A DNAzyme requiring two different metal ions at two distinct sites
Abstract
Most previously reported RNA-cleaving DNAzymes require only a single divalent metal ion for catalysis. We recently reported a general trivalent lanthanide-dependent DNAzyme named Ce13d. This work shows that Ce13d requires both Na+ and a trivalent lanthanide (e.g. Ce3+), simultaneously. This discovery is facilitated by the sequence similarity between Ce13d and a recently reported Na+-specific DNAzyme, NaA43. The Ce13d cleavage rate linearly depends on the concentration of both metal ions. Sensitized Tb3+ luminescence and DMS footprinting experiments indicate that the guanines in the enzyme loop are important for Na+-binding. The Na+ dissociation constants of Ce13d measured from the cleavage activity assay, Tb3+ luminescence and DMS footprinting are 24.6, 16.3 and 47 mM, respectively. Mutation studies indicate that the role of Ce3+ might be replaced by G(23) in NaA43. Ce3+ functions by stabilizing the transition state phosphorane, thus promoting cleavage. G(23) competes favorably with low concentration Ce3+ (below 1 mu M). The G(23)-to-hypoxanthine mutation suggests the N1 position of the guanine as a hydrogen bond donor. Together, Ce13d has two distinct metal binding sites, each fulfilling a different role. DNAzymes can be quite sophisticated in utilizing metal ions for catalysis and molecular recognition, similar to protein metalloenzymes.
Cite this version of the work
Wenhu Zhou, Yupei Zhang, Po-Jung Jimmy Huang, Jinsong Ding, Juewen Liu
(2015).
A DNAzyme requiring two different metal ions at two distinct sites. UWSpace.
http://hdl.handle.net/10012/11800
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