Oligonucleotide labeling reagents

Details Oligonucleotide labeling reagents Oligonucleotide labeling reagents

2001-06-04

2003-03-18

Fredman, Jeffrey (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025320, C558S192000, C560S155000, C428S402000

Reexamination Certificate

active

06534646

ABSTRACT:

TECHNICAL FIELD
This invention relates to methods and reagents for oligonucleotide synthesis, including for instance, reagents used to prepare labeled oligonucleotides.
BACKGROUND OF THE INVENTION
A variety of approaches exist, and in turn, a variety of reagents are available, for use in incorporating labeled molecules in synthetic oligonucleotides. For instance, the following sections repeat or paraphrase certain relevant portions of Nelson U.S. Pat. No. 5,451,463 in this regard, which itself is referred to in greater detail below.
“Methods to covalently attach labels and reporter molecules to oligonucleotides have provided valuable tools in the field of molecular biology and gene probe diagnostics. Recent advances in the preparation of non-isotopic gene probes, DNA sequencing (Connell, C. et al. [1987] Biotechniques 5:342-346; Kaiser, R., S. Mackellar, R. Vinayak, J. Sanders, R. Saavedra, L. Hood [1989] Nucleic Acids Res. 17:6087-6102), electron microscopy (Sproat, B. S., B. Beijer, P. Rider [1987] Nucleic Acids Res. 15:6181-6196), and X-ray crystallography (Sproat et al. [1987] Nucleic Acids Res. 15:4837-4848) have provided the impetus for the development and improvement of such methods. Similarly, new and emerging applications employing the polymerase chain reaction (PCR) technology (Hultman, T., S. Bergh, T. Moks, M. Uhlen [1991] Biotechniques 10:84-93; Landgraf, A., B. Reckmann, A. Pingoud [1991] Analytical Biochemistry 193:231-235; Zimran, A., C. Glass, V. Thorpe, E. Beutler [1989] Nucleic Acids Res. 17:7538) have further expanded the need for convenient and versatile reagents to chemically modify oligonucleotides.”
“Current methods to introduce chemical modifications into oligonucleotides typically employ the use of non-nucleosidic phosphoramidite reagents during automated oligonucleotide synthesis. Such methods, however, are generally limited to single modifications at only the 5′ terminus, since the 3′ terminus remains attached to the solid support. An inherent disadvantage of such methods is that the labeling reagents tend to terminate chain elongation at the point they are introduced (5′ terminus) and therefore only single modifications can be performed. Chemical modifications that have been introduced in this fashion are primary aliphatic amine (Sinha, N. D., R. M. Cook [1988] Nucleic Acids Res. 16:2659-2669) and thiol (Connolly, B. [1985] Nucleic Acids Res. 13:4485-4502) functionalities. Oligonucleotides functionalized with primary aliphatic amines or thiol groups must be subsequently derivatized with labels such as biotin, fluorescein, and enzymes. Such derivatization requires a second reaction and purification step which minimizes the convenience and practicality of this method. Cocuzza expanded this method to directly incorporate a single biotin label into an oligonucleotide at the 5′ terminus (Cocuzza, A. [1989] Tetrahedron Lett. 30:6287-6290).”
Nelson et al. introduced a new type of non-nucleosidic phosphoramidite reagent that employs a 1,2-ethanediol backbone (Nelson, P., R. Sherman-Gold, R. Leon [1989] Nucleic Acids Res. 17:7179-7186). This reagent allowed primary aliphatic amines to be incorporated multiple times and at any position of the oligonucleotide. The development of this method was said to eliminate the termination of chain elongation during synthesis, an inherent problem of the above method. Employment of the 1,2-ethanediol backbone allowed the phosphoramidite reagent to be incorporated in the same manner as a normal nucleoside phosphoramidite, at any position and multiple times. Misiura et al. expanded the use of the 1,2-ethanediol backbone derived from a glycerol intermediate, to directly incorporate multiple biotin groups into oligonucleotides (Misiura, K., I. Durrant, M. Evans, M. Gait [1990] Nucleic Acids Res. 18:4345-4354). The development of a 1,2-ethanediol backbone modification method was also said to provide better utility and versatility, especially in the field of gene probe diagnostics where multiple labels yield greater signal detection.
A more recent approach, described in U.S. Pat. No. 5,451,463 (Nelson et al.), is said to overcome the above-described disadvantages by providing improved non-nucleosidic reagents to directly modify or label oligonucleotides via automated solid phase synthesis. The '463 patent provides a trifunctional reagent possessing a primary hydroxyl, a secondary hydroxyl, and a primary amino group. This reagent is said to be useful in solid phase oligonucleotide synthesis for the convenient labeling of the 3′-terminus. The secondary hydroxyl may be a phosphoramidite derivative permitting the attachment to the solid phase support. The reporter molecule may be attached to the trifunctional molecule prior to the completion of the oligonucleotide synthesis or after the oligonucleotide is cleaved from the support.
Finally, U.S. Pat. No. 5,723,591 (Livak et al.) describes an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The reporter molecule (e.g., fluorescein dye) is separated from the quencher molecule (e.g., rhodamine dye) by at least about 15 nucleotides, more preferably at least about 18 nucleotides. In one embodiment, the oligonucleotide probe is immobilized on a solid support either directly or by a linker.
What is clearly needed however are reagents adapted to provide further options in the course of oligonucleotide synthesis, such as longer spacers and/or the ability to be used as either the support or as an amidite. Such longer spacers can be used, for instance, to optimize the binding of biotin to avidine, or to reduce steric interference of such labels when double stranded oligonucleotides (one or both of which strands may include such labels) are hybridized to each other.
SUMMARY OF THE INVENTION
The present invention provides a new 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. Depending on its conditions of preparation and/or use, a reagent of the present invention can be used to label either the 3′ or 5′ termini of a synthesized oligonucleotide, and/or for one or more positions along the oligonucleotide.
The labeling reagent is preferably prepared as the condensation product of a tritylated hydroxyacid and a diamine, in a manner that provides the resulting linear reagent backbone with one or more label attachment sites and one or more sites for attaching the reagent to a support or amidite. The labeling reagent is more preferably prepared by a reaction scheme that involves the initial preparation of a DMT-hydroxyacid intermediate in the manner provided herein.
The invention therefore provides a labeling reagent useful in making labeled oligonucleotides, the reagent preferably comprising the reaction (e.g., condensation) product of a DMT-hydroxyacid and a diamine, wherein the reaction product provides at least one secondary hydroxyl group. In one particularly preferred embodiment, the hydroxacid itself provides at least one internal amide bond prior to condensation with a diamine. The resulting reagent provides the secondary hydroxyl group residue, either attached to a solid support or converted to a phosphoramidite, and further provides one (primary or secondary) amine group residue having a suitable label or protecting group attached thereto.
In one preferred embodiment, the invention provides a reagent having the following structure selected from:
R
1
—O—CH
2
—CH(O—R
2
)—CH

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