Secondary-structure characterization of two proficient kinase deoxyribozymes.
Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.
Achenbach
John C
JC
Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Canada.
Jeffries
Greg A
GA
McManus
Simon A
SA
Billen
Lieven P
LP
Li
Yingfu
Y
D020137:Base Pair Mismatch; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D021881:DNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D015723:Gene Library; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference
D021881:DNA, Catalytic; D010770:Phosphotransferases; D004121:Dimethyl Sulfoxide