key: cord-0781042-vdpgze54
authors: Melidis, Lazaros; Hill, Harriet J.; Coltman, Nicholas J.; Davies, Scott P.; Winczura, Kinga; Chauhan, Tasha; Craig, James S.; Garai, Aditya; Hooper, Catherine A..J.; Egan, Ross T.; McKeating, Jane A.; Hodges, Nikolas J.; Stamataki, Zania; Grzechnik, Pawel; Hannon, Michael J.
title: Supramolecular cylinders target bulge structures in the 5’ UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication
date: 2021-03-31
journal: bioRxiv
DOI: 10.1101/2021.03.30.437757
sha: 866dc47c483d7a6e0f633c23a6add26f541aad50
doc_id: 781042
cord_uid: vdpgze54

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with Molecular Dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5’ UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in the stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel antiviral agents.

SARS-CoV-2 is a novel coronavirus that causes COVID-19 and as of 1st March 2021 there 44 have been 113,267,303 recorded cases and 2,520,550 deaths worldwide [1] . Emerging so These non-coding RNA regions are highly structured with multiple stem loops, bulges, 62 crosses and pseudo-knots, with common structural elements seen in many viral UTRs. 63 These structures play a role in RNA-RNA interactions (both within the viral genome and 64 with host machinery) and in protein binding for the initiation of mRNA production, 65 translation and viral replication. Moreover, these RNA structures may act as trans acting 66 elements or mediate translational frameshifting, a common feature in viruses with plus-67 strand RNA genomes. 68 Nucleic acid sensors mediate the early detection and host response to virus infections, 69 and recognise either viral nucleic acids or 'unusual ' Given this antiviral activity against HIV-1 we were interested to assess whether these 92 cylinders would bind structures in the 5' UTR of SARS-CoV-2. We now report combined 93 modelling and biophysical approaches to define the 3D structures of the SARS-CoV-2 5' 94 UTR, and demonstrate cylinder binding to specific bulge structures in the 5' UTR. 95 Furthermore, we show that cylinders inhibit SARS-CoV-2 viral replication in cells. structural importance for the UTR are more likely to be substituted. Although not 138 corrected for frequency, it is interesting to note that around 60% (19/31) of the SNP sites 139 identified to date involve replacement with a U residue, with the largest subset (11/31) 140 being a C-U mutation (Fig. S6) . As anticipated the mutations will not affect the key 141 structures of the 5' UTR.

After identifying the distinct stems loops (SLn) that were conserved throughout the 143 results from the secondary structure prediction, we attempted the more challenging step In addition to the bulge as a site of binding, in the simulations the cylinder can also insert 221 into the cavity at the central cross (4 way junction) (Fig. 4A, cylinder A) , protecting A193.

This cavity is larger than the 3-base bulge and thus although the binding site may not 223 offer as good a structural fit, it will be kinetically quite accessible. The binding also to this transcript. 253 We also tested the effect of two substituted cylinders based on ligands L' and L", to 254 confirm the key binding area of the cylinder design (Fig. 4B) . These cylinders bear 

World Health Organisation data for 1

Proc

(a) For structurally characterised binding of a metallo-intercalator inside a DNA 4-way 397 junction see

For structurally characterised binding of metallo-helicates inside DNA 3-way junctions 401 see

303-324; k) H. Crlikova