Design and switch of catalytic activity with the DNAzyme–RNAzyme combination

Abstract

Design and switch of catalytic activity in enzymology remains a subject of intense investigation. Here, we employ a DNAzyme-RNAzyme combination strategy for construction of a 10-23 deoxyribozyme-hammerhead ribozyme combination that targets different sites of the b-lactamase mRNA. The 10-23 deoxyribozyme-hammerhead ribozyme combination gene was cloned into phagemid vector pBlue-scriptIIKS (+). In vitro the single-strand recombinant phagemid vector containing the combination sequence exhibited 10-23 deoxyribozyme activity, and the linear transcript displayed hammerhead ribozyme activity. In bacteria, the 10-23 deoxyribozyme-hammerhead ribozyme combination inhibited the b-lactamase expression and repressed the growth of drug-resistant bacteria. Thus, we created a DNAzyme-RNAzyme combination strategy that provides a useful approach to design and switch of catalytic activities for nucleic acid enzymes.

Blue-white screening as a new readout for deoxyribozyme activity in bacterial cells

“…Stable knockdown effect is expected by employing methods that provide in situ production of single stranded deoxyribozymes in prokaryotic cells e.g. pBlue-scriptIIKS (+) 25 or MMLV-RT system. 7…”

“…Phosphorothioated DNA oligos were applied to cleave b-lactamase in oxacillin and methicillin resistant S. aureus, WHO-2 strains. 23 In another strategy, vectors have been designed to transform the bacterial cells with deoxyribozymes 10-23. pssXGa vector 24 and pBlue-script-II KS (+) phagemid 25 were designed for E. coli. In such strategies, the deoxyribozymes are induced to be synthesized inside bacteria and thus there is less concern for nuclease degradation.…”