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US 2023/0141860 A1Published application
Method for detecting sense and antisense strands in an oligonucleotide duplex
AssigneeMeso Scale Technologies, LLC.
InventorsSeth B. HARKINS, Timothy J. BREAK
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Abstract
Described herein is a method for detecting an oligonucleotide in a sample, and in particular, to a method for detecting sense and antisense strands of an oligonucleotide duplex in a sample.
Claims39
A method of detecting or quantifying a sense and an antisense strand 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 portion 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 a second capture oligonucleotide immobilized on the support surface, an antisense binding portion capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label,
- wherein the sense binding portion of the sense probe has a sense binding length that is shorter than a sense strand length of the sense strand, and
- wherein the antisense binding portion of the antisense strand has an antisense binding length that is shorter than an antisense strand length of the antisense strand;
- and
- (b) incubating the probes with the sample to form a hybridization mixture comprising hybridization complexes comprising:
- (i) a sense complex comprising the sense probe hybridized with the sense strand of the oligonucleotide duplex;
- and
- (ii) an antisense complex comprising the antisense probe hybridized with the antisense strand of the oligonucleotide duplex;
- and
- (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;
- and
- (d) detecting or quantifying the sense and antisense strands of the oligonucleotide duplex based on the presence of the label on the support surface.
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The method of claim, wherein (a)-(c) are performed sequentially.
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A composition comprising a set of probes, wherein 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;
- and
- (b) an antisense probe comprising a second single stranded oligonucleotide tag that is complementary to at least a portion of a second capture oligonucleotide, an antisense binding portion capable of hybridizing to a nucleotide sequence of an antisense strand of the oligonucleotide duplex, and a second label,
- wherein the sense binding portion of the sense probe has a sense binding length that is shorter than a sense strand length of the sense strand, and
- wherein the antisense binding portion of the antisense strand has an antisense binding length that is shorter than an antisense strand length of the antisense strand.
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A composition comprising:
- (a) an oligonucleotide duplex comprising a sense strand and an antisense strand;
- and
- (b) a 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, a sense binding portion 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 a second capture oligonucleotide, an antisense binding portion capable of hybridizing to a nucleotide sequence of an antisense strand of the oligonucleotide duplex, and a second label,
- wherein the sense binding portion of the sense probe has a sense binding length that is shorter than a sense strand length of the sense strand, and
- wherein the antisense binding portion of the antisense strand has an antisense binding length that is shorter than an antisense strand length of the antisense strand.
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A composition comprising one or more hybridization complexes, the hybridization complexes comprising:
- (a) a sense complex comprising a sense probe hybridized to a sense strand of an oligonucleotide duplex, wherein the sense probe comprises 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 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 a sense strand length of the sense strand;
- (b) an antisense complex comprising an antisense probe hybridized to an antisense strand of the oligonucleotide duplex, wherein the antisense probe comprises a second single stranded oligonucleotide tag that is complementary to at least a portion of a second capture oligonucleotide, an antisense binding portion capable of hybridizing to a nucleotide sequence of the antisense strand of the oligonucleotide duplex, and a second label, wherein the antisense binding portion of the antisense strand has an antisense binding length that is shorter than an antisense strand length of the antisense strand, and combinations thereof.
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Dependencies are inferred from the claim text.