is provided a kit for qualitatively and / or quantitatively detecting one or more target oligonucleotides comprising the hybridization probe described herein, one or more of the universal affinity-labeled detection probes described herein, one or more detection antibodies comprising an enzyme, and / or one or more enzyme substrates.

[0128] In embodiments, the kit further comprises a coated plate suitable for interaction with the hybridization probe.

[0129] DBl / 162941341.2 13 Attorney Docket No.: ADV-001PC / 112625-5001

[0130] In embodiments, the kit further comprises instructions for use and other information. In embodiments, each component of the kit may be in separate container, or two or more components may be in the same container.

[0131] EXAMPLES

[0132] Example 1: Oligonucleotide Detection with a Labeled Antibody

[0133] FIG. 1 shows the assay used in this Example.

[0134] Equipment used included a 96-well Plate Reader with fluorescence detection capability with Excitation at 435 nm and Detection 575 nm, PCR machine, and shaker with 96-well block.

[0135] A capture probe of the following composition was generated: TAACTAGTGACmCmAmGmAmAmAmGmAmGmUmGmUmCmUmCmAmUmCmUmUmA -Bio-3, Molecular Weight (g / mol) 10815.88.

[0136] A detection probe of the following composition was generated: 5'P-CACTAGTTA-digoxigenin 3'. Molecular Weight (g / mol) 3550.27.

[0137] A NeutrAvidin Coated 96 well Plate (ThermoFisher) was used.

[0138] Illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) standards were prepared with following concentrations in H2O, stored at -20 °C.

[0139] Buffer was added to plate / wells immediately removed. Plates were washed with buffer and shaking.

[0140] DBl / 162941341.2 14 Attorney Docket No.: ADV-001PC / 112625-5001

[0141] For hybridization, the following mixtures were prepared:

[0142] For all standards, each concentration was as follows (standards were spike-ins into the matrix (plasma, tissue extract of the same nature)).

[0143] For Negative Control:

[0144] 50 ul of each sample, standard and Negative Control tested were added into a PCR plate or PCR tubes. The standards were in the same matrix as the samples being measured. For C apture Prob e Mixture :

[0145] DBl / 162941341.2 15 Attorney Docket No.: ADV-001PC / 112625-5001

[0146] 50 ul of Capture Probe mixture was added to each well or tube containing sample or controls. A short mix with vortex was followed by quick spin to remove liquid from lid. PCR plate or tubes were placed into a PCR machine and run with the following conditions: Hybridization Conditions

[0147] 95 °C for 10 min

[0148] Ramp down at 0.1 °C per second to 25 °C

[0149] 25 °C for 20 min

[0150] Plates were activated while hybridizing. 100 uL of Hybridization Buffer was added to each well in NeutrAvidin Plate to be used followed by a wash. Sample was bound to the plate via a 60-min room temperature incubation while shaking at 3500 rpm. Rinses and washes followed.

[0151] For ligation, 50 mM Tris-HCL [pH 7.5], 10 mM MgCh, 10 mM DTT, 2 mM ATP, 75 U / mL Spilt Ligase was freshly prepared. Hybridization Buffer was removed and 100 uL of Ligation reaction was added to each well with sample. Samples were incubated overnight at 4 °C.

[0152] Blocking and antibody binding was conducted by removing ligation Reaction mixture, rinsing and washing. Block was achieved by adding 100 ul of 1% BSA Buffer for 45 min while shaking at

[0153] DBl / 162941341.2 16 Attorney Docket No.: ADV-001PC / 112625-5001

[0154] 3500 rpm while covered with removable foil, which was then removed. 100 ul of anti- digoxigenin antibody diluted 1 to 5000 in 1% BSA Buffer and incubation was undertaken.

[0155] Washing occurred with washing buffer and tween buffer.

[0156] For detection, 100 ul of AttoPhos® AP Fluorescent Substrate System was added to each well and immediately read on a Plate Reader or similar for 20 min every 30 min for kinetic data or / and at 1200 second for endpoint data (Excitation 435 nm / Detection 575 nm)

[0157] Curves were generated using standard methods.

[0158] FIG. 3 shows the efficiency of HTT siRNA detection using different ligases (HTT). To test the efficiency of ligation of siRNA with detection probe dose curves for HTT siRNA were obtained under identical hybridization conditions using different ligases.

[0159] FIG. 4 shows the effect of chemical modifications of the probe on the efficiency of siRNA detection. To measure the efficiency of siRNA detection, illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) was hybridized with probes having the same sequence but different backbone modifications.

[0160] FIG. 5 shows the detection of HTT siRNA in different buffers. To evaluate the effect of hybridization conditions the dose curves were obtained by spiking HTT siRNA in different buffers.

