Programmable DNA-Guided Artificial Restriction Enzymes.
Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research. However, they have limited sequence specificity and availability. Here we report a Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by reannealing of the cleaved single stranded DNAs. We used this platform to generate over 18 AREs for DNA fingerprinting and molecular cloning of PCR-amplified or genomic DNAs. These AREs work as efficiently as their naturally occurring counterparts, and some of them even do not have any naturally occurring counterparts, demonstrating easy programmability, generality, versatility, and high efficiency for this new technology.
Department of Chemical and Biomolecular Engineering, ‡Carl R. Woese Institute for Genomic Biology, Department of Chemistry, Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Behnam
B
Enghiad
Department of Chemical and Biomolecular Engineering, ‡Carl R. Woese Institute for Genomic Biology, Department of Chemistry, Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Huimin
0000-0002-9069-6739
H
Zhao
D001709:Biotechnology / Q000379:methods; D004247:DNA / Q000235:genetics*; D016172:DNA Fingerprinting / Q000379:methods; D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D004247:DNA; D004262:DNA Restriction Enzymes
DNA cloning; DNA profiling; recombinant DNA technology; restriction enzymes