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Matzel and Wirtz Martin et al. 1
NMR characterization and ligand binding site of the stem loop 2 motif
(s2m) from the Delta variant of SARS-CoV-2
Tobias Matzel1,‡, Maria Wirtz Martin1,‡, Alexander Herr, Anna Wacker1, Christian Richter1,
Sridhar Sreeramulu1 and Harald Schwalbe1,*
1 Institute for Organic Chemistry and Chemical Biology
Center for Biomolecular Magnetic Resonance (BMRZ)
Goethe-Universität Frankfurt
Max-von-Laue-Str. 7, 60438 Frankfurt (Germany)
‡Authors contributed equally
*To whom correspondence should be addressed. Tel: +496979829737; Fax: +496979827915;
Email: schwalbe@nmr.uni-frankfurt.de
Short title: NMR Characterization of SCoV-2 s2m Delta
Keywords: Covid-19, s2m, NMR assignment, NMR dynamics, ligand screening
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Matzel and Wirtz Martin et al. 2
ABSTRACT
The stem loop 2 motif (s2m) in SARS-CoV-2 (SCoV-2) is located in the 3’-UTR. Although s2m
has been reported to display characteristics of a mobile genomic element that might lead to an
evolutionary advantage, its function has remained unknown. The secondary structure of the
original SCoV-2 RNA sequence (Wuhan-Hu-1) was determined by NMR in late 2020,
delineating the base pairing pattern and revealing substantial differences in secondary
structure compared to SARS-CoV-1 (SCoV-1). The existence of a single G29742-A29756
mismatch in the upper stem of s2m leads to its destabilization and impedes a complete NMR
analysis. With Delta, a variant of concern has evolved with one mutation compared to the
original sequence that replaces G29742 by U29742. We show here that this mutation results
in a more defined structure at ambient temperature accompanied by a rise in melting
temperature. Consequently, we were able to identify over 90 % of the relevant NMR
resonances using a combination of selective RNA labeling and filtered 2D NOESY as well as
4D NMR experiments. We present a comprehensive NMR analysis of the secondary structure,
(sub-) nanosecond dynamics and ribose conformation of s2m Delta based on heteronuclear
13C NOE and T1 measurements and ribose carbon chemical shift-derived canonical
coordinates. We further show that the G29742U mutation in Delta has no influence on the
druggability of s2m compared to the Wuhan-Hu-1 sequence. With the assignment at hand, we
identify the flexible regions of s2m as primary site for small molecule binding.
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INTRODUCTION
The emergence of the SARS-CoV-2 (SCoV-2) virus resulted in a worldwide pandemic that not
only changed our everyday life in a way never seen before, but also has given birth to several
extensive collaborative initiatives focusing on viral research and drug development. Up to
today, the virus has infected more than 690 million people across the world with 6.9 million
deaths, and several variants have emerged. Each variant showed a change in characteristics
of the virus regarding disease severity and infectivity. The RNA genome of SCoV-2 consists
of approximately 30.000 nucleotides. It does not only encode for the viral proteins but also
harbours highly structured 3’- and 5’-untranslated regions (UTRs). The presence of these
structured regions was proposed early in the pandemic with the help of computational
predictions (Rangan et al. 2020) and experimentally verified by NMR and DMS footprinting
(Wacker and Weigand et al. 2020). It is known that the UTRs of coronaviruses play important
regulatory roles in processes like genome replication and translation (Yang & Leibowitz
2015). The stem loop 2 motif (s2m) is part of the hypervariable region (HVR) of the 3’-UTR
(Goebel et al. 2007) and found in position 29728 to 29768 in the SCoV-2 RNA genome (Wu et
al. 2020). In contrast to the entire HVR, the 43 nucleotide (nt) long s2m is more conserved
even between only distantly related viruses (Tengs et al. 2013). It was first described in 1997
in Astroviruses (Monceyron et al. 1997) and has subsequently been detected in Caliciviruses,
Picornaviruses, and Coronaviruses (Kofstad & Jonassen 2011). Its spread between different
viruses suggests that it can be transferred from one virus to another. Supporting this
hypothesis, a “MixOmicron” SCoV-2 hybrid was reported where a s2m-containing 2500-
3000 nt region from Omicron 21K/BA.1 has been transferred to Omicron 21L/BA.2. In the
resulting genome, the truncated s2m version that is present in most Omicron lineages (Frye et
al. 2023) is replaced with the full length s2m from Omicron 21K/BA.1, emphasizing the mobile
nature of this RNA-element (Colson et al. 2022). The function of s2m has remained uncertain,
but its high degree of conservation suggests that an evolutionary advantage is connected to it.
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Matzel and Wirtz Martin et al. 4
The proposed function of s2m is host-related rather than viral (Tengs et al. 2013). This
hypothesis is supported by the lack of conservation in the flanking regions of the RNA element
compared to s2m itself. In contrast to the only distantly related viruses where s2m occurs,
hosts infected by s2m-containing viruses are more closely related from an evolutionary
perspective. It has been proposed that s2m plays a role in different processes such as hijacking
of the host protein machinery (Robertson et al. 2005), RNAi-like gene regulation of host genes
(Tengs et al. 2013), viral replication (Gilbert & Tengs 2021), immune evasion through
interaction with host miRNA-1307-3p and genomic dimerization (Imperatore et al. 2022) or
protection of the viral RNA against degradation (Tengs & Jonassen 2016). Additionally, it has
been shown that s2m can bind to the viral N-protein (Padroni et al. 2023).
S2m is one of the few RNAs of betacoronaviruses for which X-ray data have been reported. In
2005, the crystal structure for SCoV-1 s2m was solved (Robertson et al. 2005). In this crystal
structure, s2m shows a unique secondary structure with two perpendicular stems, a GNRA-
like apical pentaloop, a purine-rich internal loop and a seven nt asymmetric internal loop. Close
to the apical loop, a 90° kink of the helix axis is formed that is mediated by two unpaired
nucleotides (Robertson et al. 2005). Compared to SCoV-1, s2m in SCoV-2 shows two
mutations (U29732C and G29758U). These two mutations drastically alter the secondary
structure of the RNA. 1H, 1H-NOESY, 1H, 15N-BEST-TROSY and 1H,15N-HNN-COSY NMR
experiments showed that the upper stem of SCoV-2 s2m adopts a completely different
base-pairing pattern. Here, s2m contains a nonaloop and two stems separated by a large ten
nt internal loop, whereas the upper stem contains an internal G-A mismatch (Wacker and
Weigand et al. 2020).