[0161] FIG. 6 shows the detection of an illustrative fully modified siRNA (SEQ ID NO: 5 and / or 6) in different buffers. To evaluate the effect of hybridization conditions the dose curves were obtained by spiking HTT siRNA in different buffers.

[0162] FIG. 7 shows the detection of illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in different buffers. To evaluate the effect of hybridization conditions the dose curves were obtained by spiking the illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in different buffers.

[0163] FIG. 8 shows the detection of illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in human plasma. To evaluate the effect of biological matrix the dose curves were obtained by spiking the illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in at dilutions of plasma in dilution buffer.

[0164] FIG. 9 shows the detection of illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) in liver extract. To evaluate the effect of biological matrix the dose curves were obtained by the illustrative

[0165] DBl / 162941341.2 17 Attorney Docket No.: ADV-001PC / 112625-5001 fully modified siRNA (SEQ ID NO: 1 and / or 2) in dilutions of mouse liver extract in dilution buffer.

[0166] FIG. 10 shows discrimination between full lengths and N-l, N-2 and N-3 illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2). To evaluate whether the assay can detect siRNA degradation products, illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) detection probe was hybridized with siRNAs containing full-length, and 3' N-l, N-2 and N-3 antisense strands.

[0167] Example 2: Oligonucleotide Detection with a Cy3-Labeled Detection Probe

[0168] FIG. 11 shows the assay used in this Example.

[0169] FIG. 12 shows detection of an illustrative fully modified siRNA (SEQ ID NO: 1 and / or 2) using Cy3-labeled detection probe. Detection was performed as described herein (e.g. Example 1) with the following changes: the overnight ligation with Cy3-labeled detection probe was followed by two rapid washes with 100 uL H2O followed by 100 ul of 1% Tween-20 in H2O for 10 min. After washes 100 ul of H2O was added and plate was read by the fluorescent plate reader using excitation at 522 nM and emission at 566 nm.

[0170] Accordingly, the oligonucleotide detection described here can be accomplished using a fluorescence detection probe.

[0171] Example 3: Detection of Small Interfering RNAs (siRNAs) in Liver Biopsies

[0172] In this example, mice were administered a combined dose of three human sequence siRNAs: (i) a Huntingtin (HTT) siRNA conjugated to docosanoic acid (DCA), (ii) an Illustrative siRNA #1 conjugated to N-acetylgalactosamine (Nac), and (iii) an Illustrative siRNA #2 conjugated to cholesterol (Choi). Each siRNA was delivered at dose levels of 20 mg / kg, 10 mg / kg, or 5 mg / kg. Phosphate-buffered saline (PBS) was employed as a control. Five animals were included per concentration group.

[0173] At seven days post-administration, liver biopsies were collected. Biopsies were lysed using a guanidinium-based buffer. Detection of the siRNAs was performed as described herein in Example 1. Specifically, a 10 uL aliquot of each lysate was assayed in triplicate using capture probes specific for each siRNA to determine individual siRNA concentrations.

[0174] DBl / 162941341.2 18 Attorney Docket No.: ADV-001PC / 112625-5001

[0175] Quantification was performed by reference to standard curves prepared with known quantities of the respective siRNAs diluted in lysis buffer. Each curve was fit with a five-point parametric model. Concentrations of experimental samples were interpolated against the respective curve, corrected for dilution, and normalized to total protein content measured in each sample. From the same liver extracts, total RNA was isolated, reverse transcribed, and amplified using commercially available TaqMan® PCR primers. Relative expression values were calculated, and the average fold change was determined by comparison to PBS control samples.

[0176] The results are summarized in the table below and presented in FIGs. 13A-13F. The present methods successfully detected HTT (DC A) siRNA, Illustrative siRNA #1, and Illustrative siRNA #2 in liver biopsies, the detected concentrations of which all correlate with the doses of the respective siRNA administered, as shown in FIGs. 13A-13C. Moreover, the expression levels of HTT mRNA and Illustrative siRNA #1 target mRNA in the liver biopsies, which are targets of HTT (DC A) siRNAs and Illustrative siRNA #1, respectively, also correlate with the doses of the respective siRNAs administered, as shown in FIGs. 13D-13E, which further demonstrates the effectiveness and reliability of the methods disclosed herein. Moreover, no such correlation was observed when there were mismatches between the siRNA and its target mRNA, as shown in FIG. 13F, which demonstrates the high sensitivity and specificity of the methods disclosed herein. The data further proves, inter alias, that the methods disclosed herein function properly with or without a detected Kd.

