US 11,111,527 B1Granted

Nanopore platform for DNA/RNA oligo detection using an osmium tagged complementary probe

OwnerAnastassia Kanavariotiinventor

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Abstract

Provided for herein is a method for detecting the presence of a nucleic acid target molecule in a biological sample. In certain aspects, the method comprises contacting a test sample that comprises (i) a biological sample comprising a nucleic acid target molecule and (ii) an osmylated single-stranded oligonucleotide probe comprising at least one pyrimidine residue covalently bonded to a substituted or unsubstituted Osmium tetroxide (OsO4)-2,2′-bypyridine group (OsBp group).

Claims40

Dependent claims

A method for detecting the presence of a nucleic acid target molecule in a biological sample, the method comprising the steps of:

(a) contacting a test sample that comprises (i) a biological sample comprising a nucleic acid target molecule and (ii) an osmylated single-stranded oligonucleotide probe comprising at least one pyrimidine residue covalently bonded to a substituted or unsubstituted Osmium tetroxide (OsO4)-2,2′-bypyridine group (OsBp group), wherein the sequence of the probe is at least partially complementary to the sequence of the nucleic acid target molecule, to allow the formation of a hybridized probe/target complex,
wherein at least one osmylated pyrimidine residue is a thymidine residue (T);
(b) using a nanopore device to detect in the test sample the number of events wherein unhybridized osmylated-probe traverses the nanopore;
and
(c)(i) comparing the number of events detected in the test sample to a number of corresponding probe sample events wherein unhybridized osmylated-probe traverses the nanopore in the absence of the nucleic acid target molecule, wherein a reduction in the number of events detected in the test sample relative to the number of probe sample events is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid target molecule in the test sample;
(c)(ii) comparing the number of events detected in the test sample to the noise of a corresponding baseline sample that does not contain any osmylated-probe, wherein an absence of an increase in the number of events detected in the test sample relative to the noise of the baseline sample is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid target molecule in the test sample;
and/or
(c)(iii) comparing the number of events detected in the test sample to a number of corresponding control sample events wherein unhybridized osmylated-probe traverses the nanopore in the presence of a known amount of the nucleic acid target molecule, wherein a reduction in the number of events detected in the test sample relative to the number of control sample events is indicative of increased formation of the hybridized probe/target complex in step (a) and the presence of a higher amount of the nucleic acid target molecule in the test sample over the control sample or wherein an increase in the number of events detected in the test sample relative to the number of control sample events using the same amount of probe is indicative more unhybridized probe and thus of a lower amount of the nucleic acid target molecule in the test sample compared to the control sample.

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Dependencies are inferred from the claim text.

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Abstract

A method for detecting the presence of a nucleic acid target molecule in a biological sample, the method comprising the steps of:

(a) contacting a test sample that comprises (i) a biological sample comprising a nucleic acid target molecule and (ii) an osmylated single-stranded oligonucleotide probe comprising at least one pyrimidine residue covalently bonded to a substituted or unsubstituted Osmium tetroxide (OsO4)-2,2′-bypyridine group (OsBp group), wherein the sequence of the probe is at least partially complementary to the sequence of the nucleic acid target molecule, to allow the formation of a hybridized probe/target complex,
wherein at least one osmylated pyrimidine residue is a thymidine residue (T);
(b) using a nanopore device to detect in the test sample the number of events wherein unhybridized osmylated-probe traverses the nanopore;
and
(c)(i) comparing the number of events detected in the test sample to a number of corresponding probe sample events wherein unhybridized osmylated-probe traverses the nanopore in the absence of the nucleic acid target molecule, wherein a reduction in the number of events detected in the test sample relative to the number of probe sample events is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid target molecule in the test sample;
(c)(ii) comparing the number of events detected in the test sample to the noise of a corresponding baseline sample that does not contain any osmylated-probe, wherein an absence of an increase in the number of events detected in the test sample relative to the noise of the baseline sample is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid target molecule in the test sample;
and/or
(c)(iii) comparing the number of events detected in the test sample to a number of corresponding control sample events wherein unhybridized osmylated-probe traverses the nanopore in the presence of a known amount of the nucleic acid target molecule, wherein a reduction in the number of events detected in the test sample relative to the number of control sample events is indicative of increased formation of the hybridized probe/target complex in step (a) and the presence of a higher amount of the nucleic acid target molecule in the test sample over the control sample or wherein an increase in the number of events detected in the test sample relative to the number of control sample events using the same amount of probe is indicative more unhybridized probe and thus of a lower amount of the nucleic acid target molecule in the test sample compared to the control sample.

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