Two DNAzymes targeting the telomerase mRNA with large difference in Mg2+ concentration for maximal catalytic activity.
The 10-23 RNA-cleaving DNAzymes require divalent metal ions, preferentially Mg(2+), for catalytic activity. For intracellular applications, it is important that a DNAzyme can perform maximal cleavage at physiological concentration of Mg(2+) (0.2-2mM). We designed DNAzymes targeting the mRNA of human telomere reverse transcriptase, two of them turned out to have large difference in Mg(2+) concentration requirement (1mM vs. 20mM) for maximal activity in vitro. When the two DNAzymes were transfected into HeLa cells, only the one requiring low Mg(2+) concentration showed inhibitory activity indicating that the in vitro property regarding Mg(2+) requirement was reserved in vivo. The cleavage of target RNA mainly involves two processes, that is hybridization of DNAzyme with substrate and cleavage of substrate in the DNAzyme-substrate duplex. To explore how the optimal Mg(2+) concentration was determined, we studied the effect of Mg(2+) on the two processes. For both DNAzymes, Mg(2+) enhanced hybridization over a range of concentration far beyond 1mM. Once the DNAzymes hybridized with their 19-mer substrates without flanking sequences, the cleavages showed little difference in Mg(2+) concentration-dependence. These facts suggest that the flanking sequences played a key role in determining the Mg(2+) concentration for maximal DNAzyme activity possibly via the formation of higher order structure in the DNAzyme-substrate duplex.
Yuan
Bi-Feng
BF
Laboratory of Biochemistry and Biophysics, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Xue
Yong
Y
Luo
Man
M
Hao
Yu-Hua
YH
Tan
Zheng
Z
D002384:Catalysis / Q000187:drug effects; D021881:DNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009154:Mutation; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D019098:Telomerase / Q000235:genetics*; D014162:Transfection
D021881:DNA, Catalytic; D012333:RNA, Messenger; D019098:Telomerase; D008274:Magnesium