Telomerase is a unique ribonucleoprotein complex that protects chromosomes from degradation and recombination. The defects in telomerase activity eansed by the mutations in telomerase RNA （hTER） is associated with s...Telomerase is a unique ribonucleoprotein complex that protects chromosomes from degradation and recombination. The defects in telomerase activity eansed by the mutations in telomerase RNA （hTER） is associated with several human genetic diseases, so the molecular biology and structural biology of hTER are of great significance for the treatment of related diseases and the development of corresponding drugs. In this study, hTER was obtained by in vitro transcription, and then modified with N-methylisatoic anhydride （NMIA） by SHAPE （selective 2＇-hydroxyl aeylation analyzed by primer extension） ; finally, the eDNA fragment was obtained by reverse transcription to analyze the free nucleotides and dynamic structure of hTER, which may provide experimental basis for analyzing three--di- mensional structure of hTER obtained in vitro. In addition, the SHAPE parameters for long RNA fragment （ 〉 300 nt） were optimized. The results showed that 1 μmol/L hTER and 3.9 mmol/L NMIA were optimal, and 49 ℃ was the best temperature for primer extension.展开更多
文摘Telomerase is a unique ribonucleoprotein complex that protects chromosomes from degradation and recombination. The defects in telomerase activity eansed by the mutations in telomerase RNA （hTER） is associated with several human genetic diseases, so the molecular biology and structural biology of hTER are of great significance for the treatment of related diseases and the development of corresponding drugs. In this study, hTER was obtained by in vitro transcription, and then modified with N-methylisatoic anhydride （NMIA） by SHAPE （selective 2＇-hydroxyl aeylation analyzed by primer extension） ; finally, the eDNA fragment was obtained by reverse transcription to analyze the free nucleotides and dynamic structure of hTER, which may provide experimental basis for analyzing three--di- mensional structure of hTER obtained in vitro. In addition, the SHAPE parameters for long RNA fragment （ 〉 300 nt） were optimized. The results showed that 1 μmol/L hTER and 3.9 mmol/L NMIA were optimal, and 49 ℃ was the best temperature for primer extension.
