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Stacking of G-quadruplexes: NMR structure of a G-rich oligonucleotide with potential anti-HIV and anticancer activity†

G-rich oligonucleotides T30695 (or T30923), with the sequence of (GGGT)4, and T40214, with the sequence of (GGGC)4, have been reported to exhibit anti-HIV and anticancer activity. Here we report on the structure of a dimeric G-quadruplex adopted by a derivative of these sequences in K+ solution. It comprises two identical propeller-type parallel-stranded G-quadruplex subunits each containing three G-tetrad layers that are stacked via the 5′-5′ interface. We demonstrated control over the stacking of the two monomeric subunits by sequence modifications. Our analysis of possible structures at the stacking interface provides a general principle for stacking of G-quadruplexes, which could have implications for the assembly and recognition of higher-order G-quadruplex structures.

A number of synthetic G-rich DNA oligonucleotides have been reported to exhibit anticoagulant (32,33), anti-HIV (34)(35)(36)(37)(38)(39)(40) and anticancer activities (14,(41)(42)(43)(44). These include T30695 (or T30923), with the sequence of (GGGT) 4 (41,42) and T40214, with the sequence of (GGGC) 4 (43), which have been reported as inhibitors of HIV integrase (IN), a viral enzyme responsible for the integration of viral DNA into the host-cell genome (45). In addition, T40214 has also been reported to possess anticancer properties through the inhibition of STAT3 (36). However, there remains ambiguity in the literature regarding the structure adopted by these sequences. Jing et al. (41) studied T30695 in K + solution by NMR spectroscopy and reported an antiparallel-stranded G-quadruplex, which consists of two GGGG tetrads and a GTGT tetrad, where all three loops are edgewise. However, spectral overlap could have hampered the structural interpretation, as the authors reported contrasting CD spectra of T30695 with a positive peak at 260 nm (41,42), which is a signature of parallel-stranded G-quadruplexes (23)(24)(25). This structure was thought to be an exception to the rules regarding CD spectra of G-quadruplexes (46). A related G-rich oligomer HIV IN inhibitor d(GGGGTGGGAGGAGGGT), named 93del, was later revealed to consist of an interlocked dimeric G-quadruplex with two parallel-stranded subunits (14), and prompted questions on the stoichiometry and strand orientations of T30695. Studies from different groups have concurred that single-residue loop is most favorable to adopt the double-chain-reversal configuration (47)(48)(49)(50)(51). Moreover, mass spectrometry studies have detected dimer formation for T30695 and similar oligonucleotides (12,52), and a propeller-type parallel-stranded G-quadruplex of T30695 was found to be stable in a recent molecular dynamics simulations study (53).
In this work, we present a NMR structural study on the G-quadruplex formed by T30695 and its derivative sequences in K + solution. We could significantly improve NMR spectra with a single guanine-to-inosine substitution. With unambiguous resonance assignments and stoichiometry determination, we showed that this sequence adopts a dimeric G-quadruplex, formed by the stacking of two propeller-type parallel-stranded G-quadruplex subunits at their 5 0 -ends. We present an analysis of possible structures at the stacking interface, as well as the conditions controlling this stacking.

Sample preparation
Unlabeled and site-specific labelled DNA oligonucleotides were chemically prepared using products from Glen Research and Cambridge Isotope Laboratories. Samples were purified following Glen Research protocol and then were dialyzed successively against KCl solution and water. DNA oligonucleodites were dissolved in solution containing 70 mM potassium chloride and 20 mM potassium phosphate (pH 7.0). DNA concentration was expressed in strand molarity using a nearest-neighbor approximation for the absorption coefficients of the unfolded species (54).

