| Popis: |
In miRNAs, it is known that seed pairing is an important parameter for targeting, but there are still missing links on why it targets a certain mRNA and how the decision between degradation or translation repression is made. In our work, we use liquid state NMR spectroscopy, EMSA and UV melting experiments to biophysically characterise the complex of miRNA-34a with the target mRNA of CD44 and PNUTS. Here we present the NMR sample preparation as well as the first steps for the biophysical description of the mRNA.miRNA complexes. Our EMSA data suggests both miRNA.mRNA complexes are transient and miRNA.gCD44 possibility has a higher Kd then miRNA.PNUTS. Through NMR miRNA.PNUTS complex was deemed more structurally dynamic, yet interestingly with UV melting it had a higher Tm then miRNA.gCD44 complex. Finally, we also performed several simulations on several different mRNA targets with MCFold unveiling a kink on the mRNA as a structural trend which could be a necessity for docking of the duplex into Ago. Still further studies must be conducted in order to solve the structures of both duplexes in study and a bigger population of simulated structures must be collected. |
