Compounds and methods for detecting oligonucleotides

Patent metadata

Field	Value
Publication number	US-2016281148-A1
Application number	US-201415034508-A
Country	US
Kind code	A1
Filing date	Nov 10, 2014
Priority date	Nov 8, 2013
Publication date	Sep 29, 2016
Grant date	—

Abstract

The present invention provides methods of detecting and/or quantitating a target oligonucleotide in a biological sample.

First claim

We claim:

1 . A method for detecting or quantitating a target oligonucleotide in a bodily fluid or extract wherein the target oligonucleotide is 8 to 50 nucleosides in length, comprising:
creating a test sample by contacting the bodily fluid or extract with a capture probe, wherein:
the capture probe is complementary to a first portion of the target oligonucleotide; and
the capture probe comprises a binding moiety, wherein the binding moiety is covalently bound to the capture probe;
contacting the test sample with a solid support wherein the solid support comprises a binding partner of the binding moiety of the capture probe; contacting the test sample with a detector probe, wherein:
the detector probe is complementary to a second portion of the target oligonucleotide,
wherein the first portion and the second portion of the target oligonucleotide do not overlap; and
the detector probe comprises a electrochemiluminescent moiety, wherein the electrochemiluminescent moiety is covalently bound to the detector probe;
washing the test sample to remove unbound probe; detecting the presence or amount of the electrochemiluminescent moiety in the test sample; and thereby detecting or quantitating the target oligonucleotide in the bodily fluid or extract.

2 . The method of claim 1 , wherein the target oligonucleotide is 10-40 nucleosides in length.

3 . The method of claim 1 , wherein the target oligonucleotide is 14-40 nucleosides in length.

4 . The method of claim 1 , wherein the target oligonucleotide is 18-40 nucleosides in length.

5 . The method of claim 1 , wherein the target oligonucleotide is 20-40 nucleosides in length.

6 . The method of claim 1 , wherein the target oligonucleotide is 10-30 nucleosides in length.

7 . The method of claim 1 , wherein the target oligonucleotide is 14-30 nucleosides in length.

8 . The method of claim 1 , wherein the target oligonucleotide is 18-30 nucleosides in length.

9 . The method of claim 1 , wherein the target oligonucleotide is 20-30 nucleosides in length.

10 . The method of claim 1 , wherein the target oligonucleotide is 10-22 nucleosides in length.

11 . The method of claim 1 , wherein the target oligonucleotide is 14-22 nucleosides in length.

12 . The method of claim 1 , wherein the target oligonucleotide is 18-22 nucleosides in length.

13 . The method of claim 1 , wherein the target oligonucleotide is 20-22 nucleosides in length.

14 . The method of any of claims 1 - 13 wherein the target oligonucleotide comprises at least modified nucleoside.

15 . The method of claim 14 wherein the target oligonucleotide comprises at least one modified nucleoside comprising a 2′-O-methoxyethyl modification.

16 . The method of claim 14 or 15 , wherein the target oligonucleotide comprises at least one bicyclic nucleoside.

17 . The method of claim 16 wherein the target oligonucleotide comprises at least one cEt nucleoside.

18 . The method of any of claims 14 - 17 wherein the target oligonucleotide is a gapmer.

19 . The method of any of claims 14 - 17 wherein the target oligonucleotide is a hemimer.

20 . The method of any of claims 1 - 19 wherein the target oligonucleotide comprises at least one modified base.

21 . The method of any of claims 1 - 20 wherein the target oligonucleotide comprises at least one modified internucleoside linkage.

22 . The method of any of claims 1 - 21 wherein the target oligonucleotide is an aptamer.

23 . The method of any of claims 1 - 21 , wherein the target oligonucleotide is an antisense oligonucleotide.

24 . The method of claim 23 , wherein the target oligonucleotide is an RNase H dependent antisense compound.

25 . The method of claim 24 , wherein the target oligonucleotide is a RISC dependent antisense compound.

26 . The method of any of claims 1 - 25 , wherein the target olignucleotide is single-stranded.

27 . The method of any of claims 1 - 26 , wherein the target olignucleotide is double-stranded.

28 . The method of any of claims 1 - 27 , wherein the target oligonucleotide is self-structured under physiologic conditions.

29 . The method of any of claims 1 - 28 wherein the bodily fluid is plasma.

30 . The method of any of claims 1 - 28 wherein the bodily fluid is cerebral-spinal fluid.

31 . The method of any of claims 1 - 30 wherein the binding moiety of the capture probe is conjugated to the 5′ end of the capture probe.

32 . The method of any of claims 1 - 30 wherein the binding moiety of the capture probe is conjugated to the 3′ end of the capture probe.

33 . The method of any of claims 1 - 32 wherein the capture probe comprises at least one 2′-O-methoxyethyl sugar modification.

34 . The method of any of claims 1 - 33 wherein the capture probe comprises at least one cEt sugar modification.

35 . The method of any of claims 1 - 34 wherein the capture probe comprises at least one 2′-F sugar modification.

36 . The method of any of claims 1 - 35 wherein the capture probe comprises at least one 2′-deoxy sugar.

37 . The method of any of claims 1 - 36 wherein the capture probe comprises at least one inosine.

38 . The method of any of claims 1 - 37 wherein the detector probe comprises at least one 2′-O-methoxyethyl sugar modification.

39 . The method of any of claims 1 - 38 wherein the detector probe comprises at least one cEt sugar modification.

40 . The method of any of claims 1 - 39 wherein the detector probe comprises at least one 2′-F sugar modification.

41 . The method of any of claims 1 - 40 wherein the detector probe comprises at least one 2′-deoxy sugar.

42 . The method of any of claims 1 - 41 wherein the detector probe comprises at least one inosine.

43 . The method of any of claims 1 - 42 wherein the electrochemiluminescent moiety of the detector probe is conjugated to the 3′ end of the detector probe.

44 . The method of any of claims 1 - 42 wherein the electrochemiluminescent moiety of the detector probe is conjugated to the 5′ end of the detector probe.

45 . The method of any of claims 1 - 18 wherein the capture and detector probes are each 7-10 nucleotides in length.

46 . The method of any of claims 1 - 45 wherein detecting the electrochemiluminescent moiety is a tris(2,2-bipyridine) Ruthenium (II) tag.

47 . The method of any of claims 1 - 46 wherein the method does not comprise addition of a ligase.

48 . The method of any of claims 1 - 47 wherein the method does not comprise addition of a nuclease.

49 . The method of any of claims 1 - 48 wherein the method has a sensitivity of less than 1 ng/ml of target oligonucleotide.

50 . The method of any of claims 1 - 48 wherein the method has a sensitivity of less than 500 pg/ml of target oligonucleotide.

51 . The method of any of claims 1 - 48 wherein the method has a sensitivity of less than 100 pg/ml of target oligonucleotide.

Assignees

• Ionis Pharmaceuticals Inc

Inventors

• Greenlee Sarah
• Yu Zhengrong
• Prakash Thazha P
• Matson John E

Classifications

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q1/6825

  Nucleic acid detection involving sensors · CPC title

• C12Q1/6834 Primary

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title