Covalent split protein fragment–DNA hybrids generated through N-terminus-specific modification of proteins by oligonucleotides

Abstract

Semisynthetic protein–DNA hybrid molecules have recently attracted much attention as valuable tools for bioanalytical chemistry and nanobiotechnology. Here we describe a synthetic method for conjugating oligonucleotides to the N-terminus of recombinant proteins. Our strategy involves the conversion of amine-terminated oligonucleotides to thioester-functionalized oligonucleotides by using a bifunctional reagent bearing an N-hydroxysuccinimide ester and benzyl thioester group, followed by native chemical ligation with proteins containing an N-terminal cysteine. We applied this technique to construct split luciferase fragment–DNA hybrid systems in which the catalytic activity of split luciferase is restored by the re-assembly of each fragment through a specific DNA–protein or DNA–DNA interaction. Split protein fragment–DNA hybrids will offer new opportunities to explore the potential of protein–DNA conjugates for various applications.