Covalent fluorophore labeling of oligonucleotides and generation of other oligonucleotide bioconjugates

Abstract

Oligonucleotide conjugates have already reached considerable importance in life science research and oligonucleotide drug development. Since the preparation of oligonucleotide conjugates depends critically on the chemical nature of the used ligand and linker, there is no general and universal procedure. Here, we present a detailed, quick, and facile protocol for attaching fluorescent dyes or cross-linkers of variable chemical stability to oligonucleotides at 3′- or 5′-aminoalkyl handles. Purification and removal of educts and side-products and structural verification by gel electrophoresis and mass spectrometry are presented. Aspects for adapting this protocol for other reaction sites at the oligonucleotide are discussed. We highlight important issues for generating oligonucleotide conjugates with other molecules, including peptide, proteins, and small molecules for receptor-targeting applications. The methodology is suitable for oligonucleotides with various modifications, including stabilized antisense, siRNAs, and miRNAs.