The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.

• MeSH
• 3' nepřekládaná oblast * MeSH
• chybné párování bází * MeSH
• COVID-19 virologie   MeSH
• genom virový MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• nukleotidové motivy * MeSH
• párování bází MeSH
• Plasmodium falciparum genetika   MeSH
• RNA virová * chemie   genetika   MeSH
• SARS-CoV-2 * genetika   chemie   MeSH
• simulace molekulární dynamiky MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast * MeSH
• RNA virová * MeSH

Triple resonance HCN and HCNCH experiments are reliable methods of establishing sugar-to-base connectivity in the NMR spectra of isotopicaly labeled oligonucleotides. However, with larger molecules the sensitivity of the experiments is drastically reduced due to relaxation processes. Since the polarization transfer between 13C and 15N nuclei relies on rather small heteronuclear coupling constants (11-12 Hz), the long evolution periods (up to 30-40 ms) in the pulse sequences cannot be avoided. Therefore any effort to enhance sensitivity has to concentrate on manipulating the spin system in such a way that the spin-spin relaxation rates would be minimized. In the present paper we analyze the efficiency of the two known approaches of relaxation rate control, namely the use of multiple-quantum coherence (MQ) and of the relaxation interference between chemical shift anisotropy and dipolar relaxation - TROSY. Both theoretical calculations and experimental results suggest that for the sugar moiety (H1'-C1'-N1/9) the MQ approach is clearly preferable. For the base moiety (H6/8-C6/8-N1/9), however, the TROSY shows results superior to the MQ suppression of the dipole-dipole relaxation at moderate magnetic fields (500 MHz) and the sensitivity improvement becomes dramatically more pronounced at very high fields (800 MHz). The pulse schemes of the triple-resonance HCN experiments with sensitivity optimized performance for unambiguous assignments of intra-residual sugar-to-base connectivities combining both approaches are presented.

• MeSH
• kvantová teorie MeSH
• magnetismus MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• nukleosidy chemie   MeSH
• RNA chemická syntéza   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nukleosidy MeSH
• RNA MeSH