Some homology or bioinformatic approaches used the already published secondary structure
of SCoV-1 s2m as starting point to determine the influence of the mutations in SCoV-2, stating
no significant change in secondary structure between SCoV-1 and SCoV-2 (Ryder et al. 2021).
Others used the NMR-derived secondary structure published for the Wuhan-Hu-1 version by
our group (Wacker and Weigand et al. 2020) as starting model (Kensinger et al. 2023; Rangan
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et al. 2021). Alternatively, a secondary structure deviating from SCoV-1 s2m based on RNA
structure probing data was used (Manfredonia et al. 2020). One study (Jiang et al. 2023)
functionally characterized the s2m element to assess the impact of s2m on viral growth of
SCoV-2, both in cell cultures (in vitro) and Syrian hamster models (in vivo). All these reports
feature substantially different secondary structures, warranting detailed high resolution
structural work as reported here. Although previous publications suggested an evolutionary
advantage associated with s2m, the viral fitness in Syrian hamster models did not change upon
deletion of s2m. However, the study acknowledges its limitations and leaves open the
possibility that s2m may still play a significant role concerning cell line and host dependence.
The intriguing question that remains unanswered is why the s2m element is highly conserved
across different variants, even though the RNA element appears to lack a critical functional
role. Further research is required to explore this and gain a deeper understanding of s2m's
significance in the context of viral growth and evolution.
Up to now, therapeutic strategies to combat SCoV-2 focused either on the development of
vaccines that induce the production or present (variants of the) surface receptor protein spike
to stimulate the host immune response (Pfizer-BioNTech, Moderna, Johnson & Johnson,
Astrazeneca, Novavax) or on the development of low molecular weight molecules (small
molecules) that target especially the main protease of SARS-CoV-2 (Cantrelle et al. 2021;
Günther et al. 2021; Macchiagodena et al. 2020; Zhang et al. 2020). Both strategies, while
being effective, inevitably tend to be sensitive towards evolutionary pressure to evade the
immune response or the antiviral agent. To provide a broader approach, our project
Covid19-nmr (Duchardt‐Ferner et al. 2023), has focused on screening the entire proteome
(Berg and Wirtz Martin and Altincekic et al. 2022) and RNA genome of SCoV-2 to identify
binding fragments for further medicinal chemistry campaigns. One underestimated but
promising research area is the development of drugs targeting RNA, which could potentially
revolutionize antiviral treatment. Although the function remains elusive, it is very likely that s2m
is involved in some of the proposed processes and considering its conservation this motive
presents an excellent drug target. It has successfully been targeted with antisense
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Matzel and Wirtz Martin et al. 6
oligonucleotides (ASOs) (Lulla et al. 2021), L-DNA aptamers (Li & Sczepanski 2022) and
antiviral compounds (Simba-Lahuasi et al. 2022).
We here report on the NMR characterization of the Delta variant of s2m, including near to
complete chemical shift assignment, 13C-based delineation of local sub-nanosecond dynamics
within this RNA element and the determination of binding epitopes and affinities for small
molecules from a library containing 786 compounds. For the Wuhan-Hu-1 version of s2m, there
have been ten hits with typical KD’s ranging from 64 µM – 1.3 mM, while the best binder showed
an affinity of 6 µM (Sreeramulu and Richter et al. 2021). We show that the binding affinities are
not affected by the mutation observed in the Delta variant and identify preferred sites for small
molecule binding.
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RESULTS
Comparison between secondary structures of SCoV-1, SCoV-2, and SCoV-2 (Delta)
The crystal structure for s2m SCoV-1 (FIGURE LEGENDS
Figure 1A+B) consists of two stems, a purine-rich internal loop, a seven nt internal loop and
a GNRA-like pentaloop and presents a 90° kink close to the apical loop (Robertson et al. 2005).
Two mutations (UC29732 in the lower stem and GU29758 in the upper stem) in s2m
SCoV-2 alter the secondary structure of the upper stem significantly (Wacker and Weigand et
al. 2020). The original Wuhan-Hu-1 version of s2m of SCoV-2 consists of two stems separated
by a large ten nt internal loop, an internal unstable G-A mismatch in the upper stem and a
nonaloop (FIGURE LEGENDS
Figure 1C). The mutation that occurred in the SCoV-2 Delta variant from 29742G to 29742U
suggests formation of a base pair between U29742 and A29756, leading to a stabilized six
base pair upper stem that is not disrupted by a G-A mismatch (FIGURE LEGENDS
Figure 1D). This in turn may stabilize the global structure and the loop closing non canonical
G-U base pair and should not alter the rest of the secondary structure.
NMR chemical shift assignment of s2m Delta, delineation of secondary structure, and
analysis of thermal stability
Imino proton assignment. The RNA sequence of s2m investigated in this study comprises
nucleotides 29728 to 29763. To facilitate in vitro transcription and stabilization of the lower
stem region of the RNA, two artificial GC base pairs (G-2, G-1, C+2, C+1) were added (Figure
2B). Others have reported a kissing dimer and extended duplex of this RNA in presence of
Mg2+ ions (Cunningham et al. 2023). Higher sample concentrations in our experiments
compared to this study could potentially lead to dimerization even in the absence of
magnesium ions. Since we are focusing here on the monomeric s2m Delta this state was
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Matzel and Wirtz Martin et al. 8
obtained via a folding protocol at 95 °C and verified via native polyacrylamide gel
electrophoresis (PAGE) (see Methods).
With characteristic 15N chemical shifts of Gs and Us these nucleobases can easily be
distinguished in 1H, 15N spectra, as 15N chemical shifts of imino protons (N1-H1) of G are
expected between 145 and 148 ppm and of U (N3-H3) between 157 and 162 ppm (Fürtig et
al. 2003). Due to solvent exchange, only base-paired nucleotides yield NMR peaks for the
imino protons. In total, 12 out of 13 expected 1H, 15N resonances could be detected in
1H, 15N BEST-TROSY spectra (Figure 2B) at 298 K. Five of these resonances could be
assigned to Us (N3-H3) and seven to Gs (N1-H1) based on their characteristic chemical shift.
