Bulges in left-handed G-quadruplexes

Abstract G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures.

Bulges are important structural elements of G4s, which form as protrusions of bases from the G-tetrad core. Bulges occur due to discontinuities (presence of non-guanine bases) in one or more G-tracts of the G4 forming sequences. Nearly a third of the experimentally detected G4s in the human genome (over 200 000 out of 700 000) could contain bulges (36). Unlike loops which connect two corners of the G-tetrad core, bulges connect two neighbouring guanines of the same G-tract in the G-tetrad core (37)(38)(39)(40). Allowing the presence of bulges in the genomic search increases the number of putative G4 sequence drastically, therefore expanding the library of putative G4-forming sequences (39). Bulges are also important in the context of duplexes, as they can be formed due to mismatches and thus get involved in various interactions with other nucleic acids and proteins (41,42) and be implicated in cellular processes (43)(44)(45).
Similar to the B-DNA form (right-handed) and Z-DNA form (left-handed) for a duplex DNA, the backbone progression of G4 DNA is also capable of adopting both right-and left-handed configurations (46). Right-handed G4 structures are known for decades and have been studied extensively (47,48). Left-handed G4s, on the other hand, were recently discovered and remain rather unexplored. Left-handed G4s are characterized by the anticlockwise rotation of the backbone in its helical progression. They have several unique and distinct structural features, such as: (i) ability to form stable four-layered and bi-layered dimeric structures, (ii) stacking of thymine loops with the outer tetrads (T-capping) and (iii) the local sugar orientation of each base is almost perpendicular to the overall backbone progression giving rise to the 'twisted' backbone (46,49). Note that these structures are formed by natural DNA, which are to be distinguished from the mirror-image G4 structures formed by enantiomeric L-DNA (50,51). However, the circular dichroism (CD) characteristics of lefthanded G4s are noticeably similar to that of L-DNA due to their resemblance in base stacking orientation (50,52). Interestingly, the CD spectrum of a new DNA conformation formed by G 4 C 2 repeats associated with neurodegenerative diseases exhibits similar features, although the structure has not been resolved yet (53). Characterization of intrinsic fluorescence of DNA secondary structures demonstrated enhanced fluorescence of a left-handed G4 (54). Several families of G4 ligands were reported to exhibit enantioselectivity towards right-and left-handed G4s (55,56), while another can induce right-handed G4 conformation from a sequence favouring left-handed G4 (57).
A study on the impact of sequences to the formation of left-handed G4s revealed that the 12-nt GTGGTGGTGG TG motif (previously named Block2Δ (49) and termed as LHG4motif in this paper) was a minimal sequence capable of forming a dimeric left-handed G4 on its own and convert certain adjacent G-rich sequences, such as (GGT) 4 --a sequence with four G 2 tracts forming a right-handed parallel G4 on its own--and several other derivatives to lefthanded G4s (Supplementary Figure S1) (49). LHG4motif was also shown to convert an adjacent antiparallel G4 to a right-handed parallel G4 (Supplementary Figure S1) (49,58). Bioinformatic search revealed high abundance of LHG4motif in the human genome, with over 10 000 hits, exhibiting two-order of magnitude enrichment compared to random occurrence of a 12-nt sequence (49). In this study, to understand the bulge formation in left-handed G4s, LHG4motif was attached to G-rich sequences with the potential of forming one or multiple bulges. We showed that LHG4motif could convert several adjacent sequences to left-handed G4s, including those which were unstructured on their own. We determined the crystal structure of two left-handed G4s containing one and two bulges, respectively. We have also solved the NMR solution structure of a left-handed G4 containing three bulges. Structural analysis revealed differences between bulges in right-and left-handed G4s. The high abundance of LHG4motif in the genome and the feasibility of bulged left-handed G4s driven by this motif can increase the chance of left-handed G4 formation in biological systems. Broadening our understanding of left-handed G4s should be useful for studying their biological functions, as well as their applications in engineering DNA nano-structures.

