Sequence Mutation and Structural Alteration Transform a Noncatalytic DNA Sequence into an Efficient RNA-Cleaving DNAzyme
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- 作者：Laura Chan ; Kha Tram ; Rachel Gysbers ; Jimmy Gu ; Yingfu Li
- 关键词：DNAzyme ; In vitro selection ; RNA cleavage ; Molecular evolution ; 8-7
- 刊名：Journal of Molecular Evolution
- 出版年：2015
- 出版时间：December 2015
- 年：2015
- 卷：81
- 期：5-6
- 页码：245-253
- 全文大小：2,167 KB
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Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505-10CrossRef PubMed - 作者单位：Laura Chan (1)
Kha Tram (1)
Rachel Gysbers (1) (3)
Jimmy Gu (1)
Yingfu Li (1) (2) (3)
1. Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada
3. Origins Institute, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada
2. Department of Chemistry and Chemical Biology, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada - 刊物类别：Biomedical and Life Sciences
- 刊物主题：Life Sciences
Cell Biology
Microbiology
Plant Sciences - 出版者：Springer New York
- ISSN：1432-1432
文摘
We have previously shown that through test-tube molecular evolution, an arbitrarily chosen noncatalytic DNA sequence can be evolved into a catalytic DNA (DNAzyme) with significant RNA-cleaving activity. In this study, we aim to address the question of whether the catalytic activity of such a DNAzyme can be further optimized using in vitro selection. Several cycles of selective enrichment starting with a partially randomized DNA library have resulted in the isolation of many sequence variations that show notably improved catalytic activity. Bioinformatic analysis and activity examination of several DNAzyme-substrate constructs have led to two interesting findings about sequence mutations and the secondary structure of this DNAzyme: (1) three crucial mutations have transformed the DNAzyme into 8-7, a DNAzyme that has been discovered in multiple previous in vitro selection experiments, and (2) other mutations have allowed this special 8-7 variant to make structural fine-tuning in order to cleave an arbitrarily chosen RNA-containing substrate with a defined sequence. Our study not only showcases the combined power of directed molecular evolution and in vitro selection techniques in turning a noncatalytic nucleic acid sequence into an efficient enzyme, but it also raises the question of whether mother nature has used a similar approach to evolve natural enzymes. Keywords DNAzyme In vitro selection RNA cleavage Molecular evolution 8-7