“Locked onto the target”: increasing the efficiency of antagomirzymes using locked nucleic acid modifications.
This study highlights the effect of incorporation of locked nucleic acid (LNA) on improving the functional efficacy of DNAzymes against microRNAs (antagomirzymes). DNAzymes were designed against two different sites of miR-27a, which were encompassed both within the precursor and mature form of miRNA. The cleavage and functional activities of these DNAzymes have been compared to those of LNA-modified counterparts, containing LNA modification in each of the substrate binding arms. Preliminary examination based on in vitro cleavage demonstrated LNAzyme to be much more effective in the ensuing cleavage of target miRNA under both single- and multiple-turnover conditions. Evaluation of kinetic parameters indicated almost 5-fold higher cleavage efficiency, kobs, for LNAzymes than for DNAzymes, leading to more efficient cleavage of the substrate. We attribute this enhancement in cleavage efficiency to the LNA-mediated improvement in the hybridization of the antagomirzyme·target complex. Functional validation of the relative activities was accomplished through the luciferase reporter assay and quantitative real-time polymerase chain reaction (qRT-PCR). Both the unmodified and LNA-modified antagomirzymes were very active in ensuing efficient miRNA knockdown; however, compared to the DNAzymes, the LNAzymes were almost 25% more active. A direct quantitative estimate of miRNA cleavage, conducted using qRT-PCR, further substantiated the data by indicating that LNAzyme effectively downregulated the levels of mature miRNA (up to 50%) versus the corresponding DNAzymes. Our data thus provide formative evidence of the successful employment of LNA-based antagomirzymes against miRNA.
Kaur
Harleen
H
Institute of Genomics and Integrative Biology, CSIR, Delhi, India.
Scaria
Vinod
V
Maiti
Souvik
S
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D021881:DNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity
D021881:DNA, Catalytic; D035683:MicroRNAs; D008156:Luciferases