Disintegration assay
The disintegration assay was performed essentially as described previously (14,55). The reaction mixture contained 20 mM HEPES (pH 7.5), 10 mM MnCl 2 , 30 mM NaCl, 10 mM DTT, 0.05% Nonidet-P40, 600 nM HIV-1 integrase, 200 nM DB-Y1. The DNA substrate DB-Y1 (5 0 -TGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTT GCGCC) used in the reaction was labeled with 6-FAM TM fluorescein at the 5 0 -end (1 st BASE, Singapore). After incubating at 37 C in 2 h, the reaction mixture was mixed with equal volume of 99.5% deionized formamide (Sigma), 10 mM EDTA (pH 8.0) and heated at 90 C for 3 min. For inhibition test, the inhibitors were added into the mixture and incubated in 30 min before adding DB-Y1. The reaction products were monitored by electrophoresis on 20% polyacrylamide denaturing gels with 7 M urea.

Circular dichroism
Circular dichroism CD spectra were recorded on a Jasco-815 spectropolarimeter using 1-cm path-length quartz cuvette in a reaction volume of 600 ml at 20 C. Scans from 220 to 320 nm were performed with 200 nm/ min, 1-nm pitch and 1-nm bandwidth. DNA concentration was 6 mM.

NMR spectroscopy
NMR experiments were performed on 600 and 700 MHz NMR Bruker spectrometers equipped with a cryoprobe at 25 C, unless otherwise specified. Guanine resonances were unambiguously assigned by using site-specific low-level 15 N labeling (56), site-specific 1 H-to-2 H substitutions (57), and through-bond correlations at natural abundance (58). Spectra assignments were completed by COSY, TOCSY, HSQC and NOESY experiments. Interproton distances were measured by NOESY experiments at various mixing times.

Structure calculation
Inter-proton distances for J19 (Table 1) were classified based on NOESY experiments performed in H 2 O (mixing time, 200 ms) and D 2 O (mixing times, 100, 200 and 300 ms), and were duplicated for the two monomers. In vacuum, models were generated using the XPLOR-NIH program (59) in two general steps: (i) distance geometry simulated annealing and (ii) distance-restrained molecular dynamics refinement.
Hydrogen-bond restraints, inter-proton distance restraints, dihedral restraints, planarity restraints, and non-crystallographic Table 1. DNA sequences used for structural study Name Sequence symmetry restraints were imposed during structure calculations. Ten lowest-energy structures were then subjected to distance-restrained molecular dynamics refinement in explicit solvent using the AMBER program (60), in which the dihedral, planarity and noncrystallographic symmetry restraints were removed. Detailed procedures are described in the Supplementary Data. Structures were displayed using the PyMOL program (61).

Data deposition
The coordinates of ten lowest-energy d(GIGTGGGTGG GTGGGT) (J19) dimeric G-quadruplex structures upon distance-restrained molecular dynamics refinement in explicit solvent have been deposited in the Protein Data Bank (accession code 2LE6).

RESULTS AND DISCUSSION
G-quadruplexes of T30695 and T40214 in K + solution NMR spectra including 1D spectra ( Figure 1) and 2D NOESY (Supplementary Figure S1 and Supplementary Data) indicated that the T30695 and T40214 sequences ( Table 1) form similar G-quadruplex structures in K + solution. In our hands, T30695 showed similar 1D imino proton spectra to the previously reported ones by Jing et al. (41), but very different 2D NOESY spectra. In particular, we did not observe strong intraresidue H8-H1 0 NOE cross-peaks for guanines, which would be indicative of syn glycosidic conformations. T30695 and T40214 exhibit similar CD spectra with a positive band at 260 nm ( Figure 2), a characteristic signature of parallelstranded G-quadruplexes (23).
A single guanine-to-inosine substitution improves NMR spectra of T30695 in K + solution Proton resonances in the NMR spectra of T30695 were heavily overlapped (Figure 1b; Supplementary Figure S1b and Supplementary Data), presumably due to the quasisymmetry of the structure arising from the repetitive nature of the sequence, which could have hampered detailed structural analysis by NMR (41). We found that a single guanine-to-inosine substitution at position 2 of T30695 greatly improved the spectral resolution ( Figure 1c; Supplementary Figure S1c and Supplementary Data). This modification, resulting in the sequence GIGT(GGGT) 3 (henceforth designated J19), most likely broke up some symmetry of the structure (see NMR data and structure below) and, consequently, remove the degeneracy of the NMR spectra. NMR spectra of J19 in K + solution (Figure 1c) showed eleven sharp guanine imino protons at 10.8-11.6 ppm and an inosine imino proton at 13.8 ppm, corresponding to three G-tetrads. Similar spectral characteristics, including chemical shifts and NOESY patterns (Figure 1b; Supplementary Figure S1b and Supplementary Data), indicated the same conformation for J19 and T30695 (and T40214). These were corroborated by their similar CD spectra, which showed a positive band at 260 nm ( Figure 2), consistent with the formation of parallelstranded G-quadruplexes (23). The correspondence between J19, T30695 and T40214 was also observed in gel electrophoresis experiments (see below).