Sample preparation
Unlabelled DNA oligonucleotides were purchased from IDT with standard desalting purification in the scales ranging from 100 nmol to 1 mol. Sample purity was measured with ESI-MS and was >99%. The site-specific labelled DNA oligonucleotides were chemically synthesized on an ABI 394 DNA synthesizer using reagents from Glen Research and Cambridge Isotope Laboratories. They were purified following the protocol of Glen Research (https://www.glenresearch.com/glen-pakdna-purification-cartridge.html). The purified DNA samples were further dialyzed against water, 25 mM KCl and water successively. The dialyzed oligonucleotides were frozen and lyophilized. The DNA samples (concentration, 0.1−1 mM) were dissolved in a buffer containing 70 mM KCl and 20 mM KPi (pH 7), 10% D 2 O, 20 M DSS. The DNA concentration was measured with UV absorption and expressed in strand molarity using the nearest neighbour approximation for the 260 nm molar extinction coefficient of the unfolded species. DNA samples were annealed by heating at 95 • C for 5 mins followed by cooling down slowly to room temperature before subjected to any measurement.

Circular dichroism
Circular dichroism (CD) spectra were recorded on a JASCO-815 spectropolarimeter using 1-cm path length quartz cuvettes at 25 • C. Scans were performed from 220 to 320 nm wavelength with a scanning speed of 100 nm/min, 1-nm data pitch, 2-nm bandwidth and 2 s digital integration time (DIT). Ten accumulations were obtained for each measurement, the spectral contribution of the buffer was subtracted using baseline correction, and data were zerocorrected at 320 nm. DNA samples with concentrations of 3-5 M were dissolved in a buffer containing 70 mM KCl, 20 mM KPi (pH 7), 10% D 2 O, 20 M DSS. Molar ellipticity of CD spectra was calculated and reported using the DNA concentration derived from the sample absorbance at 260 nm wavelength and the sample extinction coefficient calculated at 260 nm using nearest neighbour approximation for the unfolded sequence.

UV melting
UV melting experiments were conducted on a JASCO V-650 spectrophotometer. 3-5 M DNA samples were taken in a cuvette of a pathlength of 1-cm. UV absorption was measured at 295 and 320 nm wavelength at every 0.5 • C between 20 and 90 • C. Both the heating and cooling were performed at a rate of 0.1 • C/min to allow slow unfolding and folding of the DNA, minimizing hysteresis. The data collected at 295 nm were subtracted from those at 320 nm for background correction and further normalized. The melting temperatures (T m ) were determined from the normalized melting curves where the DNA folded fraction was 50%.

NMR spectroscopy
NMR experiments were performed on a Bruker spectrometer operating at 600 MHz at 25 • C. 0.1−1.5 mM DNA samples dissolved in 70 mM KCl, 20 mM KPi (pH 7), 10% D 2 O, 20 M DSS were used for NMR measurements. Assignments of the imino protons of guanine residues were obtained by 15 N-filtered experiments using 2% site-specific labelled samples. Assignments of guanine aromatic protons was obtained via long-range through-bond correlation between imino and aromatic protons. Assignments of other protons were determined based on through-bond (TOCSY/COSY) and through-space correlation experiments. Spectra analyses were performed using the Topspin 3.5 (Bruker) and SPARKY 3.1 (59) software.
Planarity restraints. Planarity restraints were used for the Distance-geometry simulated annealing. Initial extended conformation of 3xBulge-LHG4motif sequence was generated using the XPLOR-NIH (60) program by supplying the available standard nucleic acid topology and parameter tables. Each system was then subjected to distance-geometry simulated annealing by incorporating distance, dihedral, hydrogen bond and planarity restraints. One hundred structures were generated and subjected to further refinement.
Distance-restrained molecular dynamics refinement. The 100 structures obtained from the simulated annealing step were refined with a distance-restrained molecular dynamics protocol incorporating all distance restraints. The system was heated from 300 to 1000 K in 14 ps and allowed to equilibrate for 6 ps, during which force constants for the distance restraints were kept at 2 kcal mol −1Å−2 . The force constants for non-exchangeable proton and exchangeable proton restraints were then increased to 16 and 8 kcal mol −1Å−2 , respectively, in 20 ps before another equilibration at 1000 K for 50 ps. Next, the system was cooled down to 300 K in 42 ps, after which an equilibration was performed for 18 ps. The coordinates were saved every 0.5 ps during the last 10.0 ps and averaged. The average structure obtained was then subjected to minimization until the gradient of energy was less than 0.1 kcal mol −1 . Dihedral (50 kcal mol −1 rad −2 ) and planarity (1 kcal mol −1Å−2 for tetrads) restraints were maintained throughout the course of refinement. Ten lowest-energy structures were generated.

