Investigations toward the selection of fully-modified 4′-thioRNA aptamers: Optimization of in vitro transcription steps in the presence of 4′-thioNTPs

We describe herein a method for isolating fully-modified 4′-thioRNA aptamers against human α-thrombin using the SELEX protocol. In order to isolate the desired aptamers, in vitro transcription was examined in the presence of four kinds of 4′-thioribonucleoside triphosphates (4′-thioNTPs) in an effort to afford the fully-modified 4′-thioRNA. The transcription efficiency of the 4′-thioNTPs was first compared with that of the nucleoside 5′-(α-thio)triphosphates (αSNTPs) and found to be less effective than that of the αSNTPs, especially when GTP and/or ATP were substituted for 4′-thioGTP and/or 4′-thioATP. Further attempts to improve its efficiency, including the use of a mutant RNA polymerase instead of the wild type, various additives, and 4′-thioNTP concentrations were unsuccessful. Accordingly, the transcription was performed in the presence of 4′-thioNTPs together with the natural GTP and ATP at the appropriate concentrations. Although this attempt furnished a highly-modified 4′-thioRNA, but not a fully-modified 4′-thioRNA, we eventually succeeded in isolating the fully-modified 4′-thioRNA aptamers by SELEX using optimized transcription conditions, followed by post-modification of the resulting aptamers.

Investigation of in vitro transcription using four kinds of 4′-thioNTPs was examined. As a result, we succeeded to isolate fully-modified 4′-thioRNA aptamer.Figure optionsDownload as PowerPoint slide