Nucleic acid probes and methods of using the same
The present invention provides a composition comprising a fluorescent nucleic acid probe that can be cleaved by enzymatic and non-enzymatic means, and methods of using the same. Advantageously, the nucleic acid probe may be used to detect and quantify nucleic acid cleavage in vitro, in situ, ex vivo, and in vivo.
1. A method for detecting nucleic acid cleavage in a sample or in a subject, the method comprising:
(i) providing a nucleic acid probe selected from the group consisting of Cy5.5-AACACTCCGATGAGTGTA-Cy5.5 (SEQ ID NO: 1), Cy5.5-AACACTCCGATGAGTGTAGAATGT-Cy5.5 (SEQ ID NO: 2), and Cy5.5-AACACTCCGATGAGTGTAGAATGTACGAGT-Cy5.5 (SEQ ID NO: 3);
(ii) contacting the sample or the subject with the nucleic acid probe and detecting fluorescence in the sample or the subject, whereby fluorescence in the sample or the subject indicates nucleic acid cleavage of at least one component of the nucleic acid probe; and
(iii) optionally, quantifying the fluorescence in the sample.
2. The method of claim 1 , wherein nucleic acid cleavage is enzymatic and the enzyme is a deoxyribonuclease (DNAse) and the nucleic acid probe is a deoxyribonucleic acid (DNA) probe.
3. The method of claim 2 , wherein the DNase is further selected from the group consisting of DNase I, DNase X, DNase gamma, DNAse 2alpha, DNase 2beta, L-DNase-2, EndoG, CAD, DNAse IV, UvrABC endonuclease, RecBCD, and restriction enzyme.
4. The method of claim 1 , wherein the fluorescence is measured in vitro, in vivo, ex vivo, or in situ.
5. The method of claim 1 , wherein the sample is selected from the group consisting of a cell, a cell lysate, a protein sample, a tissue sample, a homogenized tissue sample, an organ, a homogenized organ, a biopsy sample, and bodily fluid.
6. The method of claim 1 , wherein the nucleic acid cleavage, if present, is enzymatic and the enzyme is DNase I, and the fluorophores are near-infrared fluorophores.
7. A composition comprising a nucleic acid probe, the nucleic acid probe comprising two components: A1-A2-A3 and A5-A6-A7, and optionally a third component, A4, wherein:
(a) A1 and A7 are fluorophores,
(b) A2 is adenine and A6 is a nucleic acid sequence selected from the group consisting of A, AGAATGT, and AGAATGTACGAGT (SEQ ID NO: 6),
(c) A3 is the nucleic acid sequence ACACTC and A5 is the nucleic acid sequence GAGTGT, such that A3 and A5 associate when A1-A2-A3 and A5-A6-A7 are not cleaved and disassociate when at least one of A1-A2-A3 or A5-A6-A7 is cleaved, and
(d) A4 is the nucleic acid sequence CGAT,
whereby the fluorescence of A1 and A7 is quenched when A3 and A5 associate and is detectable when A3 and A5 disassociate.
8. The composition of claim 7 , wherein the fluorophores are near-infrared fluorophores.
9. The composition of claim 7 wherein the nucleic acid probe is selected from the group consisting of Cy5.5-AACACTCCGATGAGTGTA-Cy5.5 (SEQ ID NO: 1), Cy5.5-AACACTCCGATGAGTGTAGAATGT-Cy5.5 (SEQ ID NO: 2), and Cy5.5-AACACTCCGATGAGTGTAGAATGTACGAGT-Cy5.5 (SEQ ID NO: 3).