1H, 13C and 15N chemical shift assignment of the stem-loop 5a from the 5'-UTR of SARS-CoV-2

Abstract

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive ¹H, ¹³C and ¹⁵N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.

Keywords: 5′-UTR; COVID19-NMR; SARS-CoV-2; SL5a; Solution NMR spectroscopy.

Fig. 1

Resonance assignment of aromatic protons and carbons and the linkage to the ribose. a HCCNH experiment correlating the imino protons of guanosines and uridines to the corresponding intranucleobase C8 and C6 resonances, respectively. b ¹H,¹³C-HSQC spectrum showing the aromatic H6/8–C6/8 correlations. c 2D plane of the ¹³C-detected CNC-experiment correlating C6/8 to C1′. d Transposed ¹H,¹³C-HSQC spectrum showing the H5–C5 correlations for uridines and cytidines. e ¹H,¹H-TOCSY spectrum linking H5 and H6 in pyrimidines. f Transposed ¹H,¹³C-HSQC spectrum of the H1′–C1′ region. Panel c further shows the secondary structure of SL5a with genomic numbering. Positive contours are given in black, negative contours are held in red. Experimental details are given in Table 1. Exemplary connections between the displayed spectra are demonstrated with the gray dashed lines for residues G213 and U191. Assignments of the asymmetric bulge and the apical loop are highlighted with bold font

Fig. 2

Comparison of ¹H,¹³C-CT-HSQC spectra of the ribose regions of a SL5a and b a 14 nt RNA with 5′-cUUCGg-3′ tetraloop (Fürtig et al. ; Nozinovic et al. 2010). Positive contours are given in black, negative contours in red. Experimental details are given in Table 1. The loop sequences are displayed and U202/U7 and C203/C8 resonances are highlighted in bold font. c Canonical coordinates for all residues of SL5a with a complete carbon ribose assignment. For comparison, the canonical coordinates of residues U7 and C8 of a 14 nt RNA with 5′-UUCG-3′ tetraloop are given in red