Fluorescent base analogues' usage in the characterization of nucleic acid molecules and their interactions

• US 20080020378A1
• Filed: 09/23/2005
• Published: 01/24/2008
• Est. Priority Date: 09/25/2004
• Status: Active Grant

• Current Assignee: Yaodong Chen
• Original Assignee: Yaodong Chen
• Inventors: Chen, Yaodong
• Granted Patent: US 7,851,152 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

This invention provides methods, apparatus and kits for the quantitative and qualitative characterization of the nucleic acid molecule'"'"'s behavior by modify the nucleic acid molecules to incorporate selected fluorescent base analogues (FBAs). The methods generally place one or more fluorescent base analogue into a nucleic acid molecule (e.g., an oligonucleotide) to replace the corresponding normal base(s), arrange fluorescent base analogues as intrinsic fluorescent probes by using direct excitation, indirect excitation, and excimer emission labeling schemes, introducing so modified nucleic acid molecules into the matrix with interested condition and measuring the fluorescent properties of the modified nucleic acid molecules at the specific emission wavelength of FBA(s). The apparatus is designed to irradiate the FBA(s) incorporated nucleic acid molecule at a wavelength in the range of 240 nm-280 nm and detect the fluorescent activities at the specific emission wavelength of the respective FBA(s). The kit provides oligonucleotides modified by multiple FBAs in the position of critical portions. It utilizes simultaneous indirect excitation labeling scheme for qualitative and quantitative investigation of nucleic acid molecules'"'"' interaction in vitro and in vivo.

Claims

1. A method of detecting the structures and activities of nucleic acid molecules, said method comprising the steps of:

placing or substituting the corresponding nucleotide(s) of said nucleic acid molecule with one or multiple fluorescent nucleotide analogue(s);

introducing so modified nucleic acid molecules into the into the target matrix or medium so as to subject the nucleic acid molecules to the interaction with the components in the matrix or medium;

measuring the fluorescence properties of the fluorescent base analogues incorporated at their respective emission wavelength by indirect excitation of the neighboring natural bases at a wavelength in the range of 240 nm to 280 nm.

2. A method of detecting the structure and interaction of nucleic acid molecules of claim 1, in which the fluorescent base analogues is 2-aminopurine, and the emission wavelength tested is in the range of 350 nm to 380 nm.

3. A method of detecting the structure and interaction of nucleic acid molecules of claim 1, in which the fluorescent base analogues is 4-amino-6-methyl-pteridone, and the emission wavelength tested is in the range of 410 nm to 450 nm.

4. A method of detecting the structure and interaction of nucleic acid molecules of claim 1, in which the fluorescent base analogues is 6-methyl-sioexanthopterin, and the emission wavelength tested is in the range of 410 nm to 450 nm.

5. A method of detecting the structure and interaction of nucleic acid molecules of claim 1, in which the fluorescent base analogues is pyrrolo-(d)C, and the emission wavelength tested is in the range of 440 nm to 480 nm.

6. An apparatus for measuring the fluorescent activity of fluorescent base analogue(s) incorporated nucleic acid molecules comprising:

a light-tight housing;

a radiation source illuminates a fluorescent base analogue(s) incorporated nucleic acid sample at a wavelength in the range of 240 to 280 nm;

an optical system that collects fluorescent light from the sample at the specific emission wavelength of the FBA(s) incorporated; and

a detector system that senses the collected light and provides a fluorescence spectrum as a function of time.

7. The apparatus of claim 6, in which the radiation system further comprising a monochromator fixed at a wavelength in the range of 240-280 nm.

8. The apparatus of claim 6 wherein the optical system is set to collect the fluorescent light in the range of 350 nm to 380 nm.

9. The apparatus of claim 6 wherein the optical system is set to collect the fluorescent light in the range of 410 nm to 450 nm.

10. The apparatus of claim 6 wherein the optical system is set to collect the fluorescent light in the range of 440 nm to 480 nm.

11. The apparatus of claim 6, wherein the optical system has three settings for the wavelengths at about 370 nm, 433 nm, and 460 nm.

12. The apparatus of claim 6, wherein the radiation source and the detector system are further coupled with a controller to synchronize the detection with the radiation.

13. The apparatus of claim 6, wherein the apparatus is further coupled with a computer system.

14. The apparatus of claim 6, wherein the apparatus has multiple optical systems set for specific wavelength of the respective FBA, coupled with multiple detector systems.

15. A kit, comprising, in separately packed units,

a set of oligonucleotides incorporating the fluorescent base analogue in any of the following arranged positions:

2-Aminopurines at the 5′

end portion, 6-methyl-isoxanthopterin at the middle portion and prorolo-(d)C at the 3′

end portion of the oligonucleotides;

2-Aminopurines at the 5′

end portion, 6-methyl-isoxanthopterin at the 5′

end portion and prorolo-(d)C at the middle portion of the oligonucleotides;

2-Aminopurines at the middle portion, 6-methyl-isoxanthopterin at the 5′

end portion and prorolo-(d)C at the 3′

end portion of the oligonucleotides;

2-Aminopurines at the 3′

end portion, 6-methyl-isoxanthopterin at the 5′

end portion and prorolo-(d)C at the middle portion of the oligonucleotides;

2-Aminopurines at the middle portion, 6-methyl-isoxanthopterin at the 3′

end portion and prorolo-(d)C at the 5′

end portion of the oligonucleotides;

2-Aminopurines at the 3′

end portion, 6-methyl-isoxanthopterin at the middle portion and prorolo-(d)C at the 5′

end portion of the oligonucleotides;

16. The kit in claim 15, with all the FBAs incorporated oligonucleotides indicated in claim 15 are included.

17. The kit in claim 15, wherein the FBAs incorporated oligonucleotides are double strand.

18. The kit in claim 15, further includes a suitable buffer for the stability and viability of the nucleic acid molecules.