RNA aptamers selected against DNA polymerase   inhibit the polymerase activities of DNA polymerases   and  

Abstract

DNA polymerase β (polβ), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polβ from a variable pool of 8 × 1012 individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polβ activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polβ active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polβ also inhibited DNA polymerase κ, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polβ and κ, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase actvity in cells.

Bypass of a Psoralen DNA Interstrand Cross-Link by DNA Polymerases β, ι, and κ in Vitro

“…Purified recombinant DNA polymerase beta and kappa 19 were generously provided by Dr. Leonid V. Gening at the Institute of Molecular Genetics of Russian Academy of Science, and purified human 9-1-1 complex 20 was a generous gift from Dr. Laura Lindsey-Boltz and Dr. Aziz Sancar at the University of North Carolina at Chapel Hill.…”

Aptasensors for Point-of-Care Detection of Small Molecules

“…The alternative, using the already existing functional groups further decreases the number of functional groups available for aptamer target recognition. Counter-selection steps should be carried out in SELEX to increase specificity and exclude cross-reactivity [ 79 , 80 ]. However, in an already time-consuming protocol these extra steps may drastically worsen the economics.…”

Aptamers: Problems, Solutions and Prospects

“…Four aptamers against DNApolymerase β generated in our laboratory can also bind and inhibit DNA polymerase κ, which belongs to another DNA polymerase family [73]. Aptamer cross-reactivity can be an obstacle to their therapeutic application because of the possible side effects caused by aptamer interaction with other proteins; however, this problem can be avoided by introducing a SELEX negative selection step with structurally similar molecules.…”