Antiparallel RNA G-quadruplex Formed by Human Telomere RNA Containing 8-Bromoguanosine

In this study, by combining nuclear magnetic resonance (NMR), circular dichroism (CD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and gel electrophoresis, we report an unusual topological structure of the RNA G-quadruplex motif formed by human telomere RNA r(UAGGGU) containing 8-bromoguanosine. Results showed that the RNA sequence formed an antiparallel tetramolecular G-quadruplex, in which each pair of diagonal strands run in opposite directions. Furthermore, guanosines were observed both in syn- and anti-conformations. In addition, two of these G-quadruplex subunits were found to be stacking on top of each other, forming a dimeric RNA G-quadruplex. Our findings provide a new insight into the behavior of RNA G-quadruplex structures.

conformations of the G-quadruplex structure at the 5′ end of the Rb gene 40 . The incorporation of a methyl or bromine group at the C8 position of dG causes a steric hindrance between the 8-substituent and the ribose ring that favours a syn conformation of 8 m dG or 8 Br dG. This allows the examination of the structure and thermal stability of G-quadruplexes via appropriate substitutions of dG with 8 m dG or 8 Br dG.
Here, we substituted the rG in the sequence of human telomere 6 nt RNA with 8-bromoguanosine (8 Br rG) and examined the resultant structures by using NMR, CD, LC-ESI-MS, and gel electrophoresis. For the first time, we successfully observed a new antiparallel RNA G-quadruplex formed by the modified RNA sequence, and further found that two of these G-quadruplexes as subunits, could stack on top of each other and form a dimeric RNA G-quadruplex. Multimethod approaches, CD, NMR, LC-ESI-MS, and gel electrophoresis, have been used to provide several complementary lines of evidence for the existence of antiparallel RNA G-quadruplexes. The availability of the antiparallel RNA G-quadruplex structure now allows us to reconsider the nature of RNA G-quadruplexes, and suggest that RNA may be more polymorphic than initially assumed. These results provide valuable information to allow further understanding of the structural features of RNA G-quadruplexes.

Results
CD Studies on 6 nt RNA Sequence. We substituted three rGs in each of the human telomere RNA sequences r(UAGGGU) with 8 Br rG to generate ORNs 1-3 (Table 1 and Supplementary Scheme S1 and Figures S1-S8), and examined the resultant structures and their thermal stabilities in a K + solution by CD spectroscopy. The CD spectrum of ORN-1, with a positive band at 265 nm and a negative band at 240 nm, indicated the usual parallel G-quadruplex structure, whereas the CD profile of ORN-3 was characterized as a single strand by the presence of a weak Cotton effect at 275 nm (Fig. 1a). Surprisingly, the ORN-2 showed an unusual CD profile for an RNA G-quadruplex, exhibiting a strong positive band at 295 nm and the negative band at 260 nm, which is typical of an antiparallel G-quadruplex structure (Fig. 1a). The CD spectrum of the unmodified RNA sequence UAGGGU showed a parallel G-quadruplex structure consistent with previously reported (Figure S14) 26 . The thermal stabilities of ORN-1 and 2 were examined using CD melting experiments (Fig. 1b). The Tm value of the parallel G-quadruplex (86.8 °C) suggests that the ORN-1 forms a stable G-quadruplex in presence of 100 mM KCl. Compared with the ORN-1, the melting profile of ORN-2 shows that the antiparallel RNA G-quadruplex is much more stable, with a melting temperature over 90 °C. We further examined the structures and thermal stabilities in a lower KCl concentration. We observed similar CD spectra for ORN-1, ORN-2 and ORN-3 at a concentration of 10 mM ( Figure S15). The ORN-2 formed a stable antiparallel G-quadruplex even in the low concentrated K + solution ( Figure S15). These data suggest that ORN-2 forms a very stable antiparallel G-quadruplex in K + solution. We also examined the influence of cation on the topology. We found that the antiparallel conformation is formed in sodium but is unable to form in lithium ( Figure S16).