[0177] DBl / 162941341.2 19 Attorney Docket No.: ADV-001PC / 112625-5001

[0178] SEQUENCES

[0179] SEQ ID Name Sequence Modification Pattern NO: Illustrative

[0180] UAAGAUGAGACACU [mU][Ps][fA][Ps][fA][fG][mA][fU][mG][fA][mG][fA][mC][fA][mC][fU][mC][fU][mU] 1 #1 CUUUCUGGU [fU][mC][fU][mG][Ps][mG][Ps][mU]

[0181] AntiSense „ Illustrative CAGAAAGAGUGUCU [mC][Ps][mA][Ps][mG][mA][mA][mA] [fG][mA][fG][mU][fG][mU][fC][mU][fC][mA]

[0182] 2 #1 Sense CAUCUUA [mU][mC][mU][mU][mA] „ HIT AntiSe UUAAUCUCUUUACU [5Phos][mU][Ps][fU][Ps][mA][fA][mU][fC][mU][fC][mU][fU][mU][fA][mC][fU][Ps][ 3 nse GAUAUA mG][Ps] [fA][Ps][mU] [Ps] [fA][Ps] [mU][Ps][fA]

[0183] CAGUAAAGAGAUUA

[0184] 4 HTT_Sense [fC][Ps][mA][Ps][fG][mU][fA][mA][fA][mG][fA][mG][fA][mU][fU][Ps][mA][Ps][fA] A

[0185] Illustrative

[0186] ACAAAAGCAAAACAG [mA][Ps][fC][Ps][mA][fA][fA][fA][mG][fC][mA][fA][mA][mA][mC][fA][mG][fG][mU

[0187] 5 #2_AntiSen GUCUAGAA ][fC][mU][mA][mG][Ps][rnA][Ps][mA] se Illustrative CUAGACCUGUTUUG [mC][Ps][mU][Ps][mA][mG][mA][mC][fC][mU][fG][mU][dT][mU][mU][mG][mC][ #2_Sense CUUUUGU mU][mU][mU][mU][mG][mU]

[0188] 7HTT_Captur taactagtTAUAUCAGU [dT][dA][dA][dC][dT][dA][dG][dT][dT][dA][mU][mA][mU][mC][mA][mG][mU][mA e AAAGAGAUUAA ][mA][mA][mG][mA][mG][mA][mU][mU][mA][mA][Bio-3] taactagtTTCUAGACC g Illustrative [dT][dA][dA][dC][dT][dA][dG][dT][dT][dT][mC][mU][mA][mG][mA][mC][mC][mU] UGUUUUGCUUUUG

[0189] #2_Capture [mG][mU][mU][mU][mU][mG][mC][mU][mU][mU][mU][mG][mU] [Bio-3] U

[0190] Capture_ taactagtgACCAGAAA

[0191] [dT][dA][dA][dC][dT][dA][dG][dT][dG][dA][dC][mC][mA][mG][mA][mA][mA][mG][

[0192] 9 Illustrative GAGUGUCUCAUCUU mA][mG][mU][mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA][Bio-3] #1 A Detection

[0193] 10 „ cactagtta [5Phos][dC][dA][dC][dT][dA][dG][dT][dT][dA][Dig] Probe

[0194] „ Detection

[0195] 11 ~ cactagtta [5Phos][dC][dA][dC][dT][dA][dG][dT][dT][dA][Cy3]

[0196] Probe_Cy3 Illustrative UAAGAUGAGACACU [mU][Ps][fA][Ps][fA][fG][mA][fU][mG][fA][mG][fA][mC][fA][mC][fU][mC][fU][mU] 12 #1 (-D CUUUCUGG [fU][mC][fU][mG][Ps][mG] Illustrative UAAGAUGAGACACU [mU][Ps][fA][Ps][fA][fG][mA][fU][mG][fA][mG][fA][mC][fA][mC][fU][mC][fU][mU] #1 (-2) CUUUCUG [fU][mC][fU][mG] Illustrative UAAGAUGAGACACU [mU][Ps][fA][Ps][fA][fG][mA][fU][mG][fA][mG][fA][mC][fA][mC][fU][mC][fU][mU] 14 #1 (-3) CUUUCU [fU][mC][fU] Illustrative #1 taactagtgCCAGAAAG [dT][dA][dA][dC][dT][dA][dG][dT][dG][dC][dC][mA][mG][mA][mA][mA][mG][mA][

[0197] 15 _Capture_-

[0198] AGUGUCUCAUCUUA mG][mU][mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA][Bio-3] l_Methyl+2 DNA Illustrative #1 taactagtgCAGAAAGA [dT][dA][dA][dC][dT][dA][dG][dT][dG][dC][dA][mG][mA][mA][mA][mG][mA][mG]

[0199] 16 _Capture_-

[0200] GUGUCUCAUCUUA [mU][mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA] [Bio-3]

