Capture moieties for nucleic acids and uses thereof

• US 7,230,092 B2
• Filed: 06/06/2001
• Issued: 06/12/2007
• Est. Priority Date: 06/06/2000
• Status: Expired due to Term

• Current Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Original Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Inventors: Bortolin, Susan, Zastawny, Roman L.
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/297,539
• Publication Number:
  US 20030113781A1
• Time in Patent Office: 2,197 Days
• Field of Search: 435/6, 435/91.1, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 536/24.3
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer

Abstract

A unimolecular probe for hybridization to a molecule comprising a target nucleic acid sequence, the probe includes: a first nucleic acid sequence complementary to the target sequence (target-binding sequence); a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex. In use, the target and target-binding sequence hybridize to form a duplex. A probe can be used to detect a molecule containing the target sequence, act as a primer for synthesis or amplification, etc.

Claims

1. A unimolecular probe for hybridization to a molecule comprising a target nucleic acid sequence, the probe comprising:

(i) a first nucleic acid sequence complementary to the target nuclic acid sequence and comprising a first portion and a second portion;

(ii) a second nucleic acid sequence complementary to the first portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex comprising a first hairpin structure under a defined set of conditions, the first intramolecular duplex including a portion of the first nucleic acid sequence complementary to the target nucleic acid sequence; and

(iii) third and fourth nucleic acid sequences, the third and fourth nucleic acid sequences being complementary to each other and linked to each other so as to hybridize to form a second intramolecular duplex comprising a second hairpin structure under said set of conditions,

wherein the second portion of the first nucleic acid sequence comprises a single standed region covalently attached to the third nucleic acid sequence and is located between the first and second intramolecular duplexes, and under said set of conditions and in the presence of the target nucleic acid sequence, the single standed region acts as a nucleic acid sequence such that hybrization of the target nucleic acid sequence and first nucleic acid sequence disrupts the first intramolecular duplex to form an intermolecular duplex.

2. The probe of claim 1, in combination with said target nucleic acid sequence under said set of conditions, wherein the target nucleic acid sequence is a terminal sequence of a target molecules such that upon formation of the intermolecular duplex, the intermolecular duplex and the second intramolecular duplex form a nicked duplex.

3. The probe of claim 1 wherein the third and fourth nucleic acid sequences are connected to each other by a single stranded nucleic acid sequence up to twenty bases, or up to eighteen, or up to sixteen bases, or up to fourteen, or up to twelve, or up to ten, or up to eight, or up to six bases in length, and at least two bases in length.

4. The probe of claim 1, wherein the first intramolecular duplex is no longer than the second intramolecular duplex and the second intramolecular duplex has a G:

C content greater than that of the first intramolecular duplex.

5. The probe of claim 1, wherein the first intramolecular duplex has a lower T_m than the second intramolecular duplex under said set of conditions.

6. The probe of claim 1, wherein the second intramolecular duplex is between three and thirty base pairs, or between three and twenty-five, or between six and twenty, or between ten and eighteen, or between fourteen and seventeen base pairs in length;

or wherein the second intramolecular duplex is sixteen base pairs in length.

7. The probe of claim 1 wherein the first nucleic acid sequence is up to forty, or is up to thirty, or is up to twenty-six nucleic acid bases in length, or between six and forty, or between eight and thirty-five, or between ten and thirty, or between fifteen and thirty or between eighteen and twenty-eight, or between twenty and twenty-six nucleic acid bases in length;

or wherein the first nucleic acid sequence is twenty-four nucleic acid bases in length.

8. The probe of claim 1 wherein the second nucleic acid sequence is at least four nucleic acid bases in length, or up to fourteen nucleic acid bases in length, or wherein the second nucleic acid sequence is between five and twelve, or between six and twelve, or between seven and twelve, or between eight and twelve, or between nine and twelve, or is ten nucleic acid bases in length.

9. A composition comprising a mixture of a plurality of microparticles, each microparticle having a probe attached thereto for detection of a target nucleic acid sequence, each probe comprising a single molecule comprising:

(i) a first tag sequence complementary to the target nucleic acid sequence;

(ii) a nucleic acid second sequence complementary to a portion of the first tag sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions, the first intramolecular duplex including a portion of the first tag sequence complementary to the target nucleic acid sequence;

wherein;

(a) under said set of conditions, hybridization of the target nucleic acid sequence and first tag disrupts the intramolecular duplex to form an intermolecular duplex;

(b) said mixture of a plurality of microparticles comprises at least ten different populations of microparticles, each different population of said microparticles having a said different probe attached thereto and being for detection of a said target nucleic acid sequence different from that of each other different population of microparticles; and

(c) the first tag sequence of each said probe attached to a population of microparticles is identical to the first tag sequence of every other said probe attached to that population of microparticles.

