Covalent split protein fragment-DNA hybrids generated through N-terminus-specific modification of proteins by oligonucleotides.
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.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Shuji
S
Takeda
Shinya
S
Tsukiji
Hiroshi
H
Ueda
Teruyuki
T
Nagamune
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D009841:Oligonucleotides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009841:Oligonucleotides; D011506:Proteins