[0201] 2_Methyl+2 DNA

[0202] DB1 / 162941341.2 20 Attorney Docket No.: ADV-001PC / 112625-5001

[0203] Illustrative #1 taactagtgAGAAAGAG [dT][dA][dA][dC][dT][dA][dG][dT][dG][dA][dG][mA][mA][mA][mG][mA][mG][mU]

[0204] 17 _Capture_- UGUCUCAUCUUA [mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA][Bio-3] 3_Methyl+2 DNA Illustrative taactagtgACCAGAAA

[0205] [dT][dA][dA][dC][dT][dA][dG][dT][dG][mA][mC][mC][mA][mG][mA][mA][mA][fG] is Se1n' GAGUGUCUCAUCUU se_Capt [mA][fG][mU][fG][mU][fC][mU][fC][mA][mU][mC][mU][mU][mA][Bio-3] A ure_Sense Illustrative taactagtgACCAGAAA

[0206] [dT][dA][dA][dC][dT][dA][dG][dT][dG][mA][mC][mC][mA][mG][mA][mA][mA][mG

[0207] 19 GAGUGUCUCAUCUU Se1n'se_Capt ][mA][mG][mU][mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA][Bio-3] A ure_Methyl Illustrative taactagtgACCAGAAA

[0208] [dT][dA][dA][dC][dT][dA][dG][dT][dG][dA][mC][mC][mA][mG][mA][mA][mA][mG]

[0209] 20#1' GAGUGUCUCAUCUU Capture_M [mA][mG][mU][mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA] [Bio-3] A ethyl+lDNA Illustrative taactagtgaccagaaaga [dT][dA][dA][dC][dT][dA][dG][dT][dG][dA][dC][dC][dA][dG][dA][dA][dA][dG][dA][

[0210] 21 S ce1n’se_Capt gtgtctcatctta dG][dT][dG][dT][dC][dT][dC][dA][dT][dC][dT][dT][dA][Bio-3] ure DNA

[0211] [fN] 2'Flouro base

[0212] [mN] 2'0 methyl base

[0213] [Ps] Phosphorothioate [dN] Deoxyribose

[0214] [Bio-3] Biotin 3'

[0215] [Dig] Digoxigenin NHS

[0216] [Cy3] Cyanine 3

[0217] [5Phos] 5’ Phosphorylation EQUIVALENTS

[0218] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[0219] DB1 / 162941341.2 21 Attorney Docket No.: ADV-001PC / 112625-5001

[0220] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

[0221] INCORPORATION BY REFERENCE All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[0222] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

[0223] DBl / 162941341.2 22

Claims

Attorney Docket No.: ADV-001PC / 112625-5001CLAIMSWhat is claimed is:

1. A hybridization probe defined by the formula:5' N(6-10)((Y)N)(0-5))(XN)(7-30) [binding ligand] 3' wherein:N is a nucleotide,Y is either ribose or deoxyribose, x is a 2' modification, and binding ligand is a ligand suitable for the attachment to a matrix.

2. The hybridization probe of claim 1, wherein the hybridization probe is defined by the formula:5' NNNNNNNNN (Y)N(Y)NXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXNXN- [binding ligand] 3', wherein:N is a nucleotide;;Y)N is a deoxynucleotide; and xN is 2'0Me nucleotide.

3. The hybridization probe of claim 1 or 2, wherein the hybridization probe is defined by the formula:5 ' TAACTAGTG (Y)N(Y)NxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxNxN- [binding ligand] 3', wherein:N is a nucleotide;(Y)N is a deoxynucleotide; and xN is 2'0Me nucleotide.DBl / 162941341.2 23Attorney Docket No.: ADV-001PC / 112625-50014. The hybridization probe of any one of claims 1-3, wherein Y is ribose.

5. The hybridization probe of any one of claims 1-3, wherein Y is deoxyribose.

6. The hybridization probe of any one of claims 1-5, wherein the number of (Y)N is about 1 to about 4, or about 2 nucleotides.

7. The hybridization probe of claim 6, wherein the number of Y)N is 2 nucleotides and Y is deoxyribose.

8. The hybridization probe of any one of claims 1-7, wherein the number of N is about 8 to about 10, or about 9 nucleotides.

9. The hybridization probe of any one of claims 1-8, wherein the number of xN is about 15 to about 25, or about 18 to about 22, or about 20, or about 21, or about 22 nucleotides.

10. The hybridization probe of any one of claims 1-9, wherein the 2' modification is selected from 2'-O-methyl (2'0Me), 2'-fluoro (2'F), 2'-O-methoxyethyl (2'-M0E), 2'OH, 2'H, and BNA (2-4 bridged Nucleic acid), including, by way of non-limitation, LNA, or combinations thereof.

11. The hybridization probe of claim 10, wherein the 2' modifications are solely 2'0Me.

12. The hybridization probe of any one of claims 1-11, wherein the binding ligand is biotin.

13. The hybridization probe of any one of claims 1-12, wherein the hybridization probe is suitable for specific binding to a target oligonucleotide and ligation to a detection probe.

