Labeled nucleotide composition

• US 7,524,942 B2
• Filed: 07/31/2006
• Issued: 04/28/2009
• Est. Priority Date: 07/31/2006
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Wang, Hui, Sampson, Jeffrey R.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/497,208
• Publication Number:
  US 20080124708A1
• Time in Patent Office: 1,002 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.3, 536/25.3, 536/26.6
• US Class Current: 536/23.1
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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

Abstract

Embodiments of labeled nucleotide compositions are described. Methods are described in which a sample containing RNA is contacted with an enzyme having an RNA ligation activity in the presence of a labeled nucleotide composition to provide labeled RNA. Methods of performing an array analysis of a labeled RNA sample are also described.

Claims

1. A composition having the structure (I):

P1-Nus-P2-Lnk-Obs 

 

(I)

or a salt, conjugate base, tautomer, or ionized form thereof,

wherein;

P1 is a phosphate group,

Nus is a nucleoside moiety comprising a sugar group bound to a purine or pyrimidine base;

P2 is a phosphate group;

Lnk is a linking group having the structure

2. The composition of claim 1, wherein the sugar group has a 5′

site, a 3′

site, and a 1′

site, P1 is attached to the sugar group at the 5′

site, P2 is attached to the sugar group at the 3′

site, and the purine or pyrimidine base is attached to the sugar group at the 1′

site.

3. The composition of claim 1, wherein the sugar group is ribose and the purine or pyrimidine base is selected from adenine (A), cytosine (C), guanine (G), or uracil (U).

4. The composition of claim 1, wherein the purine or pyrimidine base is selected from 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, uracil-5-oxyacetit acid methyl ester, pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, or 2,6-diaminopurine.

5. The composition of claim 1, wherein the observable label moiety is selected from a chromogenic moiety, a fluorophore, a mass label, a spin label, or a radiolabel.

6. The composition of claim 1, wherein the observable label moiety is a fluorophore selected from the group consisting Cy3, Cy5, or an Alexa dye.

7. The composition of claim 1, wherein m and n are integers independently selected from the range of 3 to about 6.

8. The composition of claim 1, wherein the sugar group is ribose and the purine or pyrimidine base is selected from adenine (A), cytosine (C), guanine (G), or uracil (U), wherein m is 6 and n is 3, and wherein the observable label moiety is a fluorophore selected from the group consisting of Cy3, Cy5, and an Alexa dye.

16. A method of labeling RNA in a sample to provide a labeled RNA, the method comprising:

contacting the RNA with a compound of claim 1 in the presence of an enzyme having an RNA ligase activity to provide the labeled RNA, said compound of claim 1 capable of being a substrate for said enzyme.

17. The method of claim 16, wherein the contacting is performed under conditions including a DMSO concentration in the range from about 20% to about 30%.

18. The method of claim 16, wherein the RNA in the sample comprises isolated RNA having length less than about 200 bases.

19. The method of claim 16, further comprising, prior to the contacting, heating the sample to at least about 80°

C. under conditions including at least about 40% DMSO.

20. The method of claim 16, wherein the enzyme is selected from T4 RNA ligase, RNA ligase II, yeast poly A polymerase, E. coli poly A polymerase, or terminal transferase.

9. A compound having the structure (II):

10. The composition of claim 9, wherein the sugar group is ribose and the purine or pyrimidine base is selected from adenine (A), cytosine (C), guanine (G), or uracil (U).

11. The composition of claim 9, wherein the purine or pyrimidine base is selected from 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, or 2,6-diaminopurine.

12. The composition of claim 9, wherein the observable label moiety is selected from a chromogenic moiety, a fluorophore, a mass label, a spin label, or a radiolabel.

13. The composition of claim 9, wherein the observable label moiety is a fluorophore selected from the group consisting Cy3, Cy5, or an Alexa dye.

14. The composition of claim 9, wherein m and n are integers independently selected from the range of 3 to about 6.

15. The composition of claim 9, wherein the purine or pyrimidine base is selected from adenine (A), cytosine (C), guanine (G), or uracil (U), wherein m is 6 and n is 3, and wherein the observable label moiety is a fluorophore selected from the group consisting of Cy3, Cy5, and an Alexa dye.