The missing G (N1-H1) resonance can be assigned to G-2 as part of the closing G-C base
pair. Missing signals are expected as the imino proton is likely in exchange with the solvent
and for this reason not detectable at 298 K. In 1H, 1H NOESY spectra NOE cross peaks of
adjacent base pairs are to be expected, where one base-paired imino proton is in close
proximity to another one. Analysis of these sequential NOE cross peaks led to a full assignment
of all imino protons of both base-paired regions. The two observable sequential walks were:
GGUGUG and GUUGUUG(G), while G-2 is only observable at low temperature
(Supplementary Figure S1), confirming the previously described secondary structure (Wacker
and Weigand et al. 2020). As for the Wuhan-Hu-1 version, the stable seven base pair lower
stem is separated from the upper stem by an unpaired dynamic internal loop region. It is formed
by six nucleotides on the 5’-strand and four nucleotides on the 3’-strand and shows no imino
proton resonances due to solvent exchange. The proposed formation of the U29742-A29756
base pair in the upper stem of the Delta mutant could be confirmed in both the 1H, 1H NOESY
and 1H, 15N BEST-TROSY spectra. (Figure 2A+B).
At low temperatures we additionally observed an NOE cross peak to G29744, which we
assigned to originate from the G29745 imino signal as part of to the GU loop closing base pair.
Typical 1H and 15N chemical shifts detected in 1H, 15N BEST-TROSY spectra confirmed this
(Error! Reference source not found.). This base pair could not be detected at 298 K. Such
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Matzel and Wirtz Martin et al. 9
loss of imino signal at higher temperatures is typically linked to accelerated base pair opening
and chemical exchange with the solvent. We conclude that the base pair is dynamic and
unstable. Additionally, the G-2 (H1-N1) resonance that could not be detected at 298 K is visible
at 283 K.
Moving on from the imino proton assignment we could identify aromatic resonances of the
base-paired Gs and Us via 1H, 13C HSQC and HCCNH experiments of a 13C, 15N uniformly
labeled sample. The latter experiment correlates the chemical shift of an H1 or H3 proton with
its C8 or C6 aromatic carbon respectively. In 1H, 13C HSQCs, we could assign peaks using this
information resulting in an assignment of the aromatic protons of Gs and Us within the two
stems (Figure 2C+D).
Melting point determination. To investigate the stability of the RNA for the different mutants,
we measured CD-melting curves of SCoV-1, SCoV-2 and SCoV-2 Delta versions of s2m. Of
all three constructs, s2m Delta showed the highest melting point of 62 °C, which is significantly
higher than the temperatures measured for the SCoV-1 (51 °C) and SCoV-2 (55 °C) versions
(Error! Reference source not found.). This is in line with our proposed secondary structure and
the finding that the upper stem now forms an uninterrupted helical segment of six base pairs.
Aromatic C6H6/C8H8 and ribose C1’H1’ assignment via 4D-NMR. Using a 4D-HMQC-
NOESY-HMQC experiment (Stanek et al. 2013), sequential assignment of 44 out of 45
C6H6/C8H8 and C1’H1’-resonances could be achieved. For this assignment, we started with
aromatic peaks (Figure 4A) that could be assigned before in HCCNH or 2D-NOESY spectra.
From there, the chemical shifts of the 13C- and 1H-dimensions were both transferred to the 4D
spectrum. The resulting HSQC-like plane then only showed C1’H1’ resonances with an NOE
cross peak to the selected aromatic proton (one peak for nucleotide (i) and one for nucleotide
(i-1) for most resonances) (Figure 4B). By comparing the 4D plane with a 1H, 13C-HSQC for
the C1’H1’ region, these resonances could easily be assigned (Figure 4C). As an example of
the sequential walk using the 4D experiment, the loop assignment is shown in (Error!
Reference source not found.). The assignment procedure was inverted for difficult
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Matzel and Wirtz Martin et al. 10
assignments starting from the C1’H1’ shifts and moving to the aromatic plane of the 4D
spectrum. The aromatic plane then showed one peak for nucleotide (i) and a second peak for
nucleotide (i+1). In addition to the 4D-HMQC-NOESY-HMQC, HCN and CNC spectra as well
as HCCNH and CPMG-NOESY experiments were used to assign C1’H1’ and C6H6/C8H8
resonances based on the imino assignment of the construct. The 4D experiment enabled us
to observe continuous NOESY connectivities for the lower stem and for the upper stem
including the loop. The most challenging region regarding the assignment was the internal loop
region between nucleotides C29733-C29738 and U29760-C29764, respectively (FIGURE
LEGENDS
Figure 1), that could only be assigned by combining all previously mentioned experiments.
Ribose assignment (C2’-C5’) based on 13C-filtered NOESY and 3D TOCSY. For the
assignment of the C2’-C5’ resonances the problem of signal overlap was particularly
challenging since the stem nucleotides showed very similar 13C chemical shifts. To alleviate
this restricted chemical shift resolution, we used 13C-filtered NOESY (Otting & Wüthrich 1989)
and 3D-TOCSY spectra of selectively labeled samples (AC and GU 13C, 15N labeled). These
combined experiments enabled a separate assignment of the 13C and 1H shifts. In turn, this
allowed unambiguous identification of most peaks in the HSQC spectra despite insufficient
resolution of the HSQCs, when considered alone. The 1H chemical shifts were obtained mainly
via the filtered NOESY spectra using both the aromatic and H1’ region in the direct dimension.
In the {F1, F2}-filtered spectrum, peaks were only visible when both protons were attached to
a 12C carbon filtering out all protons that were connected to 13C. As an example, in our AC
labeled sample, cross peaks were visible from G29757 to U29758 but not to A29756.
Consequently, this significantly diminished the number of signals and subsequently reduced
the signal overlap.
