Oligonucleotide labeling reagents

• US 6,734,297 B2
• Filed: 01/14/2003
• Issued: 05/11/2004
• Est. Priority Date: 06/04/2001
• Status: Expired due to Fees

• Current Assignee: Barrskogen, Inc.
• Original Assignee: Barrskogen, Inc.
• Inventors: Kempe, Tomas
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/341,629
• Publication Number:
  US 20030109032A1
• Time in Patent Office: 483 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.32, 435/6, 501/33, 526/72, 526/336
• US Class Current: 536/25.3
• 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: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]

Abstract

A labeling reagent for use in oligonucleotide (“oligo”) synthesis, as well as a method of preparing such a labeling reagent, a method of using such a reagent for synthesizing a labeled oligonucleotide, and an oligonucleotide prepared using such a reagent. The reagent can be used to label either the 3′ or 5′ termini of a synthesized oligo, and/or for one or more positions along the oligo. The labeling reagent can be prepared by a reaction scheme that involves the initial preparation of hydroxyacids, tritylated hydroxyacids and coupling of such derivatives to diamine, wherein the amine function serves at an attachment point for labels and the hydroxyl groups can either be used to immoblize the molecules to support or can be converted to provide a phosphorylating reagent.

Claims

1. A method of preparing a labeling reagent for use in oligonucleotide synthesis, the method comprising the steps of providing a tritylated hydroxyacid and a diamine, and condensing the hydroxyacid and diamine under conditions suitable to provide a reagent backbone having one or more label attachment sites and one or more sites for attaching the reagent to a support or amidite.

2. A method according to claim 1 wherein the tritylated hydroxyacid comprises a DMT-hydroxyacid.

3. A method according to claim 1 wherein the method is used to prepare a reagent selected from the group:

4. A method according to claim 3 wherein R₃ is a label or reporter molecule which is stable under the conditions of oligonucleotide synthesis.

5. A method according to claim 4 wherein the label or reporter molecule is selected from the group consisting of biotin, fluorescein, rhodamine, 4-(4′

-Dimethylamino-phenylazo)benzoic acid (“

Dabcyl”

);

4-(4′

-Dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride) (“

Dabsyl”

);

5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid (“

EDANS”

);

Psoralene derivatives, haptens, cyanines, acridines, fluorescent rhodol derivatives, cholesterol derivatives, ethylenediaminetetraaceticacid (“

EDTA”

), and derivatives and analogs thereof;

as well as radioactive labels.

6. A method according to claim 1 wherein the reagent is attached to a support material by means of a cleavable linkage.

7. A method according to claim 6 wherein the cleavable linkage comprises a succinyl ester linkage.

8. A method of synthesizing an oligonucleotide, comprising the steps of providing a reagent prepared according to the method of claim 1 wherein the reagent is provided in the form of a labeled support and the label is incorporated at the 3′

terminus of the oligonucleotide.

9. A method according to claim 8 wherein the reagent is provided in the form of a labeled phosphoramidite reagent and incorporated at either the 5′

terminus or along the length of the oligonucleotide.

10. A method according to claim 9 wherein a plurality of reagents are used to provide a corresponding plurality of labels in a single oligonucleotide.

11. A method according to claim 5 wherein

the fluoresceins are selected from the group consisting of;

5-carboxyfluorescein,

6-carboxyfluorescein,

5-(and

     6)carboxy-2′

,7′

-dichloro-fluorescein,

5-(and

     6)2′

,4′

,5′

,7′

,-tetrachlorofluorescein,

5-(and 6-)carboxy-4,7-dichloro-2′

,7′

,-dimethoxyfluorescein,

5-(and 6-)carboxy-4,7-dichloro-fluorescein,

5-(and 6-)carboxy-4,7,4′

,5′

,-tetrachloro-fluorescein,

5-(and 6-)carboxy-4′

,5′

-dichloro-2′

,7′

-dimethoxyfluorescein,

5-(and 6-)carboxy-4,7-dichloro-1′

,2′

,7′

,8′

-dibenzofluorescein,

5-(and 6-)carboxy-4,7,4′

,5′

-tetrachloro-1′

,2′

,7′

,8′

-dibenzofluorescein,

5-(and 6-)carboxy-4,7,4′

,5′

-tetrachloro-2′

,7′

-dimethoxyfluorescein,

5-(and 6-)aminomethylfluorescein, and

4′

,5′

-dimethoxy-5(and 6-)carboxyfluorescein;

the rhodamines are selected from the group consisting of

5-(and 6-)carboxyrhodamine,

5-(and -6)carboxytetramethylrhodamine (“

TAMRA”

),

Rhodamine Red, and

Rhodamine Green,

the psoralene derivatives are selected from the group consisting of

8-methoxypsoralene, and

4,5′

,8-trimethylpsoralene, and

the fluorescent rhodol derivatives comprise 5-(and 6-)carboxy-2′

,7′

-dimetyl-3′

-hydroxy-6′

-ethylaminospiro(isobenzofuran-1(3H), 9-(9)xanthen)-3-one.

12. A method according to claim 3 wherein R₃ is an amine protecting group selected from the group consisting of 9-fluorenylmethoxycarbonyl, monomethoxytrityl, and trifluoracetyl protecting groups, the protecting group being adapted to permit a label or other group to be incorporated post-synthesis.

13. A reagent for use in oligonucleotide synthesis, the reagent comprising a tritylated hydroxyacid of the formula

14. A method of oligonucleotide synthesis, comprising the steps of providing the reagent of claim 13, condensing the reagent to an aminated support via formation of an amide linkage between the carboxyl group of the reagent and an amine group of the support, and attaching a phosphoramidite in the course of detritylating the reagent.