title: Sequence Mutation and Structural Alteration Transform a Noncatalytic DNA Sequence into an Efficient RNA-Cleaving DNAzyme.
abstract: 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 8-17, a DNAzyme that has been discovered in multiple previous in vitro selection experiments, and (2) other mutations have allowed this special 8-17 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.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada.
authors.forename: Laura
authors.initials: L
authors.lastname: Chan
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada.
authors.forename: Kha
authors.initials: K
authors.lastname: Tram
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada.|Origins Institute, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada.
authors.forename: Rachel
authors.initials: R
authors.lastname: Gysbers
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada.
authors.forename: Jimmy
authors.initials: J
authors.lastname: Gu
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada. liying@mcmaster.ca.|Department of Chemistry and Chemical Biology, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada. liying@mcmaster.ca.|Origins Institute, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada. liying@mcmaster.ca.
authors.forename: Yingfu
authors.initials: Y
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012260:Ribonucleases / Q000378:metabolism*; D052156:SELEX Aptamer Technique
chemical_list: D021881:DNA, Catalytic; D012260:Ribonucleases
keywords: 8–17; DNAzyme; In vitro selection; Molecular evolution; RNA cleavage