Cross peaks were observed from the aromatic (or ribose H1’) protons intraresidual to all other
ribose protons (H2’-H5’’) and interresidual to H2’ and H3’ protons of the sequential nucleotide
(Figure 2A). The forward (fw)-HCCH-TOCSY (Glaser et al. 1996; Schwalbe et al. 1995) was
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Matzel and Wirtz Martin et al. 11
analyzed by selecting different 1H, 13C-planes via the H1’ chemical shifts (Figure 2B). In an
ideal case, 1H and 13C chemical shifts can be obtained for all ribose protons of a nucleotide. In
fact, most H2’C2’, H3’C3’, and H4’C4’ resonances can be assigned using this experiment. In
addition, an HCC(H)-TOCSY was recorded that enabled the assignment of all ribose 13C
chemical shifts connected to a C1’H1’ resonance (Figure 2C). In this case, the 1H, 13C-planes
were selected via the C1’ chemical shifts. Error! Reference source not found. shows which
experiments were used for the assignments of 13C and 1H chemical shifts, respectively. A
schematic representation of the assignment strategy is given in the upper right panel (Error!
Reference source not found.B).
Using the previously mentioned strategy including the 4D HMQC-NOESY-HMQC and
13C-filtered NOESY over 90 % of all structurally relevant NMR resonances were
unambiguously assigned. For the aromatic C6H6/C8H8 and the C1’H1’ chemical shifts, 44 of
45 resonances could be assigned with only nucleotide G738 missing which was overlapping
with other signals and in an adverse exchange regime leading to a missing peak in the 4D. For
the remainder of the ribose resonances, 145 out of 180 13C chemical shifts and 201 of 225 1H
chemical shifts and could be assigned with a completeness ranging from 71 % (C4’) to 98 %
(H2’). The complete assignment is reported in (Supplementary Table S1 and Supplementary
Table S2).
Canonical Coordinates
Based on the complete ribose assignment of s2m Delta, we calculated canonical coordinates
to determine the conformation of the ribofuranosyl ring of each nucleotide and the exocyclic
torsion angle γ (Error! Reference source not found.). This empirical parameterization based
on the 13C chemical shifts enables distinction between C3’ endo and C2’ endo conformation
and was carried out as described previously (Cherepanov et al. 2010; Ebrahimi et al. 2001).
Our results suggest a canonical C3’ endo conformation for both helical regions, the 3’ side of
the dynamic internal loop and the lower part of the apical loop. This is typical for canonical A-
form helices. In contrast, most of the loop nucleotides and the 5’ side of the internal loop adopt
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a C2’ endo conformation. For the internal loop nucleotides G736 and G737 we were not able
to achieve a complete ribose assignment, but characteristic C4’ chemical shifts (84.46 ppm,
84.32 ppm) indicate C2’ endo conformation for these nucleotides. In the same manner, the
loop nucleotide A752 can be considered as C2’ endo as it shows characteristic C1’
(90.86 ppm), C2’ (76.25 ppm) and C4’ (84.74 ppm) chemical shifts. For G745 and U753 the
assigned chemical shifts predict a more C3’ endo conformation which is in line with the
previously mentioned unstable G-U base pair that is formed at low temperatures and extends
the six base pair upper stem. We excluded the C+2 nucleotide as it forms a cyclic phosphate
after HDV-induced cleavage (see Material and Methods).
Dynamics of the loop and internal loop region
Heteronuclear 13C T1 relaxation and NOE. To investigate the dynamics of s2m especially in
the apical and internal loop regions, we measured heteronuclear 13C T1-relaxation rates and
NOE. All measurements were conducted for the C6H6/C8H8 aromatic resonances (Error!
Reference source not found.A) and for the C1’H1’ ribose resonances (Error! Reference source
not found.B). For the apical loop region (pink), we measured significantly higher overall
hetNOE rates. For the aromatic loop signals we measured a mean hetNOE of 1.30 compared
to a mean value of 1.14 for the stem. In addition, we observed that the hetNOE gradually
increases with distance relative to the stem e.g. U748 (1.37) and A749 (1.38) showed the
highest hetNOE. For the aromatic internal loop residues, the picture was similar although the
difference was not as prominent as for the loop (1.21 internal loop, 1.14 stem). The R1 rates
for the aromatic signals showed a similar trend with a mean rate of 1.98 s-1 for the loop
compared to 1.55 s-1 for the stem. However, it is worth noting that the R1 rates in the loop
showed reduced values for all A nucleotides (A746, A749, A751) that are symmetrically
distributed within the loop. The flexibility of the internal loop region as it is suggested from the
hetNOE could in this case not be confirmed by higher R1 rates.
In contrast to the aromatic protons, the chemical environment of H1’ was generally more similar
which led to an easier interpretation of the R1 rates and the hetNOE (Error! Reference source
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Matzel and Wirtz Martin et al. 13
not found.B). For the flexible regions, the mean hetNOEs were again significantly higher
(internal loop: 1.13, loop: 1.23) than for the stem regions (1.08). The highest hetNOEs were
detected for the nucleotides U748, A749 and C750 (1.32). These nucleotides represent the tip
of the loop and are the furthest away from the upper stem. The R1 rates for the C1’ resonances
showed the same trend with mean rates of 1.23 s-1 for the loop compared with 0.74 s-1 for the
stem. In this case, no variations are observed for A746, A749 and A751 and the internal loop
region shows slightly higher mean hetNOEs than the stem (0.89 s-1 vs 0.74 s-1). Within the loop
the same gradual increase in R1 rates was observable as for the hetNOE values. For
nucleotides G-2, G-1, and C+2, higher hetNOE values and/or R1 rates indicating increased
dynamics are observed due to the instability of closing base pairs.
Overall, the relaxation and hetNOE data confirmed the dynamic nature of the unpaired regions
of s2m. In particular, the loop must be regarded as highly flexible with an increased flexibility
for the nucleotides at the tip of the nonaloop. Structure calculations of this RNA will therefore
not yield a tight structure bundle in the loop region but show a variety of states.
