Investigating RNA-RNA interactions through computational and biophysical analysis

Abstract

Many flaviviruses such as Dengue and West Nile virus undergo essential long-range interactions of their 5' and 3' terminal regions (TRs), mediated by a conserved complementary cyclization sequence. However, we lack insights into such long-range interactions for the Japanese Encephalitis virus (JEV). Here, we utilized an extensive, multi-faceted approach involving computational and biophysical tools. We performed multi-angle light scattering (SEC-MALS) to determine absolute molecular weights of JEV TRs, and their complex concluding they form a 1:1 complex and corroborated this interaction using analytical ultracentrifugation (AUC). The microscale thermophoresis (MST) experiments demonstrated that the 5' and 3' TR of JEV interact with nM affinity, which is significantly reduced without the conserved cyclization sequence. To our knowlwege, this is the first study representing the application of three key biophysical methods (AUC, MST and SEC-MALS) to study RNA-RNA interactions. Furthermore, we performed computational kinetic analyses corroborating our MST studies showing the essential role of the cyclization sequence in the RNA-RNA interaction. The binding affinity of this biologically critical event is a vital pharmacological feature that can influence potential competitive inhibition by therapeutics. This evidence can also influence pharmaceutical interventions aimed at inhibiting the conserved flavivirus cyclization, thus, interrupting replication across the flavivirus family.

Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

“…Plasmids containing cDNA for ZIKV 5’ and 3’ NTRs were transformed and cultured in E. coli NEBα (NEB, Canada) competent cells and further recovered using GeneJET Plasmid Maxiprep Kit (ThermoFisher Scientific, Canada) as per manufacturer’s protocol, followed by a 4-hour linearization with XbaI endonuclease (NEB, Canada) at 37 °C. ZIKV NTRs RNAs were synthesized by in vitro transcription using an in-house purified T7 polymerase, as previously described (75,76). Subsequently, RNAs were purified on a Superdex TM 200 10/300 GL column (Global Life Science Solutions, USA) equilibrated with RNA buffer (10 mM Bis-tris pH 6.0, 100 mM NaCl, 15 mM KCl, 5 mM MgCl 2 , 5% glycerol).…”

Cleavage region organizes the structural architecture of the B2 SINE ribozyme

“…Therefore, SAXS is an excellent complementary approach to solving RNA structure synergistically with other biophysical methods. Numerous studies have used SAXS to help determine several RNA [31][32][33] and RNA-protein complexes structures 34 .…”