Nanoscale Structure Determination of Murray Valley Encephalitis and Powassan Virus Non-coding RNAs

Abstract

Viral infections are responsible for numerous deaths worldwide. Flaviviruses, which contain RNA as their genetic material, are one of the most pathogenic families of viruses. There is an increasing amount of evidence suggesting that their 5' and 3' non-coding terminal regions are critical for their survival. In this study, the 5' and 3' terminal regions of Murray Valley Encephalitis and Powassan virus were examined using biophysical and computational modeling methods. First, the purity of in-vitro transcribed RNAs were investigated using size exclusion chromatography and analytical ultracentrifuge methods. Next, we employed small-angle X-ray scattering techniques to study solution conformation and low-resolution structures of these RNAs, which suggested that the 3' terminal regions are highly extended, compared to the 5' terminal regions for both viruses. Using computational modeling tools, we reconstructed 3-dimensional structures of each RNA fragment and compared them with derived small-angle X-ray scattering low-resolution structures. This approach allowed us to further reinforce that the 5' terminal regions adopt more dynamic structures compared to the mainly double-stranded structures of the 3' terminal regions.

Biophysical Characterisation of Human LincRNA-p21 Sense and Antisense Alu Inverted Repeats

“…Generally, globular molecules will display a bell-shaped curve with a maximum at approximately D max /2 while elongated molecules retain non-Gaussian, asymmetrical distributions with a maximum at smaller distances which appear as shoulders. For elongated molecules, this distribution will correspond to the radius of the cross section which will generally be illustrated by a tailing of the profile at larger distances [36, 86]. This is present for both sense and antisense AluSx1 RNA whereby their respective D max /2 do not demonstrably produce an even bell-curved but rather lead into right-leaning tails reinforcing the overall elongated shape established by Figure 4C .…”

“…Furthermore, the relative foldedness of sense and antisense RNAs can be deduced from the dimensionless Kratky plot in Figure 4C . The Kratky plot depicts both sense and antisense RNAs as being elongated but relatively folded, like other ncRNA [36, 90]. Additionally, the Guinier R g and the real-space R g are close with less than 1.5% difference.…”

“…RNA transcripts were prepared using run-off in vitro transcriptions (IVT) as prepared previously [36, 37]. Briefly, concentrated plasmid samples were digested by XbaI restriction endonuclease (NEB, Canada).…”

Investigating RNA-RNA interactions through computational and biophysical analysis

“…JEV viral non-coding RNAs were purified immediately after in vitro transcription using SEC, similar to previous works (16,38,39). The elution profile for JEV 5’TR (Figure 3A) indicates that the RNA elutes at approximately 11.5 mL as a single monodispersed species, evident by the typical Gaussian distribution.…”

Biophysical characterisation of human LincRNA-p21 sense and antisense Alu inverted repeats

“…RNA transcripts were prepared using run-off in vitro transcriptions (IVT) as prepared previously ( 29 , 30 ). Briefly, concentrated plasmid samples were digested by XbaI restriction endonuclease (NEB, Canada).…”