J19 forms a stacked dimeric G-quadruplex in K + solution
Guanine imino protons of J19 were unambiguously assigned using site-specific 2% 15 N-labeled samples (56) (Figure 3a). The downfield-shifted peak at 13.8 ppm was assigned to the imino proton of I2 (62). Guanine H8 protons were assigned by through-bond correlations to the respective imino protons via 13 C5 at natural abundance (58) (Figure 3b). Some of these assignments were independently confirmed by site-specific 2 H substitutions at the H8 position (57) (Supplementary Figure S2 and Supplementary Data). Assignments of other protons were assisted by through-bond (COSY, TOCSY and HSQC), and through-space (NOESY) correlations (31). The H8/H6-H1 0 sequential NOE connectivities could be observed from G1 to G3, G5 to G7, G9 to G11 and G13 to T16, while interruptions were observed at T4, T8 and T12 (Figure 4a), which adopt the double-chain-reversal configurations (see structure below). The moderate intensity of intraresidue H8/H6-H1 0 NOEs indicated that all residues adopt anti glycosidic conformations.  Characteristic NOE patterns between imino and H8 protons pointed to the formation of three tetrads: G1 G5G9G13, I2G6G10G14 and G3G7G11G15 (Figure 4b and c). These tetrad alignments, combined with other data (see below), indicated that J19 forms a stacked dimeric G-quadruplex, in which each half of the structure is a propeller-type parallel-stranded G-quadruplex with three tetrad layers and three doublechain-reversal loops (Figure 4d). Data supporting the 5 0 -end stacking and dimeric nature of J19 include: (i) gel electrophoresis showing the migration rate of J19 to be similar to that of the interlocked dimeric G-quadruplex 93del and slower than that of J19-Q2, a monomeric propeller-type G-quadruplex containing three tetrad layers (see below); (ii) solvent-exchange data showing that the imino protons of guanines in the G1G5G9G13 tetrad at the stacking interface are protected from the exchange with D 2 O (Supplementary Figure S3); (iii) the observations of several NOE cross-peaks across the interface between two monomeric subunits (see below); and (iv) the disruption of stacking interaction when additional bases were extended from the 5 0 -end of J19 (see below).
The solution structure of J19 was calculated on the basis of NMR restraints (Table 2), including NOEs at the interface between the two subunits (discussed below), which were imposed as ambiguous distance restraints. Ten lowest-energy structures after distance-restrained molecular dynamics refinement in explicit solvent were superimposed and presented in Figure 5. The core of the G-quadruplex is well converged and there is tight packing across the interface. The hydrogen-bond directionalities of the tetrads are the same within each subunit, but opposite between the two subunits. The thymine bases T4, T8 and T12 project outwards of the core, typical of singlenucleotide double-chain-reversal loops (14).

