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Biomolecular Coupling Methods Using 1,3-dipolar Cycloaddition Chemistry

Summary

Lead Inventors: Jingyue Ju, Ph.D.Problem or Unmet Need:Synthetic DNA represents one of the most significant molecular tools for genomic research and biotechnology. Typically, modified single stranded DNA is prepared via standard phosphoramidite chemistry on solid support. A primary amine is introduced at either of the DNA termini or at an internal position, and the succinimidyl ester of a desired functionality/molecule is then coupled to this amine. However, many of the functionalities that are introduced in this fashion cannot survive the harsh conditions necessary to cleave the DNA from the solid support. Furthermore, if the coupling reaction takes place after cleavage from the support, the required aqueous conditions can hydrolyze the succinimidyl ester leading to low reaction yields. Therefore, there is a need for the development of alternative biocompatible coupling chemistries that proceed in high yield and tolerate a wide range of reaction conditions.This invention describes a reliable methodology for the covalent attachment of a biomolecule to a second moiety. A biomolecule such as DNA/RNA is modified with either an azide or an alkyne, and the corresponding functionality (for installation on the biomolecule) is modified with an azide or an alkyne. Azides and alkynes/alkynyl groups undergo the well-known Huisgen 1,3-dipolar cycloaddition reaction. When the biomolecule and corresponding functionality are brought together, the azide and alkyne react selectively and yield a robust and stable five-membered triazole ring. This reaction can also be utilized for the attachment of an azide- or alkyne-modified biomolecule to an appropriately azide- or alkyne-modified solid support.

Technology Benefits

Cycloaddition is chemoselective and proceeds in nearly quantitative yields The reaction tolerates both aqueous conditions and a wide range of pH values The triazole bond is stable under typical biological conditions and at very high temperatures The reaction is flexible since azides/alkynes can be installed at many positions on a biomolecule

Technology Application

Labeling of primers for DNA sequencing and polymerase chain reactions Development of antisense agents for chemotherapeutic applications Covalent modification of biomolecules at cell surfaces and in vivo Immobilization of biomolecules and even whole cells on a substrate or a solid support Attachment of molecular beacons for detection of genetic mutations Bioconjugation of probes to DNA/RNA for measuring gene expression at microarrays/gene chips In vitro ligation of amino acids for protein synthesis

Detailed Technology Description

This invention describes a reliable methodology for the covalent attachment of a biomolecule to a second moiety. A biomolecule such as DNA/RNA is modified with either an azide or an alkyne, and the corresponding functionality (for installation on the...

*Abstract

None

*Inquiry

Peter Golikov Columbia Technology Ventures Tel: (212) 854-8444 Email: TechTransfer@columbia.edu

*IR

1403

*Principal Investigator

*Publications

Seo TS, Li Z, Ruparel H, and Ju J. Click Chemistry to Construct Fluorescent Oligonucleotides for DNA Sequencing (2003) J. Org Chem. 68: 609-612

*Web Links

USPTO: US20050032081A1

Country/Region

USA