NMR Spectral Assignment of ORN-2.
We further investigated the structure of ORNs 1-3 by NMR. In the imino proton region of the 1 H NMR spectrum of ORN-1 obtained in the presence of K + , three well-defined signals were observed between 10.0 and 11.5 ppm (Fig. 2), which were consistent with the symmetrical parallel G-quadruplex showed by CD spectroscopy. ORN-3 did not show the imino signal, meaning that ORN-3 was not able to form a G-quadruplex, which confirms the CD spectral results. Surprisingly, with ORN-2, we observed five imino peaks with an overlap of two peaks, suggesting that the G-quadruplex structure formed by ORN-2 is far more complex than that of ORN-1, in which a twice higher intensity peak presented by two overlapped peaks (marked by asterisk). Since the strands of the UAGGGU parallel G-quadruplex are equivalent, three G-quartet rings, themselves formed by twelve G residues, should display three imino protons, as for ORN-1. The presence of these six imino peaks suggested that the strands of the G-quadruplex were not equivalent in terms of orientation, and folding in an antiparallel to yield unequivalent G residues (Fig. 3a,b). For antiparallel G-quadruplex, there are two possible patterns of G-stem orientation, "ppaa" and "papa" model structures ( Figure S9). We further performed NMR spectral assignment by a combination of hydrogen-deuterium exchange (HDX), homonuclear NOESY, and heteronuclear [ 13 C-1 H] HMBC experiments at natural abundance 24,41,42 . The imino protons assignments were based on the through-bond correlation between H8 and imino protons via 13 C5 at natural abundance of each individual rG ( Figure S10). Due to the missing H8 of the two residues (H is replaced by Br), the imino peaks (peak 3 and 5) that do not have the NOEs with the H8 can be assigned as G4 or G4′ (Fig. 3c). Both of peak 1 and peak 4 have NOEs with G4, suggesting the G residues from peak 1 and 4 are in the same strand with the G4. We can conclude that the two peaks are G3 and G5, with the same reason the peak 2 and 5 are assigned to G3′ and G5′. To accurately detect the peak 1, we assigned the peaks by the assistant of H8/H6-H1′ sequential connectivity ( Figure S11). Through the H8/H6-H1′ sequential connectivity traced in the NOESY spectrum of Figure S11, the H8-H1′ NOEs signal of G3′ could be found. Through the intratetrad cyclic NOEs connectivity (between H1 and H8), we determined the H1 of G5 and assigned the peak1 as G5. The peaks 2, 4 and 5 are further assigned using the same sequential connectivity method, respectively.
The syn-glycosidic conformations of G3′ and G5′ for the antiparallel structure is supported by the observation of the strong presence of intraresidue H8/H1′ NOEs (Fig. 3e,f). Due to the lack of H8 for G4 to detect those conformations, we used amino protons to identify glycosidic conformations. We observed NOE connectivities of both non-hydrogen-bonded and hydrogen-bonded-amino protons of G4 with itself H5′ (Fig. 3e,g), indicating that it forms a syn conformation.
Interestingly, we observed NOE peaks between G5 and G3′ (G5H1-G3′H1′, G3′H1-G5H2′) (Fig. 4a), which can only be explained by the fact that the ORN-2 forms a dimeric G-quadruplex and that the stacking effect brings the G3′, present in one subunit G-quadruplex, close enough to the G5 of the next subunit (Fig. 4b). The cross-peak between G4 imino and the A2′H2 was also observed (Fig. 4a), implying a fact that the A from UA at 5′-teminal of one subunit G-quadruplex was close to the G4-G4′-G4-G4′ tetrad of another subunit G-quadruplex (Fig. 4b). These results further confirmed the dimeric G-quadruplex structure formation. The two such structures are sandwiched on both sides of the two G-tetrads of the respective G-quadruplex subunit. These novel structural features, involving intensive stacking of adjacent G-G-G-G tetrads, should contribute to the stability of the dimer G-quadruplex.
That result is also supported by hydrogen-deuterium exchange (HDX) experiment. G3′ and G5 at the G3′-G5-G3′-G5 tetrad from the inside of dimer structure were more resistant to the solvent exchange in comparison with G3 and G5′ at the G3-G5′-G3-G5′ tetrad from the outside of dimer structure ( Figure S12), suggesting that G residues on stacking top were not easily exchanged due to the protecting effect of tetrad stacking on the inside of structure. In addition, the H1-H8 signals of G3′ and G5 at the G3′-G5-G3′-G5 tetrad showed a higher intensity than that of G3 and G5′ at the G3-G5′-G3-G5′ (Fig. 3d), suggesting that the stacking effect makes G3′ and G5 more close to each other at the tetrad, whereas due to the Br modification, G4 and G4′ do not have the H8 that leads to the absence of H1-H8 signal.
Furthermore, taking into account the presence of 5′-UA and U-3′ flanking nucleotides that might form an additional platform (U-U-U-U or U-A-U-A tetrad) to stabilize the structure, we checked the imino region around 13-14 ppm to find whether U-A Watson-Crick pairs form U-A-U-A tetrad and did not observed the peaks around the region ( Figure S13), indicating that UAUA platform is not constructed from the 5′-UA. We also did not find the NOE peaks of H3-H5, H3-H6 from the U-3′, suggesting that there is no U-tetrad formed by the U-3′ in the structure. 28,43 and DOSY 44,45 have been used for determining the molecular weight of G-quadruplex. Next, we performed the LC-ESI-MS experiment to further analyze the ORN-2. As shown in Fig. 5a, the two peaks (P1 and P2) in LC spectrum were observed with retention times of 15.5 and 19.3 min, respectively. The minor peak P1 corresponds to the molecular weight (MW = 1992.1) of ORN-2, found in the MS spectrum (Fig. 5b). The associated    (Fig. 5c) 43 . Therefore, LC-ESI-MS supports that two G-quadruplexes stack each other to form a dimeric RNA quadruplex. We performed the LC-MS experiments to compare between the unmodified, ORN-1, 2 and 3 sequences. We observed the LC-MS peaks of UAGGGU and ORN-1 corresponding to both the tetramer and monomer. Only monomer peak was found in ORN-3 ( Figure S17). These observations are consistent with the NMR and CD results.