Crystallization
DNA samples of 1xBulge-LHG4motif-TT and 2xBulge-LHG4motif-TT were prepared in 100 mM potassium cacodylate buffer (pH 7) at a concentration of ∼0.8 mM. The samples were annealed by heating at 95 • C for 5 min followed by slowly cooling down to room temperature. Initial screening for crystallization conditions were done at 24 • C using Natrix 1 and 2 sets of reagents (Hampton Research) in a 96-well sitting drop vapor diffusion setup at 1:1 sample to reagent proportions with the help of mosquito ® LCP (ttplabtech). Both the sequences produced crystals under multiple conditions. Crystals were flash frozen in liquid nitrogen before data collection. For 1xBulge-LHG4motif-TT the crystals grown in 0.08 M potassium chloride, 0.04 M sodium cacodylate trihydrate pH 6.0, 55% (v/v)-2-methyl-2,4-pentanediol and 0.012 M spermine tetrahydrochloride were seen to produce the highest resolution diffraction. For the 2xBulge-LHG4motif-TT, the crystals obtained in 0.08 M potassium chloride, 0.04 M sodium cacodylate trihydrate pH 7.0, 60% (v/v)-2-methyl-2,4-pentanediol and 0.012 M spermine tetrahydrochloride were chosen for the same reason.

X-ray diffraction data collection and refinement
Crystal diffraction data were collected at PROXIMA 2 beamline for 1xBulge-LHG4motif-TT and at PROXIMA 1 beamline for 2xBulge-LHG4motif-TT of SOLEIL synchrotron, France. Native datasets were collected over 360 • rotation ranges at 0.1 • oscillation range. Data were processed in P2 1 space group using the XDS software package (61). Molecular replacement was done using the Z-G4 crystal structure (PDB ID: 4U5M) as a search model to obtain initial phases. One and two copies per unit cell were found respectively for 1xBulge-LHG4motif-TT and 2xBulge-LHG4motif-TT. The model was iteratively built through cycles of refinement using phenix (62,63) or Ref-mac5 (ccp4) and manual rebuilding in coot (64,65). Spermines were fitted into the structures using LigandFit function in Phenix package.

LHG4motif converts various adjacent sequences to lefthanded G4s
Previously, LHG4motif was shown to convert an adjacent sequence (TGGTGGTGGTGG) or (GGTGGTGGTGG) containing four G 2 tracts separated by single thymines to a left-handed G4 ((46,49) and data not shown). To explore the bulge formation in left-handed G4s, additional thymines were added in these G 2 tracts. Four adjacent sequences which potentially can form one or multiple bulges (Supplementary Table S1) were designed as potential bulgeforming motifs wherein thymines were inserted in the Gtracts as the following: (i) second G-tract (T5), named 1xBulge; (ii) second and third G-tracts (T5, T9), named 2xBulge; (iii) second, third and fourth G-tracts (T5, T9, T13), named 3xBulge and (iv) first, second, third and fourth G-tract (T2, T6, T10, T14), named 4xBulge. It is to be noted that this nomenclature does not indicate the corresponding bulge formation in these sequences individually. The first sequence on its own formed an unidentified structure, as indicated by six peaks observed in the imino proton region (10-12 ppm) of the one-dimensional (1D) 1 H NMR spectrum; all the other three sequences were essentially unstructured, as no peaks were observed in the imino proton region (Supplementary Figure S2A). The results were also supported by the CD spectroscopy data (Supplementary Figure S2B). These four bulge-forming motifs were attached with LHG4motif at their 3 -end via a single thymine linker, resulting in sequences named as 1xBulge-LHG4motif, 2xBulge-LHG4motif, 3xBulge-LHG4motif and 4xBulge-LHG4motif, respectively ( Figure 1A). Upon combining with LHG4motif, the first three sequences exhibited the NMR and CD spectral signatures of left-handed G4s: 1D 1 H NMR spectra displayed sixteen major imino protons at 10.5-12.0 ppm regrouped into two regions with eight peaks each ( Figure 1B); CD spectra showed positive and negative peaks at 240 and 265 nm, respectively ( Figure 1C). The fourth sequence, 4xBulge-LHG4motif, remained unstructured as indicated by NMR and CD spectra ( Figure  1B, C). We also investigated the structural driving ability of LHG4motif when it is attached to the 5 -end of the designed sequences (Supplementary Table S2). The NMR and CD results suggested that two sequences, LHG4motif-1xBulge and LHG4motif-3xBulge, but not the sequence LHG4motif-2xBulge, were able to form left-handed G4 structures (Supplementary Figure S3). Thus, the structural driving ability of LHG4motif was context dependent and appeared to be stronger when attached to the 3 -end rather than the 5end of a G-rich sequence. Given the previous observation that a small disruption to the minimal left-handed motif (LHG4motif), such as a single thymine addition in a loop, could abolish the left-handed G4 structure (49), we always kept the LHG4motif intact in this study.
Crystal structure of a left-handed G4 with one bulge X-ray crystallography was employed to investigate the structure of 1xBulge-LHG4motif. The sequence 1xBulge-LHG4motif-TT (Supplementary Table S3) with two additional thymines (TT) at the 3 -end was crystallized. Addition of 3 -end thymines (TT) has improved the crystal packing to provide high-quality X-ray diffraction data. The NMR and CD spectra of 1xBulge-LHG4motif and 1xBulge-LHG4motif-TT displayed highly similar spectral characteristics (Supplementary Figure S4), indicating the formation of the same structural conformation by these two sequences. 1xBulge-LHG4motif-TT was found to crystallize in the P2 1 space group with high-resolution diffraction to 1.18Å (Table 1). The model fits the electron density excellently as shown in Figure 2A. The crystal structure of 1xBulge-LHG4motif-TT features a four-layered unimolecular G4 structure separated into two blocks connected by a thymine linker. Each block has parallel left-handed backbone progression similar to the previously reported lefthanded G4 structure of Z-G4 (46). The 5 -5 stacking interface between guanine bases in successive blocks is in the opposite-polarity mode with partial 5/6-membered ring overlap. The thymines capping of the outer tetrads of the G4 structure (T-capping)--a distinctive feature of left-handed G4s--are also conserved in this structure (46). The upper block has three T-capping residues, while the lower block has four. The thymine residue T5 forming a bulge between the G4 and G6 stacked guanines, projecting out of the Gtetrad core, yielding the first left-handed G4 structure accommodating a bulge ( Figure 2B-D). The positions of the potassium ions in between the Gtetrads are consistent with those of Z-G4 and other reported left-handed G4 structures (46,49). The overhang additional 3 -thymines (TT) stack on the capping thymines (Supplementary Figure S5A), while a spermine molecule (from the crystallization solution) stacks over the outer tetrad in each block squeezing itself between the capping thymines (Figure 2). The accommodation of spermine stacking over the tetrads despite the presence of capping thymines establishes the possibility of ligands binding to left-handed G4s.