14. The hybridization probe of any one of claims 1-13, wherein the hybridization probe is suitable for binding a matrix via interaction of the binding ligand with the matrix.

15. The hybridization probe of claim 14, wherein the matrix is a solid substrate.

16. The hybridization probe of claim 14, wherein the matrix is a well plate.

17. The hybridization probe of claim 14, wherein the matrix is a 96-well plate or a bead.

18. The hybridization probe of any one of claims 1-17, wherein the hybridization probe is suitable for adhering to a 96-well plate or bead via biotin / avidin binding.

19. A universal affinity-labeled detection probe defined by the formula:5'P CACTAGTTA-[label] 3',DBl / 162941341.2 24Attorney Docket No.: ADV-001PC / 112625-5001 wherein the label is detectable by specific binding by a binding partner or by direct detection.

20. The universal affinity-labeled detection probe of claim 19, wherein the label is selected from digoxigenin (DIG), biotin, fluorescein isothiocyanate (FITC), and dinitrophenol (DNP).

21. The universal affinity-labeled detection probe of claim 19, wherein the binding partner is an antibody or other protein-based binding partner.

22. The universal affinity-labeled detection probe of any one of claims 19-21, wherein the binding partner is selected from avidin, anti-FITC antibody, anti-DNP antibody, and anti-DIG antibody.

23. The universal affinity -labeled detection probe of any one of claims 19-22, wherein: the label comprises biotin and the binding partner comprises avidin; the label comprises biotin and the binding partner comprises streptavidin; the label comprises fluorescein isothiocyanate (FITC) and the binding partner comprises anti-FITC antibody; the label comprises dinitrophenol (DNP) and the binding partner comprises anti-DNP antibody; or the label comprises digoxigenin (DIG) and the binding partner comprises anti-DIG antibody.

24. The universal affinity-labeled detection probe of any one of claims 19-23, wherein the binding partner comprises an enzyme-conjugated antibody, and wherein the enzyme-conjugated antibody comprises an enzyme that is suitable for detection, optionally wherein the enzyme is selected from alkaline phosphatase, 13-galactosidase, urease, and peroxidase.

25. The universal affinity-labeled detection probe of any one of claims 19-24, wherein the label is fluorescent and allows direct detection, optionally wherein the label is Cy3.

26. The universal affinity -lab eled detection probe of any one of claims 19-25, wherein the probe is a branched oligonucleotide probe, optionally a branched DNA probe.DBl / 162941341.2 25Attorney Docket No.: ADV-001PC / 112625-500127. The hybridization probe of any one of claims 1-18 or universal affinity -labeled detection probe of any one of claims 19-26, wherein the one or more target oligonucleotides are selected from DNA and RNA, optionally antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), microRNAs (miRNAs), and aptamers.

28. The hybridization probe of any one of claims 1-18 or universal affinity-labeled detection probe of any one of claims 19-26, as shown in FIG. 2 or substantially similar to any of one those shown in FIG. 2.

29. A composition comprising the hybridization probe of any one of claims 1-18, universal affinity-labeled detection probe of any one of claims 19-26, and / or a suitable buffer.

30. A nucleic acid complex comprising:(a) the hybridization probe of any one of claims 1-18; and(b) the universal affinity-labeled detection probe of any one of claims 19-26.

31. The nucleic acid complex of claim 30, further comprising one or more target oligonucleotides.

32. The nucleic acid complex of claim 31, wherein the one or more target oligonucleotides is hybridized to the hybridization probe.

33. The nucleic acid complex of any one of claims 30-32, wherein the universal affinity-labeled detection probe is hybridized to the hybridization probe.

