Optimized UV Melting as a Tool for Conformational Studies on Virla [i.e. Viral] RNA Motifs

UV melting study has been instrumental in investigating the thermodynamic characteristics of various biologically important RNA conformations. In my master’s degree thesis research, I have optimized the in-house UV melting experimental system by establishing a calibration method which addresses the issue of temperature variation between different UV cuvettes. Using the optimized melting method, I have investigated the thermodynamic characteristics of two important viral RNA motifs – Subgenomic RNA promoter of Brome Mosaic Virus (BMV) and the kissing complex of H2 and H3 RNA motifs of Moloney Murine Leukemia Virus (MMLV). My results have shown that 1) BMV subgenomic promoter seems to form an usually flexible C•G base pair in its loop region. 2) The conformational stability of kissing complexes of both H2 and H3 RNA motifs of MMLV are significantly affected by the identity of its loop-closing base pair only when the wild-type base pair is changed to 5’C•G3’. In addition, both the base composition and the length of its stem region also seem to contribute to its conformational stability. I have also established a protocol for purification of T7 RNA polymerase as a part of my thesis work since preparation of pure, active T7 RNA polymerase was crucial in preparing RNA samples in a large scale for my thesis work.