The 5′-terminal stem–loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH

Abstract We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5′-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5′- and 3′-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12–C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5′-UTR of SARS-CoV-2.


Figure S1, related to figure 4
Figure S1, related to figure 4 pKa plot based on the pH-induced 13 C-CSPs of the A12 C2 resonance reveals a pKa of ~5.8 for the A12N1.The carbon chemical shift was measured using an 1 H, 13 C HSQC optimized for the aromatic resonances as described (1) at 600 MHz and 298 K.

Figure
Figure S2 pH-dependent A12N1 shifts: The A12 H2N1 resonance was monitored in 2 J-1 H, 15 N-HSQC spectra at pH values from 5.0 (green) to 4.7 (purple).The A12N1 resonance shifts towards the 15 N spectral region of canonical H8,N9 chemical shift ranges for adenosine.The experiments were recorded at 800 MHz with 16 scans and a spectral width of 9.8 x 32 ppm and 1024 x 192 complex points in the direct and indirect dimension, respectively.

Figure S3
Figure S3 Chemical shift signature of C28 at pH <5.NMR data were acquired at 600 MHz and 298 K. Correlations are represented by the respective color code highlighted within the cytosine structures: loop cytidines: green; stem cytidines: black; C28: purple; C34: light grey (2-5).

Figure S4
Figure S4 Supercooled NMR experiment of an unlabeled SL1 RNA sample allows the detection of an additional imino signal at a pH of 4.8.A Overlay of the imino region of 1D 1 H spectra recorded at pH 4.8 and different temperatures.Chemical shift assignments are annotated.An additional imino signal is observed around 11.2 ppm, indicated by an asterisk.B Stacked view into the imino 1 H chemical shift region at different temperatures.Asterisk highlight the novel detected imino signal.A total concentration of 840 μM SL1 was measured in 1 mm capillaries at pH 4.8.NMR data were acquired at 700 MHz and in a temperature range between 262 and 278 K (6, 7).

Figure S5
Figure S5 Detection of the U imino signal appearing at lower pH.A 2D-15 N, 1 H-HSQC spectrum leads to the detection of a new non-canonical U imino signal at 10.97/158.28 δ 1 H/δ 15 N ppm at lower pH.B 2D-1 H, 1 H-NOESY (mixing time = 100 ms) shows that the novel imino signal only has exchange peak to water.This exchange is less pronounced than exchange peaks of G8 and G23 indicating reduced solvent exchange rates for this new peak under the applied conditions compared to unstructured residues.NMR spectra were acquired at 800 MHz, 278 K and a buffer pH of 4.2.

Figure S6, related to figure 7
Figure S6, related to figure 7 pH-dependent melting point determination of SL1 by using derivations of the CD melting curves.Data were smoothened by applying a Savitzky-Golay filter with 15 points before the first and second derivation.Melting points were taken at the minimum of the 1 st derivation curve at that point where the curve if the 2 nd derivation crosses zero.Standard deviation was calculated from ± two points around the melting point.Data were recorded at pH 5.2 (A), pH 6.2 (B) and pH 7.2 (C).

Figure S7, related to figure 8
Figure S7, related to figure 8 Correlation plots comparing replicates of DMS probing for pH 8.0 (top, blue) and 5.0 (bottom, red) sets.

Figure S8, related to figure 8
Figure S8, related to figure 8 Raw mutation rates for the control (-DMS), pH 8 (+DMS), and pH 5 (+DMS) data for each replicate with median mutation rates for each set listed in the upper left-hand corner.Control samples generally exhibit lower mutation rates compared to +DMS samples at either pH, with pH 8 exhibiting the greatest median reactivity in each replicate set.

Figure S10, related to figure 9
Figure S10, related to figure 9 Targeting of SARS-CoV-2 SL1.A 1D-1 H spectra of C11 at different pH values highlighting the pH sensitive resonances of C11.NMR spectra were acquired at 500 MHz and 298 K. B 2D-1 H, 1 H-TOCSY overlay of SL1 alone and SL1 after C11 addition reveals CSPs mapped onto the secondary structure of SL1.C11 was added in excess with an [RNA]:[ligand]-ratio of 1:7 (150 μM:1 mM).NMR spectra were acquired at 600 MHz and 298 K. C 1D-1 H NMR spectra of the ligandbased titrations with C11 and SL1 at pH 7.2.NMR spectra were acquired at 600 MHz and 298 K with 100 μM C11 and 0-250 μM SL1.A+C Spectra were calibrated on DMSO-d6 (2.50 ppm) and B on DSS (0.00 ppm); all samples were measured in 25 mM KPi (pH 5.2-7.2),50 mM KCl and 5% DMSO-d6.D 2-Aminopurine modification of SL1 A27 and the assay set-up.Increasing compound concentration induces a decrease of the intrinsic 2AP fluorescence due to binding of the labelled RNA (highlighted by the greyed star).

Figure
Figure S11 hetNOE ratios for the aromatic resonances C2H2 (black) and C8H8 (blue) of adenosines of SL1 measured at 298 K and pH 7.2 and pH 5.2 plotted against the respective residues.The hetNOE ratio for A31, which is located most distant from any observed protonation event, was set to 1 and deviations from 1 given.Values above 0 correspond to greater flexibility at pH 7.2 compared to 5.2, whereas ratios blow 0 correspond to greater flexibility at pH 5.2 versus pH 7.2.Experiments were recorded at 800 MHz with a selectively 13 C A/C labeled sample.

Figure S12
Figure S12 Motif search; comparison of lowest-energy NMR structure of SL1 (blue sticks) with a set of different internal 1:2 loops form an RNA secondary structure motif search (https://www.rnacossmos.com/search.php)(8).Salmon sticks) showing the preferential arrangement of A12 and C28 isosteric to a G•U wobble base pair as present in the 1:2 internal loop of 5MC6.The sequentially identical internal loops (A:AC; present in 2AAr, 5MMJ, 6YW5, 7NRC) show greater structure

Table S1 , related to figure 2
Comparison of Rg and Dmax from prediction for NMR ensemble and SAXS

Table S4 , related to figure 5
The distribution of distances of the A + •C wobble base pair between A12N6and C28O3, A12N6 and C28O2, and A12N1 and C26O2, respectively.The distances representing a perfect A + •C-wobble geometry are shown for comparison.

Table S5 , related to figure
8 Template and primer sequences used in PD-MaP experiments.Underlined portion of template denotes T7 RNA Polymerase promotor region.

Table S6 , related to figure 8
Read depths per probing condition per replicate.