34. The nucleic acid complex of any one of claims 30-33, further comprising an antibody bound to the label.

35. A composition comprising an oligonucleotide selected from any one of the following or a variant thereof:[dT][dA][dA][dC][dT][dA][dG][dT][dT][dA][mU][mA][mU][mC][mA][mG][mU][mA][ mA][mA][mG][mA][mG][mA][mU][mU][mA][mA][Bio-3] (SEQ ID NO: 7),[dT] [dA] [dA] [dC] [dT] [dA][dG] [dT][dT] [dT] [mC] [mU] [mA] [mG] [mA] [mC] [mC] [mU] [ mG] [mU] [mU] [mU] [mU] [mG] [mC] [mU][mU] [mU] [mU] [mG][mU] [Bio-3 ] (SEQ ID NO: 8),DBl / 162941341.2 26Attorney Docket No.: ADV-001PC / 112625-5001[dT] [dA] [dA] [dC] [dT] [d A] [dG] [dT] [dG] [dA] [dC] [mC] [mA] [mG] [mA] [mA] [mA] [mG] [ mA] [mG] [mU] [mG] [mU] [mC] [mU] [mC][mA] [mU][mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 9),[5Phos][dC][dA][dC][dT][dA][dG][dT][dT][dA][Dig] (SEQ ID NO: 10),[5Phos][dC][dA][dC][dT][dA][dG][dT][dT][dA][Cy3] (SEQ ID NO: 11),[mU] [Ps] [f A] [Ps] [f A] [fG] [mA] [fU] [mG] [f ] [mG] [f A] [mC] [fA] [mC] [fU] [mC] [fU] [mU] [fU][mC][fU][mG][Ps][mG] (SEQ ID NO: 12),[mU] [Ps] [f A] [Ps] [f A] [fG] [mA] [fU] [mG] [fA] [mG] [f A] [mC] [f A] [mC] [fU] [mC] [fU] [mU] [fU][mC][fU][mG] (SEQ ID NO: 13),[mU] [Ps] [f A] [Ps] [f A] [fG] [mA] [fU] [mG] [f ] [mG] [f A] [mC] [fA] [mC] [fU] [mC ] [fU] [mU] [IU][mC][fU] (SEQ ID NO: 14),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [dC] [dC] [m A] [mG] [mA] [mA] [mA] [mG] [mA] [ mG] [mU] [mG] [mU] [mC] [mU] [mC][mA] [mU] [mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 15),[dT] [dA] [dA] [dC] [dT] [d A] [dG] [dT] [dG] [dC] [dA] [mG] [mA] [mA] [mA] [mG] [mA] [mG] [ mU][mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA][Bio-3] (SEQ ID NO: 16),[dT][dA][dA][dC][dT][dA][dG][dT][dG][dA][dG][mA][mA][mA][mG][mA][mG][mU][ mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA][Bio-3] (SEQ ID NO: 17),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [mA] [mC] [mC] [mA] [mG] [mA] [mA] [mA] [fG] [ mA] [fG] [mU] [fG] [mU] [fC] [mU] [fC] [mA] [mU] [mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 18),[dT] [dA] [dA] [dC] [dT] [dA][dG] [dT][dG] [mA] [mC] [mC] [mA] [mG] [mA] [mA] [mA] [mG] [mA] [mG] [mU] [mG] [mU] [mC] [mU] [mC] [mA] [mU] [mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 19),[dT][dA][dA][dC][dT][dA][dG][dT][dG][dA][mC][mC][mA][mG][mA][mA][mA][mG] [mA] [mG] [mU] [mG] [mU] [mC] [mU] [mC] [mA] [mU] [mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 20), andDBl / 162941341.2 27Attorney Docket No.: ADV-001PC / 112625-5001[dT] [dA] [dA] [dC] [dT] [d A] [dG] [dT] [dG] [dA] [dC] [dC] [dA] [dG] [dA] [d A] [dA] [dG] [dA] [ dG][dT][dG][dT][dC][dT][dC][dA][dT][dC][dT][dT][dA][Bio-3] (SEQ ID NO: 21), wherein[fN] is 2'Flouro base,[mN] is 2'0 methyl base,[Ps] is Phosphorothioate,[dN] is Deoxyribose,[Bio-3] is Biotin 3',[Dig] is Digoxigenin NHS,[Cy3] is Cyanine 3, and[5Phos] is 5’ Phosphorylation.

36. A method for detecting one or more target oligonucleotides, comprising:(a) contacting one or more target oligonucleotides with the hybridization probe of any one of claims 1-18 to yield a duplex; and(b) detecting the presence or absence of the one or more oligonucleotides.

37. The method of claim 36, further comprising separating the duplex from unbound oligonucleotide by the binding to matrix.

38. The method of any one of claims 37, wherein the matrix is a solid substrate.

39. The method of any one of claims 37, wherein the matrix is a well plate.

40. The method of any one of claims 37, wherein the matrix is a 96-well plate or bead.

41. The method of any one of claims 36-40, wherein the hybridization probe is suitable for adhering to a 96-well plate or bead via biotin / avidin binding.

42. The method of any one of claims 36-41, further comprising ligating the matrix-bound duplex with a universal affinity -labeled detection probe, optionally of any one of claims 19-26, comprising a label to yield a labeled duplex.

43. The method of any one of claims 36-42, further comprising one or more washing steps.DBl / 162941341.2 28Attorney Docket No.: ADV-001PC / 112625-500144. The method of any one of claims 36-43, further comprising contacting the labeled duplex with one or more binding partners.

45. The method of claim 44, wherein the one or more binding partners are detection antibodies.

46. The method of claim 45, wherein the detection antibodies are enzyme-conjugated antibodies directed against the label.