Structure of the stacking interface
Stacking between G-quadruplexes has been proposed (8-13), but so far there have been limited discussions regarding the structural details at the stacking interface. For a pair of G-quadruplex monomers that stack through planar tetrads, we can envisage a range of isomers, wherein the two subunits are rotated with respect to each other about the common helical axis. We might suppose that certain orientations of the two subunits are most favorable, corresponding to states of lowest energies, which will be determined by many factors, including van der Waals contacts, orbital overlap, ion coordination and steric hindrance. For the current case involving two parallel-stranded G-quadruplexes, one major mode of stacking was observed: the two monomers stack in a 5 0 -5 0 manner, with G1, G5, G9, and G13 from one subunit directly above G9*, G5*, G1* and G13*, respectively, from the other subunit ( Figure 5; Supplementary Figure S4 and Supplementary Data). The sugars from the two tetrads are immediately adjacent to one another (with the faces containing the H1 0 and H4 0 atoms facing each other), the backbones of the two subunits align in a staggered manner, and the five-and six-membered rings of the guanines display partial overlap across the interface (Supplementary Figure S5iii, iii 0 and Supplementary Data). This dimeric arrangement was supported by the observation of NOEs among base and sugar protons of the G1G5G9G13 tetrad, including those between imino-H8 of G5-G5* and G13-G13*, as well as reciprocal NOEs between H8-H1 0 , H1 0 -H1 0 and H1 0 -H3 0 of G1 and G9* (or G9 and G1*). Some of these NOEs were unambiguously confirmed in samples where G(H8) were site-specifically substituted by deuterium ( Figure 6  will be prudent not to preclude them entirely. In a recent RNA G-quadruplex structure consisting of a dimer of dimers, a major mode of stacking was also observed (11). We note that in these two examples, the terminal 5 0 -OH groups of the component strands are positioned furthest away from each other in the major conformation (Supplementary Figure S7 and Supplementary Data), which might explain why this isomer is more stable than the rest.

Patterns of overlap at the interface for different stacking orientations
Various patterns of overlap of the guanine bases at the interface have been observed across a number of G-quadruplex structures involving a pair of tetrads with opposing hydrogen-bond directionalities: (i) maximum overlap of the five-membered rings of guanines (9,14,63,64); (ii) maximum overlap of the six-membered rings of guanines (65,66); and (iii) partial overlap of the five-and sixmembered rings of guanines (67)(68)(69). Comparing against the current case, i.e. pattern (iii) (Supplementary Figure S5 and Supplementary Data), (i) is related by a 25 clockwise rotation of the bottom subunit with respect to the top, while (ii) is related by a 15 anticlockwise rotation of the bottom subunit with respect to the top. Pattern (i) was observed at the interface of the interlocked dimeric G-quadruplex 93del (14), and is typically observed within an antiparallel G-quadruplex. Pattern (ii) was seen in crystal structures of tetrameric quadruplex assemblies where the interface involves the intersection of eight strands, with octad formation at the sandwiching layers immediately adjacent to the interface (65,66). Pattern (iii) was observed across a number of dimeric structures that involve stacking of intramolecular G-quadruplexes (67)(68)(69), including the present case of J19. It is interesting to note that regardless if J19 is twisted into orientation The characteristic guanine imino-H8 cross-peaks for G-tetrads are framed and labeled with the imino proton assignment in the first position and that of the H8 proton in the second position. (c) Specific imino-H8 connectivity pattern around a G-tetrad (G a G b G g G d ) indicated with arrows (connectivity between G d and G a implied). The connectivities observed for G1G5G9G13 (purple), I2G6G10G14 (red) and G3G7G11G15 (green) tetrads are shown below. (d) The schematic structure of J19 dimeric parallel-stranded G-quadruplex.
(i) or (ii), it reverted to (iii) upon unrestrained molecular dynamics simulations in explicit solvent (data not shown).

