Hybridization-triggered fluorescent detection of nucleic acids

• US 20030113765A1
• Filed: 08/21/2002
• Published: 06/19/2003
• Est. Priority Date: 10/26/1999
• Status: Active Grant

• Current Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Vermeulen, Nicolaas M.J., Dempcy, Robert O., Afonina, Irina Aleksandrovna
• Granted Patent: US 6,951,930 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

Compositions and methods for fluorescent detection of nucleic acids are provided. The compositions can be detected by fluorescence when hybridized to a nucleic acid containing a target sequence, but are non-fluorescent in the non-hybridized state. Alternatively, the fluorescence properties of the compositions change in a detectable manner upon hybridization to a nucleic acid containing a target sequence. Methods for synthesis and methods of use of the compositions are also provided.

Claims

1. A composition comprising:

(a) an oligonucleotide (ODN), wherein the oligonucleotide comprises a plurality of nucleotides, a 3′

end and a 5′

end;

(b) a minor groove binder (MGB), wherein the minor groove binder is a radical of a molecule having a molecular weight of approximately 150 to approximately 5000 Daltons which molecule binds in a non-intercalating manner into the minor groove of non-single-stranded DNA, RNA or hybrids thereof with an association constant greater than approximately 10³M<sup>−

1</sup>; and

(c) a latent fluorophore (LF);

wherein the latent fluorophore is a radical of a molecule having a molecular weight of approximately 150 to approximately 5000 Daltons which binds in an intercalating manner into non-single-stranded DNA, RNA or hybrids thereof, or alternatively lies in the minor groove, or in another manner is oriented to the DNA molecule by the minor groove binder moiety so that it becomes fluorescent or its fluorescence properties are changed in a detectable way;

wherein the minor groove binder is covalently attached to the oligonucleotide through a first linking group, and the latent fluorophore is covalently attached to the minor groove binder through a second linking group.

2. The composition of claim 1, wherein the minor groove binder moiety is covalently linked to the 5′

end of the oligonucleotide.

3. The composition of claim 1, wherein the minor groove binder moiety is covalently linked to the 3′

end of the oligonucleotide.

4. The composition of claim 1, wherein the minor groove binder moiety is covalently linked to the oligonucleotide at one or more of the nucleotide units.

5. The composition of claim 1, wherein the first linking group comprises a chain having a backbone of no more than about 100 atoms.

6. The composition of claim 1, wherein the second linking group comprises a chain having a backbone of no more than about 50 atoms.

7. The composition of claim 1, wherein the composition does not exhibit substantial fluorescence.

8. The composition of claim 1, wherein the composition does not exhibit detectable fluorescence emission at a particular wavelength.

9. The composition of claim 8, wherein fluorescence emission is increased at the particular wavelength after hybridization of the oligonucleotide to a target sequence.

10. The composition of claim 1, wherein the fluorescence emission maximum shifts from a first wavelength to a second wavelength after hybridization of the oligonucleotide to a target sequence.

11. The composition of claim 1, wherein the oligonucleotide comprises one or more nucleotide analogues.

12. The composition of claim 11, wherein the oligonucleotide is a PNA/DNA chimera, wherein PNA is a polyamide (peptide) nucleic acid.

13. The composition of claim 11, wherein inosine is substituted for guanosine.

14. The composition of claim 11, wherein the oligonucleotide comprises one or more pyrazolopyrimidine nucleotide residues.

15. The composition of claim 14, wherein 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one is substituted for guanine.

16. The composition of claim 14, wherein 4-amino-1H-pyrazolo[3,4-d]pyrimidine is substituted for adenine.

17. The composition of claim 14, wherein 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione is substituted for adenine.

18. A method for detecting a target sequence in a polynucleotide, wherein the method comprises:

(a) combining a composition according to claim 1 with a sample containing a polynucleotide, wherein the oligonucleotide portion of the composition comprises a sequence which hybridizes to the target sequence, to form a hybridization mixture;

(b) incubating the hybridization mixture under conditions which yield specific hybridization; and

(c) thereafter measuring fluorescence of the hybridization mixture, wherein fluorescence is indicative of the presence of the target sequence.

19. The method according to claim 18, wherein the polynucleotide is in a sample comprising a plurality of other polynucleotides having different sequences.

