The advantages of being locked. Assessing the cleavage of short and long RNAs by locked nucleic acid-containing 8-17 deoxyribozymes.

摘要: RNA-cleaving deoxyribozymes can be used for the sequence-specific knockdown of mRNAs. It was previously shown that activity these is enhanced when their substrate-binding arms include some locked nucleic acid (LNA) residues, but mechanistic basis this enhancement not explored. Here we dissected kinetics and thermodynamics underlying reaction LNA-containing 8-17 deoxyribozymes. Four constructs were designed to target sequences within E6 mRNA from human papillomavirus type 16. When one (DNAzymes) corresponding LNA-armed enzyme (LNAzyme) tested against a minimal RNA substrate, they showed similar rates substrate binding intramolecular cleavage, LNAzyme released its more slowly. The superior thermodynamic stability LNAzyme-substrate complex led improved performances in reactions carried out at low catalyst concentrations. four DNAzymes LNAzymes then extended transcripts (>500 nucleotides long). With structured substrates, retained full activity, whereas cleaved extremely poorly, unless allowed pre-anneal targets. These results imply easily overcome kinetic barrier represented by local structure bind folded targets with faster association rate as compared DNAzymes. Such annealing explained model whereby LNA monomers favor initial hybridization short stretches unpaired residues ("nucleation"), which precedes disruption completion process.