TY - JOUR
T1 - PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity
AU - Lyu, Mingkuan
AU - Kong, Linggen
AU - Yang, Zhenglin
AU - Wu, Yuting
AU - Mcghee, Claire E.
AU - Lu, Yi
N1 - Funding Information:
This work was funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award DE-SC0018420), U.S. National Institute of Health (GM141931) and a Chemistry Discovery Fund at the University of Illinois at Urbana–Champaign. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the U.S. Department of Energy. We thank Prof. Dawei Li for providing help with the initial design, and Prof. Danith Ly for providing help with the PNA gel shift assay. We thank Ryan Lake and Greg Pawel for the help with data analysis.
Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/7/7
Y1 - 2021/7/7
N2 - DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, we herein report peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.
AB - DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, we herein report peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.
UR - http://www.scopus.com/inward/record.url?scp=85110071857&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85110071857&partnerID=8YFLogxK
U2 - 10.1021/jacs.1c03129
DO - 10.1021/jacs.1c03129
M3 - Article
C2 - 34156847
SN - 0002-7863
VL - 143
SP - 9724
EP - 9728
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 26
ER -