20. The method according to claim 18, wherein the sample comprises a signal amplification hybridization reaction.

21. A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, and wherein one or more of the other polynucleotides in the mixture comprise sequences that are related but not identical to the target sequence, the method comprising the steps of:

(a) contacting the mixture of polynucleotides with a composition according to claim 1, wherein the composition forms a stable hybrid only with said target sequence that is perfectly complementary to the oligonucleotide portion of the composition and wherein the composition does not form a stable hybrid with any of the related sequences; and

(b) measuring fluorescence, wherein fluorescence is indicative of the presence of the target sequence.

22. The method according to claim 21, wherein one or more of the related sequences has a single-nucleotide mismatch with respect to the ODN portion of the composition.

23. A method for detecting one or more sequences related to a target sequence, wherein the one or more related sequences are present in a sample of polynucleotides, the method comprising:

(a) contacting the sample with a composition according to claim 1, wherein the oligonucleotide portion of the composition has a sequence that is complementary to the target sequence, and wherein the composition forms stable hybrids with the related sequences; and

(b) measuring fluorescence, wherein fluorescence is indicative of the presence of the one or more related sequences.

24. The method according to claim 18, wherein a plurality of target sequences are detected;

wherein, for each target sequence, there is a unique ODN-MGB-LF comprising an ODN portion that is complementary to the target sequence and a LF whose fluorescence is distinguishable from that of other LFs conjugated to ODNs complementary to different target sequences.

25. A method for primer extension, wherein the method comprises the steps of:

(a) providing a sample containing a target sequence;

(b) providing one or more oligonucleotide primers, wherein each primer is complementary to a region of the target sequence;

(c) providing a polymerizing enzyme and nucleotide substrates; and

(d) incubating the sample, the oligonucleotide primers, the enzyme and the substrates under conditions favorable for polymerization;

wherein at least one of the primers comprises a ODN-MGB-LF conjugate according to claim 1.

26. The method according to claim 25, wherein the products of polymerization are detected in real time.

27. A ODN-MGB-LF according to claim 1 that is immobilized on a solid support.

28. A method for detecting a target sequence, wherein the method comprises:

(a) immobilizing a ODN-MGB-LF according to claim 1 at an address on a solid support, wherein the ODN is complementary to the target sequence;

(b) exposing the support to a solution potentially containing the target sequence under conditions favorable to hybridization; and

(c) thereafter measuring fluorescence of the support, wherein fluorescence at the address is indicative of the presence of the target sequence in the solution.

29. The method according to claim 28, wherein a plurality of ODN-MGB-LFs, having different sequences in their ODN portions, are immobilized at distinct addresses on the support in an ordered array.

37. A kit for hybridization analysis, wherein the kit comprises a composition according to claim 1.

39. The composition of claim 1, wherein the LF is covalently attached to the ODN through a third linking group and the MGB is attached to the LF through a fourth linking group.

40. The composition of claim 39, wherein the third linking group comprises a chain having a backbone of no more than about 50 atoms.

41. The composition of claim 39, wherein the fourth linking group comprises a chain having a backbone of no more than about 50 atoms.

30. A composition comprising:

31. The compound of claim 30, wherein the leaving group forms part of an activated ester.

32. The compound of claim 31, wherein the activated ester is selected from the group consisting of hydroxysulfosuccinimide esters, hydroxysucciniminde esters, tetrafluorophenyl esters and anhydrides.

33. A method for the synthesis of a ODN-MGB-LF conjugate, wherein the method comprises:

(a) treating the composition of claim 30 with an acid; and

(b) reacting the product of step (a) with an amino-tailed ODN and with a LF, wherein the LF comprises a linking group which terminates in a reactive group.

34. A composition comprising:

35. The composition of claim 34, wherein K is

36. A method for the synthesis of a ODN-MGB-LF conjugate, wherein the method comprises:

(a) removing the DMTr group from the composition of claim 34;

(b) using the product of step (a) for oligonucleotide synthesis to generate a CPG-MGB-ODN;

(c) removing the MGB-ODN of step (b) from the CPG by cleavage at the cleavable linker K; and

(d) reacting the product of step (c) with a LF, wherein the LF comprises a linking group which terminates in a reactive group, to produce a ODN-MGB-LF.

38. A composition having the properties of a latent fluorophore wherein the composition comprises an electron donating group and an electron accepting group which are covalently joined to each other by a resonance linker.