Nuclease-based method for detecting and quantitating oligonucleotides
View Patent ↗A method for quantitating an oligonucleotide in a sample of bodily fluid and/or extract is provided. The method provides for the detection and/or localization of oligonucleotides, including administered modified oligonucleotides, for therapeutic and/or pharmacokinetic purposes.
1. A method for detecting or quantitating an oligonucleotide in a bodily fluid or extract wherein the oligonucleotide is 8 to 50 nucleosides in length, comprising:
creating a test sample by contacting the bodily fluid or extract with a probe, wherein:
the probe is complementary to the oligonucleotide; and
the probe comprises a detectable marker and a binding moiety, wherein the detectable marker and the binding moiety are each covalently bound to the probe;
contacting the test sample with a solid support wherein the solid support comprises a binding partner of the binding moiety;
contacting the test sample with a single-strand-specific nuclease;
washing the test sample to remove detectable marker that is not associated with the solid support;
detecting the presence or amount of detectable marker in the test sample; and
thereby detecting or quantitating the oligonucleotide in the bodily fluid or extract.
2. The method of claim 1 wherein the oligonucleotide is 20-30 nucleosides in length.
3. The method of claim 1 wherein the bodily fluid is plasma.
4. The method of claim 1 wherein the oligonucleotide comprises at least one phosphorothioate linkage.
5. The method of claim 1 wherein the oligonucleotide comprises at least one 2′ sugar modification.
6. The method of claim 5 wherein the 2′ sugar modification is a 2′-O-methoxyethyl modification.
7. The method of claim 5 wherein the oligonucleotide is a gapmer.
8. The method of claim 5 wherein the oligonucleotide is a hemimer.
9. The method of claim 1 wherein the oligonucleotide comprises at least one modified base.
10. The method of claim 9 wherein the modified base is 5-methylcytosine.
11. The method of claim 1 wherein detecting the detectable marker comprises detecting a detectable label selected from among a colorimetric, radioactive, chemiluminescent, enzymatic or fluorescent label.
12. The method of claim 1 wherein the detectable label is digoxigenin.
13. The method of claim 1 wherein the single-strand-specific nuclease is S1 nuclease.
14. The method of claim 1 wherein the single-strand-specific nuclease is mung bean nuclease.