Druggability of s2m Delta
To investigate the druggability of s2m, ligand- and RNA-based titrations were carried out. In
an earlier fragment-based NMR screening approach, ten hits out of 768 fragments were
identified for SCoV-2 s2m and KD values (KDest) were estimated in ligand-based titrations
(Sreeramulu and Richter et al. 2021). We now determined KDest values for s2m Delta with
seven of these initial hits using 1H 1D-NMR titrations with constant ligand concentration
(100 µM) and increasing RNA concentration (0 – 250 µM). An overview of the determined
affinities is given in Error! Reference source not found.E. 1D spectra and KDest fits for all
ligands are given in Error! Reference source not found.. A comparison of all affinities
between s2m SCoV-2 and s2m Delta showed an overall good agreement except for ligand
P5H08 where a significantly lower affinity was detected for the Delta version (KDest > 1 mM vs
200 µM). An explanation for this could be that the ligand might interact with the upper stem of
the motif close to the G-A mismatch that is present in the Wuhan-Hu-1 construct. For both s2m
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Matzel and Wirtz Martin et al. 14
versions the ligands P6E06 and P4C11 showed the highest affinity (KDest = 120 - 212 µM). We
conclude that the druggability of s2m does not change significantly between the two mutants.
The binding behavior of P4C11 and P6E06 was further analyzed via analysis of chemical shift
perturbations (CSP mapping) in RNA detected 1H, 13C HSQC spectra of the aromatic region
using a 5-fold excess of ligands (Error! Reference source not found. and Error! Reference
source not found.). Here, the chemical shift differences between spectra with and without
added ligand were determined and plotted in histograms (Error! Reference source not found.B
and Error! Reference source not found.B). To visualize the largest changes in chemical shift,
CSPs were mapped on the secondary structure of s2m Delta (Error! Reference source not
found.D and Error! Reference source not found.D). Nucleotides that revealed the largest CSPs
were considered to be part of the binding pocket. Using this approach, the binding site of both
ligands could be localized in the internal loop region of the RNA. For P4C11, more prominent
effects were observed on the 3’ side of the internal loop where all four nucleotides (UACA)
showed significant shifts with C762 and A763 shifting the most. Similarly, for P6E06, C762 and
A763 were shifting the most but also the 5’ site of the internal loop shows significant CSPs at
position A735 and G736. Residues further away from this region including the nonaloop
showed significantly lower CSPs of the aromatic resonances. The same procedure was carried
out for the C1’H1’ resonances upon addition of P4C11 (Error! Reference source not found.)
and P6E06 Error! Reference source not found.). As for the aromatic resonances the ribose
H1’C1’ resonances corresponding to the internal loop nucleotides show the strongest shifts. In
contrast to the aromatics, the lower part of the nonaloop (G745, A746 and U753) showed also
clear effects. We interpret this as a second binding event in the lower part of the nonaloop.
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DISCUSSION
Understanding the structure and function of untranslated regions in viruses is an important
step in understanding the virus since these regions are known to fulfill regulatory roles (Yang
& Leibowitz 2015). The s2m element is of particular interest due to its high degree of
conservation and potential ability to be transferred horizontally between different viruses
(Colson et al. 2022; Tengs et al. 2013; Tengs & Jonassen 2016). It is important to understand
the structure and function of elements like s2m considering future pandemics or viral
outbreaks, since it is likely that these viruses also contain similar motifs.
As of November 2023, there are more than 1000 structures deposited in pdb containing the
main protease nsp5 and over 1500 structures deposited in pdb containing Spike and its
variants of concern (RCSB.org, (Berman 2000)). By stark contrast, empirical structural studies
delineating the impact of mutations on structure and dynamics of conserved RNA elements
are very sparse. We focus on the 3D structure determination of these viral RNAs (Vögele et
al. 2023) (Toews, Wacker et al., in revision, Vögele et al., in revision) and report here on the
detailed characterization of the impact of the Delta mutation in the s2m RNA element located
in the 3’-UTR. This element stands out as it is the one of the few viral elements for which a
crystal structure was published for SCoV-1. To our knowledge, there is no experimental RNA
structure available for SCoV-2 s2m. It has been shown by our lab that the secondary structure
of s2m significantly differs between SCoV-1 and SCoV-2 (Wacker and Weigand et al. 2020).
Therefore, computational data that uses SCoV-1 as a starting point could potentially lead to
false conclusion. In addition, any biochemical experimental data that rely on the secondary
structure of SCoV-1 will inevitably lead to wrong conclusions.
The secondary structure presented here for the Delta variant of s2m analyzes the impact of
the G29742U mutation appearing in the Delta variant. In CD melting experiments, we detected
a significantly higher melting temperature of s2m Delta (62 °C) compared to SCoV-2 (55 °C)
or SCoV-1 (51 °C). These results regarding the stability of the s2m Delta are in agreement with
UV thermal denaturing experiments published by others (Cunningham et al. 2023).
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Matzel and Wirtz Martin et al. 16
In line with the increase in melting temperature, we detected an additional U29742-A29756
base pair in NOESY spectra in contrast to the internal unstable G-A mismatch that is present
in SCoV-2. With this new base pair, an uninterrupted six base pair upper helix is formed that
likely leads to a global stabilization against thermal denaturation. With a complete imino proton
assignment of the upper stem we unambiguously show that s2m consists of two stems
separated by a ten nt asymmetric internal loop with a large apical nonaloop. In addition, as
indicated by higher R1 rates and higher hetNOE values for the unpaired regions our dynamic
NMR data confirm this secondary structure.
Previously, partial NOESY data of s2m Delta recorded at 292 K, at lower magnetic fields, with
lower sample concentration and in a different buffer system were reported (Cunningham et al.
2023). The imino proton assignment reported there is incomplete and was not augmented by
substantial assignment experiments, including 4D NOESY as reported here. Incomplete NMR
data for an RNA can lead to ambiguous assignments, as is likely the case in the previous
report. The reason for this may likely stem from the lower chemical shift resolution and fewer
NOE cross peaks due to lower signal-to-noise compared to our spectra (Cunningham et al.
2023). These ambiguities, however, do not lead to a wrong secondary structure. In fact, follow-
up MD simulations are in agreement with our dynamic NMR data (Makowski et al. 2023).
(personal communication).
