Cenk Andac
Istinye University, Turkey
Title: Synthesis, HPLC purification, NMR characterization and AMBER molecular dynamics structure of the anticodon stem-loop region of a t-RNA with a modified 3-methyl uridine
Biography
Biography: Cenk Andac
Abstract
A specific H-bond between U33 and the phosphate group between C36 and G34 in the anticodon loop region of t-RNA renders a very compact anticodon loop structure, hence called a closed-loop structure. It is known that the closed loop structure is in exchange with an open-loop structure in which the aforementioned specific H-bond in the anticodon loop region is broken. In order to shed light on to the dynamics of the open-loop structure of the transfer RNA, a 17-mer RNA hairpin molecule, representing the open-loop structure anticodon region of a transfer RNA, with the sequence 5'GGGAGUXAGCGGCUCCC 3' (X=3-N-methyl uridine) was synthesized using the dimethoxytrityl, tert-butyldimethylsilyl and 2-cyanoethyl diisopropylphosphoamidite. The crude product was then purified by reversed phase HPLC using an 8 mm C-18 Radial-Pak column (by Waters Assoc., Inc) on a Beckman (Fullerton, CA, USA) System Gold HPLC instrument with ultra violet detection at 254 nm. Optimal conditions for the separation of the 17-mer RNA were provided by using an isocratic elution system based on 0.05 M ammonium acetate (NH4OAc) solution in RNase-free dd.H2O (buffered at pH=7.0). The retention time was 9 min and the elution speed was 0.7 ml/min. The purified RNA hairpin was further studied by NMR and it was found out that two major conformationally different structures of the RNA exist in slow exchange with concentrations ~51% and ~43% on NMR time scale. AMBER molecular dynamics and cluster analysis studies indicate reveal two open-loop structures of the RNA hairpin.