10. A repertoire of probes comprising a plurality of populations of unimolecular probes for hybridization to molecules, each molecule comprising a target nucleic acid sequence, each probe comprising:

(i) a first nucleic acid sequence complementary to the target nucleic acid sequence; and

(ii) a second nucleic acid sequence complementary to a first portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions, the first intramolecular duplex including a portion of the first nucleic acid sequence complementary to the target nucleic acid sequence; and

wherein;

(a) under said set of conditions and in the presence of the target nucleic acid sequence, hybridization of the target nucleic acid sequence and first nucleic acid sequence disrupts the first intramolecular duplex to form an intermolecular duplex;

(b) each probe of a said population is the same as each other probe in that population;

(c) for each probe of the repertoire, the first nucleic acid sequence of the probe is the same length, and;

(d) the first nucleic acid sequence of each probe comprises a tag complement that is complementary to a tag sequence of the target nucleic acid and wherein there are at least ten populations of said tag complements, in the repertoire of probes, each said tag complement of one population of said probes having a sequence different from the tag complement sequence of tags in every other population of said probes in the repertoire.

11. A unimolecular probe for hybridization to a molecule comprising a target nucleic acid sequence, the probe comprising:

(i) a first nucleic acid sequence complementary to the target nucleic acid sequence;

(ii) a second nucleic acid sequence complementary to a first portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex comprising a first hairpin structure under a defined set of conditions, the first intramolecular duplex including a portion of the first nucleic acid sequence complementary to the target nucleic acid sequence; and

(iii) third and fourth nucleic acid sequences being complementary to each other and linked to each other so as to hybridize to form a second intramolecular duplex comprising a second hairpin structure under said set of conditions;

wherein one end of the first nucleic acid sequence is covalently attached to the third nucleic acid sequence, and under said set of conditions and in the presence of the target nucleic acid sequence, hybridization of the target nucleic acid sequence and first nucleic acid sequence disrupts the first intramolecular duplex to form an intermolecular duplex.

12. The unimolecular probe of claim 11, wherein the first nucleic acid sequence includes a single-stranded region located between the first and second intramolecular duplexes.

13. The unimolecular probe of claim 12, wherein the single-stranded region acts as a nucleation site of intermolecular duplex formation between the target nucleic acid sequence and the first nucleic acid sequence.

14. The unimolecular probe of claim 13, in combination with said target nucleic acid sequence under said set of conditions, wherein the target nucleic acid sequence is a terminal sequence of a target molecule, such that upon formation of the intermolecular duplex, the intermolecular duplex and the second intramolecular duplex form a nicked duplex.

15. The unimolecular probe of claim 11 wherein the third and fourth nucleic acid sequences are connected to each other by a single stranded nucleic acid sequence up to twenty bases, or up to eighteen, or up to sixteen bases, or up to fourteen, or up to twelve, or up to ten, or up to eight, or up to six bases in length, and at least two bases in length.

16. The unimolecular probe of claim 11, wherein the first intramolecular duplex is no longer than the second intramolecular duplex and the second intramolecular duplex has a G:

C content greater than that of the first intramolecular duplex.

17. The unimolecular probe of claim 11, wherein the first intramolecular duplex has a lower T_m than the second intramolecular duplex under said set of conditions.

18. The unimolecular probe of claim 11, wherein the second intramolecular duplex is between three and thirty base pairs, or between three and twenty-five, or between six and twenty, or between ten and eighteen, or between fourteen and seventeen base pairs in length;

or wherein the second intramolecular duplex is sixteen base pairs in length.

19. The unimolecular probe of claim 11 wherein the first nucleic acid sequence is up to forty, or is up to thirty, or is up to twenty-six nucleic acid bases in length, or between six and forty, or between eight and thirty-five, or between ten and thirty, or between fifteen and thirty or between eighteen and twenty-eight, or between twenty and twenty-six nucleic acid bases in length;

or wherein the first nucleic acid sequence is twenty-four nucleic acid bases in length.

20. The unimolecular probe of claim 11 wherein the second nucleic acid sequence is at least four nucleic acid bases in length, or up to fourteen nucleic acid bases in length, or wherein the second nucleic acid sequence is between five and twelve, or between six and twelve, or between seven and twelve, or between eight and twelve, or between nine and twelve, or is ten nucleic acid bases in length.