Fast and efficient chemical ligation of oligonucleotides using diels-alder reaction.

Abstract

Diels-Alder chemical ligation was applied to the ligation of two and three oligonucleotides to form a single, continuous strand with an unnatural DNA backbone at the ligation site. The reaction occurred between a maleimide-labelled oligonucleotide and a furan-labelled oligonucleotide to form two types of Diels-Alder linkage (one of each directionality). A different type of Diels-Alder ligated oligonucleotide was formed via the reaction between dual-furan labelled oligonucleotide and two maleimide-labelled oligonucleotide. The template-mediated reaction occurred rapidly and efficiently, with most product formation occurring by zero hour reaction time. An approximately drop in 10 °C was found for the melting temperature of oligonucleotides with a single unnatural backbone as compared to native DNA strand, indicative of distortion of the duplex by the linker.