1H, 13C, 15N and 31P chemical shift assignment for stem-loop 4 from the 5′-UTR of SARS-CoV-2

Abstract

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5′-untranslated region (5′-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.

The preference signature of the SARS-CoV-2 Nucleocapsid NTD for its 5’-genomic RNA elements

“…Not of a large surprise, we found the SL4 RNA to be a stable enlarged stem-loop satisfying the anticipated and suggested geometric parameters (Table 2, Fig. 6e) 54 . We also found the Ext RNA to comprise a visible degree of structure (in good agreement with the model RNA) in line with the suggested equilibrium based on its TM around room temperature.…”

“…We, however, found a less expressed stacking within the model based on SAXS at RT, which yet more is reasonable through the presence of ssRNA conformations in Ext. In support of this, in-detail-comparison of imino spectra acquired from SL4 and SL4ext using available assignments 36,54 revealed only minor CS differences of SL4 resonances (Fig. 6c and Supplementary Fig.…”

NMR Reveals Specific Tracts within the Intrinsically Disordered Regions of the SARS-CoV-2 Nucleocapsid Protein Involved in RNA Encountering

“…The 40 nucleotides (nt) SARS-CoV-2 genomic RNA element stem loop 4 (SL4) located within the 5 UTR (nt 86 to 125), extended 5 by two guanine residues and 3 by two cytidine residues, yielded the 44-nt sequence 5 -GGGUG UGG CUG UCA CUC GGC UGC AUG CUU AGU GCA CUC ACGC CC-3 [19]. The DNA template for 5_SL4 was kindly provided in a HDV ribozyme vector by the COVID19-nmr consortium.…”

“…This allows us to focus on the IDRs while linked to the NTD, that is the domain deputed to bind gRNA [1]. The interaction between this N construct (1-248, referred to as N-terminal region, NTR) with RNA was studied by selecting a highly conserved cis element of the gRNA, namely the 5 -UTR-contained stem-loop 4 (5_SL4) [19]. This is centrally located within the 5 -UTR, has very recently been found targetable by small molecules [20] and thus represents a potential drug target to disrupt its interactions with abundant viral proteins such as N. It is described as stable [5] and is chemically versatile comprising a pentaloop, two internal loops, a bulge, and a good mix of nucleotides and types of base pairs (Supplementary Figure S1).…”

NMR 1H,19F-based screening of the four stem-looped structure 5_SL1–SL4 located in the 5′-untranslated region of SARS-CoV 2 RNA

“…1) using an RNA-dedicated library 32 (DRTL-F library) and a non-RNA-dedicated fragment library (DSI-PL). 33,34 The DSI-PL and the DRTL-F library consist of 768 compound fragments (200-250 Da) and 49 compounds (222-439 Da), respectively.…”