Modified nucleotides

• US 8,536,323 B2
• Filed: 04/20/2011
• Issued: 09/17/2013
• Est. Priority Date: 04/21/2010
• Status: Active Grant

• Current Assignee: Pierce Biotechnology Incorporated (Perspecta, Inc.)
• Original Assignee: Pierce Biotechnology Incorporated (Perspecta, Inc.)
• Inventors: Opperman, Kay, Kaboord, Barbara J., Schultz, Jean-Samuel, Etienne, Christopher L., Hermanson, Greg
• Primary Examiner(s): OLSON, ERIC
• Application Number:
  US13/090,729
• Publication Number:
  US 20110262917A1
• Time in Patent Office: 881 Days
• Field of Search: None
• US Class Current: 536/26.3
• CPC Class Codes: C07H 19/067 with ribosyl as the saccharide radical
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/167 with ribosyl as the saccharide radical
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Modified nucleotides, and methods to modify nucleotides with a moiety or label, such as biotin, that permits their detection and results in a modified nucleotide, and methods of use of the modified nucleotide in quantitative and qualitative assays.

Claims

1. A compound having the structure (I):

2. The compound of claim 1 wherein

the sugar is ribose having a 5′

site, a 3′

site, and a 1′

site,

P1 is attached to ribose at the 5′

site, P2 is attached to ribose at the 3′

site,

and the purine or pyrimidine base is selected from cytosine (C), uracil (U), adenine (A), guanine (G), or inosine (I) and is attached to ribose at the 1′

site.

3. The compound of claim 1 wherein the purine or pyridine base is selected from 1-methyladenine, N6-methyladenine, N6-isopentyladenine, N,N-dimethyladenine, 7-deazaadenine, 2-thiocytosine, 3-methylcytosine, N4-acetylcytosine, 2-thiocytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, N2,N2-dimethylguanine, 7-deazaguanine, 2-thiouracil, 6-thiopurine, or 2,6-diaminopurine.

4. The compound of claim 1 wherein the observable label is a chromogen, a fluorophore, a mass label, a spin label, a streptavidin-binding label, or a secondary detection label.

5. The compound of claim 1 wherein n is an integer ranging from 2 to 24 inclusive.

6. The compound of claim 1 wherein the sugar is ribose, the purine or pyrimidine base is selected from adenine (A), cytosine (C), guanine (G), uracil (U), or inosine (I), m is 3, n is 4, and the observable label is a streptavidin-binding compound selected from biotin, desthiobiotin, or iminobiotin.

7. The compound of claim 1 wherein the sugar is ribose, the purine or pyrimidine base is cytosine (C),

m is 3, Lnk is

8. A method of labeling a ribonucleic acid (RNA), the method comprising

heating RNA in a solution, the solution optionally containing dimethylsulfoxide at a concentration up to 25%, to at least 75°

C. up to 95°

C. then cooling the heated RNA for at least one minute to less than 10°

C., and,

contacting the heated and cooled RNA with the compound of claim 1 under reaction conditions using T4 RNA ligase and including PEG having molecular weight between about 1500 and 24,000 inclusive and at a concentration ranging from 5% PEG to 20% PEG inclusive to ligate the compound of claim 1 to the RNA to result in a modified RNA.

9. The method of claim 8 wherein the concentration of PEG is about 15%.

10. The method of claim 8 wherein the molecular weight of PEG is 20,000.

11. The method of claim 8 using the compound of claim 1.

12. A method of synthesizing the compound of claim 1, wherein the compound is biotin-polyethylene glycol (PEG)-alkane-3′

,5′

-cytidine-bisphosphate, the method comprising

reacting propargyltrifluoroacetamide with 5-iodocytidine to result in 5-[3-(trifluoroacetamido)propynyl]cytidine,

converting 5-[3-(trifluoroacetamido)propynyl]cytidine to 5-[3-(trifluoroacetamido)propyl]cytidine, converting 5-[3-(trifluoroacetamido)propyl]cytidine to 5-(3-aminopropyl)cytidine,

reacting 5-(3-aminopropyl)cytidine with NHS-PEG-biotin to result in biotin-PEG-alkane-cytidine,

and

reacting biotin-PEG-alkane-cytidine with diphosphoryl chloride to result in biotin-polyethylene glycol (PEG)-alkane-3′

,5′

-cytidine-bisphosphate.

13. A kit comprising the compound of claim 1 and instructions for labeling a ribonucleic acid (RNA) with the compound.

14. A kit comprising the compound of claim 1, wherein Obs is biotin, and instructions for capturing and/or using a ribonucleic acid (RNA) labeled with the compound on an array or chip.

15. A method for assaying a ribonucleic acid (RNA) analyte, the method comprising

labeling an RNA with the compound of claim 1 to result in a modified RNA probe, and

contacting the modified RNA probe with a sample containing the RNA analyte under conditions to hybridize the modified RNA probe with the RNA analyte, and

detecting the RNA analyte hybridized with the modified RNA probe, wherein hybridization and detection of the modified RNA probe assays the RNA analyte.

16. The method of claim 15 where the assay is at least one of mobility shift, Northern blot, pull-down assay, or in situ hybridization.

17. The method of claim 15 where the detection uses a streptavidin-conjugated reporter molecule.

18. The method of claim 15 where the reporter molecule is selected from the group consisting of enzymes, fluorescent compounds, isotopes, gold particles, and combinations thereof.

19. The method of claim 15 using the compound of claim 1.

20. A compound having the structure