Nucleic acid labeling compounds

• US 20050003496A1
• Filed: 12/23/2003
• Published: 01/06/2005
• Est. Priority Date: 01/23/1996
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: McGall, Glenn H., Barone, Anthony D., Li, Handong
• Granted Patent: US 7,291,463 B2
• Time in Patent Office: Days
• US Class Current: 435/117
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• 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
• CPC Class Codes: C12P 17/00 Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms C12P13/04 - C12P13/24 take precedence

Abstract

Nucleic acid labeling compounds containing heterocyclic derivatives are disclosed. Methods for making such heterocyclic compounds are also disclosed. The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.

Claims

1. A method for converting a pseudoisocytidine base to a pseuodouridine base, said method comprising the steps of

providing a pseudoisocytidine molecule having the formula

wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;

X is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl;

Y is H, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl;

Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl;

L is a linker group selected from the group consisting of

—

CH═

CH—

R₁₁, or C≡

C—

R₁₁ wherein R₁₁ comprises a moiety selected from the group consisting of alkyl, functionalized alkyl, alkenyl alkyl, alkynal alkyl, amido alkyl, amino alkyl, alkoxy, amino, aryl, and

thio;

Q is a detectable moiety; and

, M is a connecting group, and wherein m is an integer ranging from 0 to about 3;

incubating said pseudoisocytidine molecule with EDC to provide a pseudouridine molecule having the formula;

wherein Q, (M)m, L, A, X, Y and Z have the meaning ascribed above.

2. A method according to claim 1 wherein A is selected from the group consisting of H, α

-thiotriphosphate and H₄O₉P₃—

;

X is O;

Y is H or OH;

Z is H or OR₁₀, wherein R₁₀ is H, alkyl or aryl.

3. A method according to claim 2 wherein Q is selected from the group consisting of colloidal gold, a fluorescent group and biotin.

4. A method according to claim 3 wherein Q is a biotin.

5. A method according to claim 3 having a first M and a second M, wherein said first M is —

NH(CH₂)_nNH—

, wherein n is an integer from about 2 to about 10, and a second M is —

CO(CH₂)_pNH—

, wherein p is an integer from about 2 to about 10 and m is 2.

6. A method according to claim 5 wherein said first M is —

NH(CH₂)₂NH— and

said second M is —

CO(CH₂)₅NH—

.

7. A method according to claim 1 wherein A is a triphosphate group having appropriate counterions and Y is OH and Z is OH.

8. A method according to claim 7 wherein said counterions are selected from the group consisting of H⁺, Na⁺, Li⁺, K⁺, (CH₃CH₂)₃NH⁺ and NH₄⁺.

9. A method for converting a pseudoisocytidine base to a biotinylated pseuodouridine, said method comprising the steps of

providing a pseudoisocytidine base having the formula

condensing said pseudoisocytidine molecule with one equivalent of (biotin-ε

-aminocaprolamido)ethylamine using excess EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to form a biotinylated pseudouridine compound having the formula;

10. A method according to claim 9 wherein said step of condensing is carried out at pH 5.5 in MES buffer.