LC-ESI-MS and Gel Electrophoresis studies on ORN-2. Several methods such as LC-ESI-MS
Furthermore, we carried out non-denaturing gel electrophoresis to investigate the structure of ORN-2. Contrary to the control RNA r(UAGGGU), the ORN-2 showed a reduced mobility compared with the reference DNA oligonucleotide dT24 (Fig. 6), indicating a higher order G-quadruplex formation.

Conclusions
Although there is no report that confirmed any RNA sequences can form an antiparallel G-quadruplex conformation, recently, accumulated evidence showed that RNA G-quadruplexes present some antiparallel features (such as the complex loops and the antiparallel characteristic CD signature), suggesting that the RNA maybe more polymorphic than initially assumed. For example, Gabelica and coauthors have reported that the relative intensity of CD band at 295 nm was observed for some RNA sequences, suggestive of a mixed arrangement of the guanine steps in RNA G-quadruplexes 27 . The crystal structure study of RNA aptamer (PDB code: 3IVK) revealed the complex loops of G-quadruplex structure, which were described as "non-parallel" 21 . To find the typical antiparallel RNA G-quadruplex with different orientations in G-stem, Mergny et al. applied the hybrid duplex-quadruplex structure and tried to induce the formation of a RNA antiparallel orientation in G-stem 22 . Furthermore, Defrancq et al. applied a template strategy for constraining RNA to adopt an antiparallel topology, they were unable to form antiparallel RNA 46 . The DNA G-quadruplex was successfully transformed into an antiparallel conformation, unlike the RNA G-quadruplex that was found difficult to form an antiparallel orientation in G-stem. The results suggested that the G-stem structure itself, and not the loops, maybe the key element when it comes to forcing RNA G-quadruplexes into a parallel conformation. Accordingly, the syn/anti-glycosidic conformation of rGs is an important factor in the G-stem orientation of RNA G-quadruplexes 47 . Consequently, stabilizing the syn-glycosidic conformation of rGs in G-stem can drive the formation of antiparallel G-quadruplexes.
In this study, by using the 8-substituted purine 8 Br rG to stabilize the syn conformation, we found that the modified ORN-2 RNA sequence was able to form an antiparallel RNA G-quadruplex, which had never been reported before, and further observed a dimeric RNA G-quadruplex, adopted by stacking two of these G-quadruplexes. Interestingly, the replacement of the central rG in the G-stem results in the formation of an antiparallel G-quadruplex (ORN-2), while the replacement of the first rG in the sequence does not affect the parallel (ORN-1) and the third rG substitution does not induce any folding (ORN-3), suggesting that the rG at different positions of G-stem exerted differential effects on G-quadruplex folding. A recent report also suggested the similar position-dependent effect 48 . The G at central position has more effect on G-quadruplex structure than the G at the other positions. Antiparallel RNA G-quadruplex formation provides new insights into the RNA structure and leads to a better understanding of the essential biological role of RNA molecules.

Methods
RNA Synthesis and Purification. The syntheses of the 8-bromoguanosine-containing RNAs were carried out by phosphoramidite chemistry. All RNAs were synthesized on the 1 μmol scale with an automatic DNA/RNA synthesizer (Nihon Techno Service Co., LTD.). After automated synthesis, the oligonucleotides were detached from the support and deprotected according to the manufacturer's protocol. All oligonucleotides were purified by Reverse phase-HPLC (JASCO). All synthetic procedures and compound characterizations are described in the Supporting Information. RNA sequences are Table 1. Scientific RepoRTs | 7: 6695 | DOI:10.1038/s41598-017-07050-w CD Measurements and Analysis of CD Melting Profile. CD spectra were measured using a Jasco model J-810 CD spectrophotometer. Samples were prepared by heating the oligonucleotides at 90 °C for 5 min and gradually cooling them to room temperature. The melting curves were obtained by monitoring a 265 and 295 nm CD band. Solutions for CD spectra were prepared as 0.3 mL samples at 0.01 and 0.1 mM concentrations in the presence of 10 and 100 mM KCl or in the absence of KCl, 10 mM potassium phosphate buffer (pH 6.8).
NMR Experiments. NMR experiments were performed a BRUKER (AV-400M) magnetic resonance spectrometer. Spectra were recorded at 25 °C. RNA samples (0.6-5.2 mM) were dissolved in 0.15 mL of 90% H 2 O/10% D 2 O, 10 mM potassium phosphate, pH 6.8, 100 mM KCl. Assignments of the proton resonances were initially made by using the methods previously described for assignments of G-quadruplex RNA structures. These assignments were confirmed by comparison of NOESY spectra to those of the related telomere RNA 20,23,26,49,50 . HDX Experiments: RNA samples (5.2 mM in 0.15 mL H 2 O) were lyophilized overnight prior to the HDX experiment. 0.15 mL D 2 O was added to resolve the sample just before measurement. Three samples were prepared, and the 1 H NMR spectra were recorded, respectively. For each sample, the spectrum was recorded after the specific D 2 O exchange time, measurement time is 10 min.