To characterize an NMR structure at single-residue level, and to evaluate NMR dynamics data
or map binding sites of an RNA, it is essential to assign as many resonances as possible. NMR
resonance assignments of large RNAs is inherently challenging due to signal overlap,
increased line widths and intermediate exchange. In the case of s2m, in addition the
palindromic sequences flanking the ten nt internal loop make a close to complete assignment
difficult to achieve. We here present an assignment strategy using a 4D HMQC-NOESY-
HMQC published before (Stanek et al. 2013) in combination with {13C, 15N}-filtered NOESY
and 3D TOCSY of selectively labeled samples. Even with severe signal overlap in
1H, 13C HSQC spectra, especially in the ribose regions, assigning resonances is enabled by
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Matzel and Wirtz Martin et al. 17
separate assignment of the 1H and 13C chemical shifts using a combination of the mentioned
experiments. Over 90 % of the relevant NMR signals were assigned in this manner.
Based on the 13C assignment, we calculated canonical coordinates to analyze the ribose
conformation of each nucleotide. For larger RNAs this is of particular interest as it is often
challenging to determine torsion angles based on J-coupling constants, cross-correlated
relaxation rates (CCR) or chemical shift anisotropy (CSA) (Marino et al. 1996; Nozinovic et al.
2010; Rinnenthal et al. 2007).
Folded RNAs often show conformational dynamics, and especially loop or internal loop regions
tend to be flexible. Already for small tetraloops dynamic data shows some degree of flexibility
and an ensemble is required to correctly describe the RNA structure (Oxenfarth et al. 2023).
For larger RNAs containing larger unpaired regions this is even more relevant. To quantify the
dynamics in s2m we conducted 1H, 13C HSQC based T1 and hetNOE data of the aromatic
H6C6/H8C8 and ribose H1’C1’ resonances that clearly confirm the flexibility of both
unstructured regions while the tip of the nonaloop shows the biggest effects. By comparing the
hetNOE and T1 values within the RNA, we also concluded that the dynamics of the internal
loop nucleotides occur on slower time scales than those of the loop nucleotides, and express
overall less flexibility. Judging from the overall high degree of flexibility of this RNA, we
conclude that a single structure is not sufficient to describe this motif and that an ensemble of
structures is required to correctly describe s2m.
In the recent years, more and more different RNA drug targets have been identified
(Campagne et al. 2019; Kelly et al. 2021; Meyer et al. 2020; Umuhire Juru & Hargrove 2021;
Warner et al. 2018). Considering the regulatory roles of the UTRs of viruses, it is interesting to
explore the druggability of these regions. Performing initial screens with small molecules can
be the starting point for the development of new antiviral drugs. Except for classical
intercalators, typical RNA-small molecule interactions involve conformationally flexible RNA
regions with elevated dynamics. Ligand binding is often characterized by conformational
adaption of these RNA motifs during the recognition and binding process (Hermann 2016;
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Matzel and Wirtz Martin et al. 18
Noeske et al. 2006; Stelzer et al. 2011; Zafferani et al. 2021). We have shown here that seven
out of the ten hits identified in our previous work (Sreeramulu and Richter et al. 2021) also bind
to s2m Delta. The two best binders show an estimated KD below 200 µM. Mapping of the
largest binding-induced changes to individual RNA resonances confirmed the regions of s2m
with most pronounced hetNOE- and T1-derived dynamics as binding sites: the ten nt dynamic
internal loop and the apical nonaloop. It has been shown before that flexible regions of RNAs
like the internal loop or the apical nonaloop of s2m can provide specific binding sites for small
molecules (Stelzer et al. 2011). We assume therefore that especially the internal loop of s2m
provides a perfect balance between flexibility and rigidity to facilitate specific binding events.
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Matzel and Wirtz Martin et al. 19
MATERIAL AND METHODS
DNA templates
Plasmids were used as DNA templates for in vitro transcription containing the different s2m
RNA sequences from the SCoV-2 RNA genome. For 3’-end homogeneity the HDV ribozyme
sequence was positioned at the 3’-end of the s2m sequence (Ferre-D’Amare & Doudna 1996).
This ribozyme induces self-cleavage at the 3’-end resulting in a cyclic phosphate. The
production of the DNA plasmids with T7 promoter was done via hybridization of complementary
oligonucleotides and ligation into the EcoRI and NcoI sites in the pSP64 vector (Promega)
containing the HDV ribozyme (the s2m SCoV-2 plasmid was provided by the group of Prof. Dr.
Julia Weigand as part of the covid19-NMR consortium). For RNA production DNA plasmids
containing the cloned sequence were transformed and amplified in Escherichia coli DH5α
competent cells in SB medium. Purification of plasmids was performed using the Gigaprep
(Qiagen) purification kit with yields between 2-10 mg plasmid per liter SB medium. Plasmids
were linearized with HindIII before being used for in vitro transcription. S2m RNA sequences
are summarized in Error! Reference source not found. and plasmid vector cards can be
obtained upon request.
In vitro transcription
RNA synthesis was done with in-house expressed and purified T7-polymerase in in vitro
transcriptions. Preparative-scale in vitro transcriptions (10-15 mL) were performed.
Nucleotides (13C, 15N labeled or natural abundance) were purchased at (Silantes GmbH) and
used according to different isotope labelling schemes. All prepared RNA constructs and the
yields after purification are listed in Error! Reference source not found..
Purification
Preparative-scale in vitro transcriptions were terminated after incubation at 37 °C for 6 h by
addition of ethylenediaminetetraacetic acid (EDTA) with a final concentration of 80 mM at
pH 8.0. Precipitation was done via addition of 0.3 M (final concentration) NaOAc and
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Matzel and Wirtz Martin et al. 20
isopropanol. RNA was isolated by performing gel electrophoresis to separate RNA bands. The
respective RNA band was excised with a scalpel under UV-illumination at 254 nm. RNA was
eluted from gel pieces by diffusion into 0.3 M NaOAc solution. Eluted RNA was precipitated
with EtOH and residual PAA was removed by reversed-phase HPLC using a Kromasil RP 18
column and a gradient of 0-40 % 0.1 M acetonitrile/triethylammonium acetate. RNA containing
fractions were freeze dried and precipitated with LiOCl4 solution (2 % in acetone) to exchange
cations. Folding of RNA was achieved by heating to 95 °C for 5 min and cooling to room
temperature before exchanging to NMR buffer (25 mM K2HPO4/KH2PO4, pH 6.2, 50 mM KCl)
via centrifugal concentrators with molecular weight cut-off of 2-3 kDa. The purity of RNA
samples was verified through analytical denaturing polyacrylamide gel electrophoresis. Special
are was taken to verify the monomeric state and homogeneity of the RNA samples as other
have reported kissing dimer and duplex formation of s2m Delta (Cunningham et al.. 2023).