47. The method of any one of claims 36-46, wherein: the label comprises biotin and the binding partner comprises avidin; the label comprises biotin and the binding partner comprises streptavidin; the label comprises fluorescein isothiocyanate (FITC) and the binding partner comprises anti-FITC antibody; the label comprises dinitrophenol (DNP) and the binding partner comprises anti-DNP antibody; or the label comprises digoxigenin (DIG) and the binding partner comprises anti-DIG antibody.

48. The method of claim 46, wherein the enzyme-conjugated antibody comprises an enzyme that is suitable for detection, optionally wherein the enzyme is selected from alkaline phosphatase, 13-galactosidase, urease, and peroxidase.

49. The method of any one of claims 36-48, further comprising detecting bound duplex by measuring enzyme activity.

50. The method of any one of claims 36-49, wherein the detecting of bound duplex provides detection of the one or more target oligonucleotides.

51. The method of any one of claims 36-50, wherein the label is fluorescent and allows direct detection.

52. The method of claim 51, wherein the label is Cy3.

53. The method of any one of claims 42-52, wherein the universal affinity -labeled detection probe is a branched oligonucleotide probe, optionally a branched DNA probe.

54. A method for detecting one or more target oligonucleotides, comprising:DBl / 162941341.2 29Attorney Docket No.: ADV-001PC / 112625-5001(a) contacting one or more target oligonucleotides with a hybridization probe to form a duplex;(b) separating the duplex from unbound oligonucleotides by the binding to matrix;(c) ligating the duplex with a universal affinity-labeled detection probe to yield a labelled duplex;(d) contacting the labelled duplex with one or more detection antibodies, optionally wherein the one or more detection antibodies are enzyme-conjugated antibodies; and(e) detecting the presence or absence of the one or more target oligonucleotides.

55. A method for detecting one or more target oligonucleotides, comprising:(a) contacting one or more target oligonucleotides with a hybridization probe to form a duplex;(b) separating the duplex from unbound oligonucleotides by the binding to matrix;(c) ligating the duplex with a universal affinity -lab eled detection probe to yield a labelled duplex; and(d) detecting the presence or absence of the one or more target oligonucleotides, optionally wherein the detecting is achieved by fluorescent detection.

56. The method of claim 54 or 55, wherein the hybridization probe comprises biotin and is bound to a streptavidin-coated matrix.

57. The method of any one of claims 54-56, wherein the universal affinity-labeled detection probe comprises a label selected from digoxigenin, DNP, and FITC58. The method of any one of claims 54-57, wherein the method uses the composition of any one of those shown in FIG. 2 or substantially similar to any one of those shown in FIG. 2.

59. The method of any one of claims 54-58, wherein the method comprises the steps of FIG. 1 or FIG. 11 or substantially similar to the steps of FIG. 1 or FIG. 11.

60. The method of any one of claims 54-59, wherein the method provides detection in about 1 to about 1000 fmol range.DBl / 162941341.2 30Attorney Docket No.: ADV-001PC / 112625-500161. The method of any one of claims 54-60, wherein the method is adaptable to detection of any target short oligonucleotide sequences.

62. The method of any one of claims 54-61, wherein the one or more target oligonucleotides are selected from DNA and RNA, optionally antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), microRNAs (miRNAs), and aptamers.

