Characterization of a catalytically efficient acidic RNA-cleaving deoxyribozyme.
We previously demonstrated--through the isolation of RNA-cleaving deoxyribozymes by in vitro selection that are catalytically active in highly acidic solutions--that DNA, despite its chemical simplicity, could perform catalysis under challenging chemical conditions [Liu,Z., Mei,S.H., Brennan,J.D. and Li,Y. (2003) J. Am. Chem. Soc. 125, 7539-7545]. One remarkable DNA molecule therefrom is pH4DZ1, a self-cleaving deoxyribozyme that exhibits a k(obs) of approximately 1 min(-1) at pH 3.8. In this study, we carried out a series of experiments to examine the sequence and catalytic properties of this acidic deoxyribozyme. Extensive nucleotide truncation experiments indicated that pH4DZ1 was a considerably large deoxyribozyme, requiring approximately 80 out of the original 123 nt for the optimal catalytic activity. A reselection experiment identified ten absolutely conserved nucleotides that are distributed in three catalytically crucial sequence elements. In addition, a trans deoxyribozyme was successfully designed. Comparison of the observed rate constant of pH4DZ1 with experimentally determined rate constant for the uncatalyzed reaction revealed that pH4DZ1 achieved a rate enhancement of approximately 10(6)-fold. This study provides valuable information about this low-pH-functional deoxyribozyme and paves way for further structural and mechanistic characterization of this unique catalytic DNA.
Kandadai
Srinivas A
SA
Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
Li
Yingfu
Y
D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D021881:DNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013463:Sulfuric Acid Esters / Q000737:chemistry
D021881:DNA, Catalytic; D009711:Nucleotides; D013463:Sulfuric Acid Esters; C506887:deoxyribozyme pH4DZ1; D012313:RNA; C007482:dimethyl sulfate