After folding and rebuffering, the monomeric fold was verified by native polyacrylamide gel
electrophoresis (Supplementary Figure S7).
CD
Stability differences between different construct variants were investigated by CD melting
experiments on a spectropolarimeter (Jasco AKS J-810). 8 µM RNA were measured in 25 mM
potassium phosphate buffer with a pH of 6.2. A quartz cuvette with a path length of 1 mm was
used. Melting curves were recorded at a wavelength of 261 nm from 15 °C to 95 °C and a
heating rate of 1 °C/min. All melting curves were measured from low to high temperature
(forward) and from high to low temperature (reverse). Melting points were determined
graphically via the intersection of the forward curve and the median of linearly fitted baselines.
NMR experiments
A summary of all measured NMR spectra for the resonance assignment of s2m Delta is given
in Error! Reference source not found.. Spectrometers that were used to record NMR spectra
were 600, 800, 900, 950 MHz Bruker Avance NMR-spectrometers equipped with 5-mm
cryogenic triple resonance TCI-N probes, 700 MHz spectrometer which was equipped with a
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Matzel and Wirtz Martin et al. 21
QCI-31P probe and another 800 MHz spectrometer that was equipped with a TXO cryogenic
probe. Sample specifications varied but for exchangeable protons measurements were
performed in 5 % D2O / 95 % H2O and for non-exchangeable protons in 100 % D2O. All
samples were measured in 5 mm Shigemi tubes in 25 mM K2HPO4/KH2PO4, at pH 6.2 and
50 mM KCl with Sodium 2,2-Dimethyl-2-silapentane-5-sulfonate (DSS) as external reference.
NMR spectra were analyzed using TopSpin 4.1 and resonance assignment was done with
NMRFAM-SPARKY (Lee et al. 2015). HetNOE data were recorded to analyze structural
dynamics at 308 K and evaluated via peak heights in Sparky.
Canonical Coordinates
Canonical Coordinates (can1* and can2*) were calculated based on 13C ribose chemical shifts
as published before (Cherepanov et al. 2010; Ebrahimi et al. 2001):
can1* = PFit = -14.7δC1’ + 22.1δC2’ + 13.2δC3’ + 6.5δC4’ – 2.9 δC5’ – 1595
can2* = γFit = 9.8δC1’ + 16.5 δC2’ – 0.5δC3’ -1.7δC4’ + 13.5 δC5’ -2781
where δ is 13C the chemical shift in ppm with indices for the ribose carbon, P is the
pseudorotation phase and γ the torsion angle.
T1 measurements
R1 rates were measured via an HSQC-based experiment as pseudo 3D using 8 different T1
delays ranging from 20 ms to 1.5 s at constant time with constant time delays of 12.5 ms
(aromatics) and 8.8 ms (riboses) and with a relaxation delay of 2.6 s. Errors were calculated
based on the fitting with NMRFAM-SPARKY (Lee et al. 2015). For optimized spectral quality
all experiments were recorded at 308 K.
HetNOE measurements
Heteronuclear NOE experiments were performed as pseudo 3D experiments measuring the
NOE and the reference experiment without NOE in an interleaved manner with an interscan
delay of 5 s. For the NOE experiment 3 s presaturation were used and an off-resonant pulse
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Matzel and Wirtz Martin et al. 22
at -1000 ppm was used for temperature compensation in the reference experiment. Errors
were calculated based on the signal/noise ratios. To decrease spectral overlap, two selectively
13C, 15N labeled samples were used (AC labeled: 290 µM, GU labeled: 360 µM). All
measurements were carried out at 308 K and 800 MHz in 25 mM potassium phosphate
(pH 6.2), 50 mM KCl, in 100 % D2O.
Ligand binding measurements
For the determination of the estimated KD (KDest) individual samples with varying RNA
concentrations of RNA (50-250 µM) and constant ligand concentration (100 µM) were
measured in 1.7 mm tubes. 1H-1D spectra were recorded and the chemical shifts of reporter
signals of the ligands were monitored. KDest values were fitted with Origin
(© OriginLab Corporation) based on Michaelis Menten kinetics. All samples were measured in
25 mM potassium phosphate (pH 6.2), 50 mM KCl and 5 % DMSO-d6. Sodium
trimethylsilylpropanesulfonate (DSS) was used as internal reference. The binding site was
determined in 1H, 13C HSQC experiments with 100 µM RNA and 0 or 500 µM ligand. Chemical
shift changes were monitored and euclidean distances (d) were determined via:
d =
ට1
2൫δH
2+α×δC
2൯
Where δ is the chemical shift change of 1H and 13C in ppm and α is the weighting factor for 13C
based on the ratio of gyromagnetic ratios (α = 0.25) as described by the Al-Hashimi lab (Getz
et al. 2007).
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Matzel and Wirtz Martin et al. 23
DATA DEPOSITION
Assignment data can be found in the BMRB (bmrb.io) with the code 52215.
SUPPLEMENTAL MATERIAL
Supplemental material is available for this article.
ACKNOWLEDGEMENT
Work was supported by the Goethe Corona Funds, German funding agency (DFG) in
Collaborative Research Center 902: “Molecular principles of RNA-based regulation”, and by
European Union’s Horizon 2020 research and innovation program iNEXT-discovery. Work at
BMRZ was supported by the state of Hesse.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Matzel and Wirtz Martin et al. 24
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Matzel and Wirtz Martin et al. 30
FIGURE LEGENDS
Figure 1. (A) Crystal structure of s2m SCoV-1 and secondary structures of s2m SCoV-1
(B), SCoV-2 (C) and SCoV-2 Delta (D). The crystal structure for SCoV-1 (Robertson et al.