63. The method of any one of claims 54-62, wherein the method comprises use of an oligonucleotide selected from any one of the following or a variant thereof:[dT][dA][dA][dC][dT][dA][dG][dT][dT][dA][mU][mA][mU][mC][mA][mG][mU][mA][ mA][mA][mG][mA][mG][mA][mU][mU][mA][mA][Bio-3] (SEQ ID NO: 7),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dT] [dT] [mC] [mU] [mA] [mG] [mA] [mC] [mC] [mU] [ mG] [mU] [mU] [mU] [mU] [mG] [mC] [mU][mU] [mU] [mU] [mG][mU] [Bio-3 ] (SEQ ID NO: 8),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [dA] [dC] [mC] [mA] [mG] [mA] [mA] [mA] [mG] [ mA] [mG] [mU] [mG] [mU] [mC] [mU] [mC][mA] [mU][mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 9),[5Phos][dC][dA][dC][dT][dA][dG][dT][dT][dA][Dig] (SEQ ID NO: 10),[5Phos][dC][dA][dC][dT][dA][dG][dT][dT][dA][Cy3] (SEQ ID NO: 11),[mU] [Ps] [f A] [Ps] [f A] [fG] [mA] [fU] [mG] [fA] [mG] [f A] [mC] [fA] [mC] [fU] [mC ] [fU] [mU] [fU][mC][fU][mG][Ps][mG] (SEQ ID NO: 12),[mU] [Ps] [f A] [Ps] [f A] [fG] [mA] [fU] [mG] [fA] [mG] [f A] [mC] [f A] [mC] [fU] [mC] [fU] [mU] [fU][mC][fU][mG] (SEQ ID NO: 13),[mU] [Ps] [f A] [Ps] [f A] [fG] [mA] [fU] [mG] [fA] [mG] [f A] [mC] [f A] [mC] [fU] [mC ] [fU] [mU] [fU][mC][fU] (SEQ ID NO: 14),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [dC] [dC] [m A] [mG] [mA] [mA] [mA] [mG] [mA] [ m G] [mU] [m G] [mU] [m C] [mU] [m C] [m A] [mU] [mC] [mU] [mU] [m A] [Bi o-3 ] (SEQ ID NO: 15),DBl / 162941341.2 31Attorney Docket No.: ADV-001PC / 112625-5001[dT] [dA] [dA] [dC] [dT] [d A] [dG] [dT] [dG] [dC] [dA] [mG] [mA] [mA] [mA] [mG] [mA] [mG] [ mU][mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA][Bio-3] (SEQ ID NO: 16),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [d A] [dG] [mA] [mA] [mA] [mG] [mA] [mG] [mU] [ mG][mU][mC][mU][mC][mA][mU][mC][mU][mU][mA][Bio-3] (SEQ ID NO: 17),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [mA] [mC] [mC] [mA] [mG] [mA] [mA] [mA] [fG] [ mA] [fG] [mU] [fG] [mU] [fC] [mU] [fC] [mA] [mU] [mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 18),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [m A] [mC] [mC] [mA] [mG] [mA] [mA] [mA] [mG] [mA] [mG] [mU] [mG] [mU] [mC] [mU] [mC] [mA] [mU] [mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 19),[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [d A] [mC] [mC] [mA] [mG] [mA] [mA] [mA] [mG] [mA] [mG] [mU] [mG] [mU] [mC] [mU] [mC] [mA] [mU] [mC] [mU] [mU] [mA] [Bio-3 ] (SEQ ID NO: 20), and[dT] [dA] [d A] [dC] [dT] [d A] [dG] [dT] [dG] [dA] [dC] [dC] [dA] [dG] [dA] [d A] [dA] [dG] [d A] [ dG][dT][dG][dT][dC][dT][dC][dA][dT][dC][dT][dT][dA][Bio-3] (SEQ ID NO: 21), wherein[fN] is 2'Flouro base,[mN] is 2'0 methyl base,[Ps] is Phosphorothioate,[dN] is Deoxyribose,[Bio-3] is Biotin 3',[Dig] is Digoxigenin NHS,[Cy3] is Cyanine 3, and[5Phos] is 5’ Phosphorylation.

64. The method of any one of claims 36-63, wherein the one or more target oligonucleotides are in a biological sample.DBl / 162941341.2 32Attorney Docket No.: ADV-001PC / 112625-500165. The method of claim 64, wherein the biological sample is selected from cells, tissue, blood, plasma, saliva, sweat, urine, and tears.

66. The method of claim 65, wherein the tissue is from an organ selected from liver, adrenal glands, anus, appendix, arteries, bladder, blood cells, bone marrow, bones, brain, bronchi, bulbourethral glands, capillaries, cerebellum, diaphragm, ears, esophagus, eyes, fallopian tubes, gallbladder, genitals, hair, heart, human skeleton, hypothalamus, joints, kidneys, large intestine, larynx, ligaments, lungs, lymph nodes, lymphatic vessels, mammary glands, mesentery, mouth, nails, nasal cavity, nerves, nose, olfactory epithelium, ovaries, pancreas, parathyroid glands, parathyroid glands, penis, pharynx, pineal gland, pituitary gland, placenta, prostate, rectum, salivary glands, scrotum, seminal vesicles, skeletal muscles, skin, small intestine, spinal cord, spleen, stomach, subcutaneous tissue, teeth, tendons, testes, the vestibular system of the ear, thoracic ducts, thymus gland, thyroid, tongue, tonsils, trachea, ureters, urethra, uterus, vagina, vas deferens, and veins.

67. The method of any one of claims 36-66, wherein the method is used for detecting the biodistribution of the one or more target oligonucleotides in an organism, optionally wherein the organism is selected from a human, a mouse, and a non-human primate.

68. The method of any one of claims 36-67, wherein the method is for qualitatively and / or quantitatively detecting.

69. A kit for detecting one or more target oligonucleotides, comprising: the hybridization probe of any one of claims 1-18; one or more of the universal affinity-labeled detection probe of any one of claims 19-26; one or more detection antibodies comprising an enzyme; and / or one or more enzyme substrates.

70. The kit of claim 69, further comprising a coated plate suitable for interaction with the hybridization probe.

71. The kit of claim 69 or 70, further comprising instructions for use.DBl / 162941341.2 33

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