期刊
ACS CHEMICAL BIOLOGY
卷 12, 期 12, 页码 2940-2945出版社
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.7b00621
关键词
-
资金
- Okinawa Institute of Science and Technology Graduate University
Deoxyribozyrnes are catalytic DNA sequences whose atomic structures are generally difficult to elucidate. Mutational analysis remains a principal approach for understanding and engineering deoxyribozymes with diverse catalytic activities. However, laborious preparation and biochemical characterization of individual sequences severely limit the number of mutants that can be studied biochemically. Here, we applied deep sequencing to directly measure the activities of self-hydrolyzing deoxyribozyme sequences in high throughput. First, all single and double mutants within the 15-base catalytic core of the deoxyribozyme I-R3 were assayed to unambiguously determine the tolerated and untolerated mutations at each position. Subsequently, 4096 deoxyribozyme variants with tolerated base substitutions at seven positions were kinetically assayed in parallel. We identified 533 active mutants whose first-order rate constants and activation energies were determined. The results indicate an isolated and narrow peak in the deoxyribozyme sequence space and provide a quantitative view of the effects of multiple mutations on the deoxyribozyme activity for the first time.
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