2005) was taken from the pdb: 1XJR. Nucleotides that differ between SCoV-1 and SCoV-2 are
highlighted in red and the Delta mutation is highlighted in magenta. The new base pair that
can be formed in Delta is shown in a grey box. The numbering of nucleotides corresponds to
the position in the genome -29000. The nucleotide notation for SCoV-1 is adjusted to match
the notation in SCoV-2. (E) Sequence alignment of s2m SCoV-1, SCoV-2, and SCoV-2 Delta.
Nucleotides that differ compared to s2m SCoV-2 are highlighted in red or magenta with a grey
box.
Figure 2. Secondary structure, imino proton assignment and transfer to aromatic
protons of s2m Delta. The assignment is annotated in each spectrum. The numbering of
nucleotides corresponds to the position in the genome -29000. Additionally introduced terminal
G-C base pairs are annotated as G-1/2 or C+1/2, respectively. Nucleotides labeled in grey
belong to the lower stem and nucleotides labeled in magenta belong to the upper stem. (A)
1H, 1H NOESY spectrum of the imino proton region. (B) 1H, 15N BEST-TROSY of base-paired
region of s2m Delta. A schematic representation of the secondary structure is shown in the
lower right corner. (C) HCCNH spectrum showing H1-C6 and H3-C8 correlations for base-
paired Us and Gs. (D) 1H, 13C HSQC of the aromatic region showing C6-H6 and C8-H8
correlations for base-paired Us and Gs respectively. Resonances that could be assigned in
the HCCNH spectrum are annotated.
Figure 3. CD melting curves of s2m SCoV-1 (A), SCoV-2 (B) and Delta (C). All melting
points are annotated. Black and grey curves correspond to measurements with increasing
(forward) or decreasing (reverse) temperatures respectively. Baselines and medians are
shown as red lines.
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Matzel and Wirtz Martin et al. 31
Figure 4. Aromatic C6H6/C8H8 and ribose C1’H1’ assignment via 4D-HMQC-NOESY-
HMQC. (A) 1H, 13C HSQC of the aromatic region of s2m Delta. The annotation and chemical
shifts of nucleotide A735 are highlighted in blue. (B) 4D-HMQC-NOESY-HMQC C1’H1’ plane
of A735. By transferring the aromatic chemical shifts (blue box) to the 4D a 1H, 13C HSQC like
plane of the C1’H1’ region is displayed. (C) Overview of relevant magnetization transfers and
secondary structure. (D) Overlay of the 1H, 13C HSQC (ribose C1’H1’ region) of s2m Delta with
the 4D plane of nucleotide A735. The resonance assignment for nucleotide A735 is highlighted
in red. The resonance assignment of all nucleotides is annotated and the notation of
nucleotides corresponds to the position in the genome -29000.
Figure 5. Assignment of C2’ – C5’ resonances via 13C-filtered NOESY and TOCSY
spectra of selectively labeled s2m Delta samples. The resonance assignments for
nucleotide C732 are annotated as an example. The notation of nucleotides corresponds to the
position in the genome -29000. (A) 13C-filtered NOESY of the aromatic to ribose region. (B)
Overview of applied experiments for both selectively labeled samples and assignments derived
from this. (C) fw-HCCH-TOCSY plane of nucleotide C732 (red) in overlay with a constant time
HSQC (black) of the ribose region. The plane was selected via the H1’ chemical shift of
nucleotide C732 (F1 = 5.3 ppm).
Figure 6. Canonical Coordinates based on ribose carbon shifts of s2m. (A) Each square
corresponds to a can1*/can2* combination of one nucleotide derived from its ribose 13C
chemical shifts. Specific nucleotides are annotated. Red squares indicate C2’ endo and black
squares indicate C3’ endo conformation. Loop and internal loop nucleotides are annotated. (B)
Secondary structure of s2m Delta.
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Matzel and Wirtz Martin et al. 32
Figure 7. 1H, 13C R1 rates (upper panels) and heteronuclear NOE of s2m Delta (lower
panels). For all measurements separate experiments were recorded for (A) aromatic
C6H6/C8H8 and (B) ribose C1’H1’ resonances. The secondary structure of the RNA as
determined by NMR is given on the top. The loop region (pink) and internal loop region (green)
are highlighted in the plots. The notation of nucleotides corresponds to the position in the
genome -29000.
Figure 8. (A) Determination of estimated KD (KDest) via ligand-detected 1H-1D NMR titration.
(B) RNA-based euclidean distance plot to determine the binding site of P4C11 to s2m. The
structural formular of the ligand is shown in the upper right corner. Internal loop residues are
highlighted in green and loop residues are highlighted in magenta. A threshold of twofold the
standard deviation was used. (C) 1H, 13C HSQC spectra of the aromatic region of s2m Delta
(100 µM) with 0 µM (black) and 500 µM (yellow) P4C11. Resonance assignments are
annotated and the notation of nucleotides corresponds to the position in the genome -29000.
Loop resonances are labeled in magenta and internal loop resonances are labeled in green.
(D) Secondary structure of s2m Delta colored with HSQC-based CSP heatmap (strong: red,
weak; blue) while red corresponds to the maximum CSP detected. (E) Overview of KDest values
of different ligands for s2m SCoV-2 and s2m Delta.
Figure 9. (A) Determination of estimated KD (KDest) via ligand detected 1H-1D NMR titration.
(B) RNA-based euclidean distance plot to determine the binding site of P6E06 to s2m Delta.
The structural formular of the ligand is shown in the middle. Internal loop residues are
highlighted in green and loop residues are highlighted in magenta. A threshold of twofold the
standard deviation was used. (C) 1H, 13C HSQC spectra of the aromatic region of s2m (100 µM)
with 0 µM (black) and 500 µM (yellow) P6E06. Resonance assignments are annotated and the
notation of nucleotides corresponds to the position in the genome -29000. Loop resonances
are labeled in magenta and internal loop resonances are labeled in green. (D) Secondary
structure of s2m Delta colored with HSQC based CSP heatmap (strong: red, weak; blue) while
red corresponds to the maximum CSP detected.
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published online April 2, 2024RNA
Tobias Matzel, Maria Wirtz Martin, Alexander Herr, et al.
(s2m) from the Delta variant of SARS-CoV-2
NMR characterization and ligand binding site of the stem loop 2 motif
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