Crystal structure of a left-handed G4 with two bulges
The sequence 2xBulge-LHG4motif-TT with two thymines at the 3 -end was crystallized in the P2 1 space group with two molecules in the asymmetric unit ( Supplementary Fig-ure S6). The two molecules were related by a translation. Hence, the crystals of 2xBulge-LHG4motif-TT and 1xBulge-LHG4motif-TT were packed in a similar fashion. The overall structures are noticeably close to each other with an rmsd of 0.112Å for all non-H superimposed atoms excluding the bulges. Both crystal structures feature fourlayered G4s separated into two blocks connected by one thymine linker. In case of 2xBulge-LHG4motif-TT, two thymine residues T5 and T9 are projected outward between guanines G4 and G6 as well as G8 and G10, respectively, in the upper block ( Figure 3). The overhang 3thymines (TT) stack on the capping thymines (Supplementary Figure S5B). A spermine molecule (from the crystallization solution) stacks over the outer tetrad in each block.
The crystal structure of 2xBulge-LHG4motif-TT also displays three potassium ions coordinated in the central channel of the G4. The two molecules (A and B) in the asymmetric unit are closely similar with an rmsd of 0.132Å for all non-H superimposed atoms. The molecule B was observed to contain one additional potassium ion in close proximity to thymine T22 and one of the spermine molecule (Supplementary Figure S6). In addition, there is a sodium ion nearby the second bulge T9 in molecule B that interacts with O2 and O4' of the bulge residue, potentially stabilizing the structure. The sodium ion was modelled to best fit the map, while other molecules such as water or potassium generated significant difference peaks in the Fo -Fc map. The data collection and refinement statistics of the crystal structure 2xBulge-LHG4motif-TT are summarised in Table 1.

NMR solution structure of a left-handed G4 with three bulges
As we were unable to obtain good crystals for 3xBulge-LHG4motif and its derivatives, we proceeded to further characterize the structure of the 3xBulge-LHG4motif sequence by NMR spectroscopy. The observation of sixteen imino proton peaks supported the formation of a fourlayered left-handed G4 by this sequence. These sixteen guanine imino protons were assigned unambiguously by the site-specific 2% 15 Figure S10). The glycosidic conformations of all the participating guanine residues were found to be anti, deduced from the intensities of intra-residue H8-H1' cross-peaks in a NOESY spectrum performed in D 2 O at a mixing time of 100 ms, which showed similar moderate intensity for all residues. NOE sequential walk between H8 (n) -H1' (n) -H8 (n+1/n+2) observed in a NOESY spectrum performed in D 2 O at a mixing time of 300 ms (Supplementary Figure S11) revealed the stacking pattern of the Gtetrads, as well as the thymine loop and bulge arrangements (see caption of Figure S11). The structural schematics of 3xBulge-LHG4motif is proposed in Figure 4D.
Finally, the NMR solution structure of 3xBulge-LHG4motif was computed based on NMR constraints obtained from analyses of the NOESY spectra. The structure calculation provided 100 structures based on the distance, angle, hydrogen-bond and planarity constraints, out of which 10 lowest-energy structures were superimposed and presented in Figure 4 with the statistics of the computed structure shown in Table 2. The solution structure of 3xBulge-LHG4motif revealed a very similar structural characteristics as 1xBulge-LHG4motif and 2xBulge-LHG4motif with a four-layered G4 comprising of two blocks connected by a thymine linker. Both the blocks, the lower LHG4motif block and the upper block with three bulges, feature parallel left-handed G4 backbone progression. The three bulges T5, T9 and T13 are projecting out between the two stacked guanines in the upper block, while the thymine loops capping at the two ends distinctive to left-handed G4 structures are maintained (Supplementary Figure S12). The three bulges (T5, T9 and T13) and the thymine linker (T15) are not well defined due to lack of constraints. These residues could also be dynamic. While the glycosidic conformations of these residues were not imposed in the structure calculation, diverse conformations were observed among the ensemble of 10 lowest-energy structures, with the majority adopting an anti-conformation and a few in syn-conformation. The LHG4motif block adopts the same structure as reported previously including the thymine loops capping the bottom tetrad.

The destabilizing effect of additional bulges: left-handed versus right-handed G4s
Previously, simultaneous accommodation of up to three bulges in different positions of a right-handed G4 was demonstrated (39). In that study, G4 structures with more than three bulges could not be formed, coincident with our current study where 4xBulge-LHG4motif was unable to form any stable structure. The comparison between the costs in the thermal stability upon increasing the number of bulges is summarized (Supplementary Table S4) (39). In the right-handed G4 system, on average, the melting temperature decreased by ∼17 • C upon addition of a second bulge in a different G-tract with respect to the first bulge (39). It further decreased by ∼21 • C due to the addition of a third bulge in a different G-tract (39). Comparatively, in the current left-handed G4 system, the melting temperature decreased by ∼16 • C when the second bulge (T9) was introduced in 2xBulge-LHG4motif in addition to the first one (T5) in 1xBulge-LHG4motif. The stability was further decreased by ∼12 • C when the third bulge was added in 3xBulge-LHG4motif (Supplementary Figure S13). Taken together, we found a diminished destabilizing effect of additional bulges in left-handed G4s compared to that of righthanded G4s. The comparison with no-bulge left-handed G4 structures was not attempted due to the presence of more than one conformation in the no-bulge construct.

Effect of bulges in altering the backbone dihedral angles in the G-tetrad core: left-handed versus right-handed G4s
The dihedral angles of left-and right-handed G4 backbones were previously compared, showing significant differences between them (46). Here, we compare the same set of stepwise dihedral angles alterations (ε, ζ , α+1, β+1 and γ +1) upon addition of bulges. Specifically, we quantified the dihedral angle adjustments on the core guanine residues on the immediate 5 -side of the bulge (termed 'previous residue') as well as the core guanine residue on the immediate 3 -side of the bulge (termed 'next residue') in the systems of left-and right-handed G4s. The dihedral angle alterations are compared with the standard set of dihedral angle values of parallel G4s without bulges obtained from selected crystal structures deposited in PDB for both left-(4U5M, 6GZ6) (46,49) and right-handed systems (1KF1, 244D, 352D) (8,67,68).
The comparison results are summarized in Figure 5. The standard dihedral angle values are represented in black open circle, solid red symbols represent the dihedral angle On the other hand, the bulge in a right-handed G4 affected the angles ε and γ +1 for the 'previous residue' by ∼90 • and ∼135 • respectively, as well as the angles ζ , α+1 and γ +1 for the 'next residue' by ∼180 • , ∼135 • and ∼120 • respectively. We conclude that the bulges in left-handed G4s are less disruptive to the neighbouring residues in terms of dihedral angle values than their right-handed counterparts. The data of bulged left-handed G4s were taken from the crystal structures of 1xBulge-LHG4motif-TT and 2xBulge-LHG4motif-TT in this paper, while the bulged right-handed G4 data were taken from the deposited crystal structure 5UA3 (40).