Control of stacking between G-quadruplexes
Gel electrophoresis, which can provide information on the molecular sizes of G-quadruplexes, was used to examine the possibility of stacking of parallel-stranded G-quadruplex blocks to form dimeric structures. Sequences beginning with G at the 5 0 -end, such as T30695 and J19, were found to favor stacking of G-quadruplexes at this end. The migration of these stacked dimeric G-quadruplexes is comparable to that of the reference interlocked dimeric G-quadruplex 93del (14) (Figure 7), which also consists of six G-tetrad layers. It is conceivable that the addition of terminal non-G residue(s) would reduce the stacking propensity of these G-quadruplexes (8,10,11). Indeed, the extension of two thymines at the 5 0 -end of J19 (giving J19-Q2; Table 3) led to the sequence migrating faster than 93del ( Figure  7). We have shown that this sequence forms in a similar condition a propeller-type G-quadruplex with three G-tetrad layers (unpublished results). The extension of three thymines instead of two only marginally increased the mobility of the structure (Supplementary Figure S8 and Supplementary Data). On the other hand, sequences with only one additional T at the 5 0 -end migrated faster than the original sequences (T30695 and J19), but slower  Restraints were duplicated for the two monomers, and include those within each subunit, as well as those at the interface between the two subunits.
c These distance restraints were imposed as ambiguous restraints and specified with the sum-averaging option.
than those containing two thymines (Figure 7). There could be equilibrium between the monomeric and stacked dimeric forms, resulting in the intermediate migra-  Figure S10) results were obtained for T40214, consistent with the formation of a similar stacked dimeric G-quadruplex for this sequence.

HIV-1 IN inhibition activity of various G-rich sequences
In order to probe if the various G-rich oligonucleotides retain anti-HIV IN activity, we performed assays on the reverse 'disintegration' reaction by HIV- 1 IN (14,55). The results are presented in Figure 8. T30695, J19 and their derivative sequences with thymines at the 3 0 -end exhibited comparable inhibition activity as 93del, whereas derivative sequences containing thymines at the 5 0 -end were somewhat less active, which might be attributed to their lower potentials to form stacked dimeric structures.
Combined with other studies (14,70), this work suggested dimeric parallel-stranded G-quadruplexes comprising a total of six G-tetrad layers as potential HIV-1 IN inhibitors. Note however that the conclusion from the assay would be more qualitative than quantitative, as the concentrations here are in the micromolar range, which are around two orders of magnitude larger than the reported IC 50 for the inhibition activity of T30695 against HIV-1 IN (71).

CONCLUSION
A DNA sequence derived from the oligonucleotides T30695 and T40214 was shown to adopt a dimeric G-quadruplex, formed by the stacking of two identical propeller-type parallel-stranded G-quadruplex subunits at their 5 0 -ends. The structure of the stacking interface was analyzed in detail. We showed that stacking interaction between the two subunits was disfavored by extension of non-G residues from the terminals. Figure 6. Determination of the stacking pattern of two J19 subunits. Imino-H8 proton region of NOESY spectra (mixing time, 400 ms) of (a) J19 and (b-d) modified sequences with site-specific 2 H substitution at position H8 of (b) G9, (c) G13 and (d) both G9/G13 are shown. The peaks involving substituted guanines (framed) are missing for modified sequences. The inter-subunit NOE, whose intensity is significantly decreased for the G13-substituted sequence (marked with asterisk on the projection), is framed in red.     T GGG T GGG T GGG T  T30695-Q1 T  GGG T GGG T GGG T GGG T  T30695-Q2 TT  GGG T GGG T GGG T GGG T  T30695-Q3 TTT GGG T GGG T GGG T GGG T  T30695-Q4  GGG T GGG T GGG T GGG TT  T30695-Q5 TT  GGG T GGG T GGG T GGG  T30695-Q6 TT  GGG T GGG T GGG T GGG TT  T30695-Q7 TT  GGG T GGG T GGG T GGG TTT  J19  GIG  T GGG T GGG T GGG T  J19-Q1  T  GIG  T GGG T GGG T