Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography. Nucleic Acids Res

Knowledge of the three-dimensional structures of RNA and its complexes is important for understand-ing the molecular mechanism of RNA recognition by proteins or ligands. Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallo-graphy. However, this run-off transcription method results in heterogeneity at the RNA 3-terminus. For structural studies, RNA purification requires a single nucleotide resolution. Usually PAGE purification is used, but it is tedious, time-consuming and cost ineffective. To overcome these problems in high-throughput RNA synthesis, we devised a method of RNA preparation that uses trans-acting DNAzyme and sequence-specific affinity column chromatography. A tag sequence is added at the 30 end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence. The 30 endtag is thenremovedbysequence-specificcleav-age using trans-acting DNAzyme, the arm lengths of which are optimized for turnover number. This purific-ation method is simpler and faster than the conven-tional method