Effect of bulges in altering the sugar puckering in the Gtetrad core: left-handed versus right-handed G4s
The presence of a bulge also affects the sugar puckering of the core guanine residues immediately adjacent to the bulge. Here, we quantified the alteration effect of the sugar puckers of the 'previous residue' and 'next residue' by the standard pseudorotation phase angle parameter (P) ( Figure 6A) (69). Standard values of P were measured using the same set of crystal structures (PDB ID: 4U5M, 6GZ6, 1KF1, 244D and 352D) (8,46,49,67,68) for both left-and right-handed G4 systems. The P values were obtained from 1xBulge-LHG4motif-TT and 2xBulge-LHG4motif-TT structures for left-handed G4s and the crystal structure 5UA3 (40) for a right-handed G4. The summary of the comparison is presented in Figure 6B.
In non-bulged left-handed G4 systems, all the core guanine residues adopt C2'-endo conformations (135 • < P < 180 • ). Upon introduction of a bulge, both the 'previous residue' and 'next residue' adjacent to the bulge do not undergo changes in sugar pucker conformation, staying in C2'-endo conformations. Comparatively, in non-bulged right-handed G4 systems, the majority of the core guanine residues similarly adopt C2'-endo conformations. However, in the presence of a bulge, the 'next residue' sugar pucker conformation was altered to C3'-endo, with the P values around 0 • . This analysis showed that the presence of a bulge in left-handed G4s requires less alteration in terms of sugar puckering compared to a bulge in right-handed G4s.
To complement the pseudorotation phase angle which can be difficult to visualize, we introduced the term zdeviation as an alternative parameter which is defined as the perpendicular distance between the plane containing the atoms C4', O4' and C1' to either C2' or C3' (Supplementary Figure S14A). Analogous comparisons between nonbulged and bulged left-and right-handed G4s are presented (Supplementary Figure S14B). Like for the P values, the only significant change was observed for the C2' z-deviation values of the 'next residue' in right-handed bulged G4 which was negative instead of the positive values for the standard G4s. As expected, both the C3' and C2' z-deviation values of bulged left-handed G4 structures are unchanged from the standard left-handed G4s.

Bulges and loops in left-and right-handed G4s
Outside of the G-tetrad core formed by four G-columns (or G-tracts), the two significant structural elements of a G4 structure are the loops and bulges. By definition, a loop is a nucleotide chain ranging from zero (only a single phosphate group) to several bases that connects two separate G-columns, while a bulge is a nucleotide chain of at least one base between two stacking guanines in the same Gcolumn. In the context of sequences with alternating guanines and thymines, the concepts of loops and bulges might be undistinguishable based on the sequences alone, whereby the structures are required to recognize them. To do that, the stacking guanines are to be identified first. For example, in left-handed G4s the stacking guanines are defined to have a left-handed helical progression with a base rotation magnitude of ∼27 • (46). In this definition, bulges and loops of a G4 structure can be readily distinguished.
This study revealed the detailed similarities and differences between the conformations of single-nucleotide bulges in left-and right-handed parallel G4s. Among the four structural elements (single-nucleotide bulges and loops in left-and right-handed parallel G4s), the loop in lefthanded G4 has the most distinct feature allowing to pack a tight T-cap in an inward direction covering the ends of the G-tetrad core, whilst all the other three elements have outward projections (Supplementary Figure S15). Another distinguishing feature is the local sugar orientation (from 5 to 3 ) on each of the elements. Using the crystal structures of a bulged parallel left-handed G4 (1xBulge-LHG4motif-TT) and a bulged parallel right-handed G4 (PDB ID: 5UA3), we can see that: the local sugar orientations of the left-handed loop and the right-handed bulge are parallel to (same direction of) those of the G-tetrad guanines; while those of the left-handed bulge and the right-handed loop are anti-   (8,46,49,67,68), with the mean and standard deviations denoted; red and green circles indicate the P values of the 'previous residue' and 'next residue' before and after the bulge, respectively (structures of 1xBulge-LHG4motif-TT,2xBulge-LHG4motif-TT and PDB ID: 5UA3) (40).