Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats.
Bottom Line: We also found that each G-quadruplex fold is different: A:(G:G:G:G)A hexads were found for (AGG)2A, whereas mixed G:C:G:C tetrads and U-tetrads were observed in the NMR spectra of G(CGG)2C and p(UGG)2U, respectively.Finally, our NMR study highlights the influence of the strand sequence on the structure formed, and the influence of the intracellular environment on the folding.Importantly, we highlight that although potassium ions are prevalent in cells, the structures observed in the HeLa cell extract are not always the same as those prevailing in biophysical studies in the presence of K(+) ions.
Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland.Show MeSH
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Mentions: Another method that we used to assess G-quadruplex formation was NMR spectroscopy. We have used the same RNA solutions for NMR measurements as for CD and UV experiments. Consequently, the very low concentration of NMR samples (0.01 mM) entailed long acquisition times, typically about 14 h, in order to obtain a satisfactory signal-to-noise (S/N) ratio. Additionally, we have recorded 1H NMR spectra at higher concentrations for each RNA sequence, in order to determine the effect of strand concentration on RNA structure. A comparison of 1H NMR spectra of (AGG)2A recorded in solutions containing K+, Na+ or NH4+ ions is depicted in Figure 3A (a–c). A spectrum recorded in K+ solution revealed the presence of two major and one minor forms. However, when the sample concentration was increased (1.14 mM) only one of these two major species was observed (Figure 3Ad). In NMR spectra of both concentrated and diluted samples, a characteristic signal appeared around 10 ppm. Signals in this region were previously observed in the NMR spectra of oligoribonucleotides containing an A:(G:G:G:G):A hexad motif (Figure 4A) and were assigned to guanosine amino protons involved in hydrogen bonding with adenosine (34,51,52). We recorded the 1H-15N HSQC spectrum for concentrated samples, in order to confirm that this high-field signal is due to amino proton (Supplementary Figure S5). The imino and amino protons can be easily distinguished based on the analysis of 15N chemical shifts in 1H-15N HSQC spectra (53). Indeed, the chemical shift of the nitrogen atom corresponding to the proton at 10.14 ppm allowed us to unambiguously attribute this signal to the guanosine amino group. Finally, taking into account the results from the ESI-MS, we assumed that (AGG)2A folds into a dimer of bimolecular G-quadruplexes (Figure 4E), similar to that previously published (51).
Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland.