Letter pubs.acs.org/JPCL
Nonconventional C−H···F Hydrogen Bonds Support a Tetrad Flip in Modified G‑Quadruplexes Jonathan Dickerhoff and Klaus Weisz* Institute of Biochemistry, Ernst-Moritz-Arndt University Greifswald, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany S Supporting Information *
ABSTRACT: A G-quadruplex adopting a (3 + 1)-hybrid structure was substituted at its 5′-tetrad by 2′-deoxy-2′-fluoroarabinoguanosine (FaraG) analogs. Incorporation of antifavoring FaraG at syn-positions of the 5′-outer tetrad induced a reversal of the tetrad polarity without noticeably compromising the quadruplex stability. This conformational change is shown to be promoted by nonconventional C−H···F hydrogen bonds acting within the anti-FaraG residues.
G
folds.14−16 Here we report on conformational rearrangements with the formation of nonconventional C−H···F hydrogen bonds by selective substitution of FaraG for dG exclusively at nonmatching positions of a (3 + 1)-hybrid quadruplex (Figure 1).17 Seven quadruplexes with all possible substitution patterns involving the three syn-residues within the tetrad at the 5′terminus (5′-tetrad) were evaluated. The impact of substitu-
-quadruplexes (G4) formed by guanine-rich DNA or RNA sequences have been recognized as an important noncanonical structural motif of nucleic acids. On the basis of their involvement in biological processes such as gene expression, replication, or telomere maintenance, quadruplexes are considered promising targets for novel anticancer therapeutics.1,2 In addition, many G4 structures constitute scaffolds for various biosensors, aptamers, or catalytic nucleic acids.3,4 Owing to their considerable structural variability that depends on the sequence context but also on environmental conditions, quadruplexes of many different topologies have been reported in the past.5 In order to mitigate G4 polymorphism for isolating a single species, the selective incorporation of modified nucleotides has proved a successful strategy.6,7 On the other hand, the substitution by guanosine surrogates may also be used to explore the conformational landscape of quadruplexes to provide for novel topologies.8,9 Here, underlying mechanisms usually involve a shift in glycosidic torsion angles by substituting positions with analogs that favor divergent glycosidic conformations. Recently, a concerted transition of all four glycosidic torsion angles within a tetrad could be induced while maintaining the global fold of monomolecular G4s.10−12 Typically, anti and syn conformations can be enforced by guanosine derivatives with appropriate sugar and base modifications, respectively. However, a combination of alternative bases such as 8-oxoguanine and xanthine was also successfully employed for inducing a complete tetrad flip.13 2′-Deoxy-2′-fluoro-riboguanosine (FrG) and 2′-deoxy-2′fluoro-arabinoguanosine (FaraG) are known for their propensity to adopt an anti conformation. On the basis of these conformational preferences, both FrG and FaraG analogs have been exploited for the stabilization of particular quadruplex © XXXX American Chemical Society
Figure 1. Chemical structure of the FaraG and FrG nucleoside (top). Sequence and topology of the (3 + 1)-hybrid quadruplex showing a 5′tetrad flip (bottom). Received: September 12, 2017 Accepted: October 4, 2017 Published: October 4, 2017 5148
DOI: 10.1021/acs.jpclett.7b02428 J. Phys. Chem. Lett. 2017, 8, 5148−5152
Letter
The Journal of Physical Chemistry Letters
H2′-F2′ heteronuclear couplings for the FaraG analogs (Figure S3).10,12 A comparison of 1H−13C HSQC spectra reveals similar patterns as found for the unmodified G4 (Figure S4). On the other hand, a polarity reversal of its 5′-tetrad is indicated by striking spectral similarities with the FrG substituted sequence and additionally confirmed by H8−H1′ nuclear Overhauser effect (NOE) crosspeak intensities used as indicator for syn and anti glycosidic torsion angles. Chemical shifts of C6/C8 base carbons have previously been shown to also constitute excellent markers of syn−anti transitions with chemical shift changes amounting to about 4 ppm.10,19 As shown in Figure 3, C8 resonances of the 5′-tetrad
tions on thermal stability and structure was analyzed by ultraviolet melting and circular dichroism (CD) spectroscopy. In analogy to previous studies employing FrG analogs incorporated into the same hybrid quadruplex, an increase of the CD band at 260 nm and an accompanying decrease of the band at 295 nm suggest a polarity reversal of the G4 5′-tetrad without disruption of the overall fold (Figure S1).10,12 On the basis of the observation of two isoelliptic points at about 250 and 280 nm, the population of coexisting species was assessed based on a simple two-state equilibrium. In Figure 2 the
Figure 3. C8 13C chemical shift differences of FaraG and FrG 1,6,20trisubstituted quadruplexes referenced against the unmodified G4.
exhibit 13C chemical shift changes in line with concerted syn− anti interconversions. However, whereas deshielding effects at unmodified position 16 matches expectations for an anti → syn transition, shielding effects at FaraG-modified positions 1, 6, and 20 are clearly smaller than expected and suggest the presence of additional opposing effects. Because both H1′ and H2′ sugar protons are scalar coupled to fluorine, a typical exclusive correlation spectroscopy (E.COSY) pattern is observed for all intranucleotide FaraG H8−H1′ and H8−H2′ NOE crosspeaks in the absence of any pulses or decoupling schemes on 19F (Figure 4). This pattern clearly reveals an additional intraresidual coupling of H8 to F2′ for all three FaraG analogs. Couplings increase from 0.8 Hz for F araG1 to 2.0 Hz for FaraG6 and 3.0 Hz in FaraG20 with uncertainties of ±0.4 Hz. Scalar couplings of 1−3 Hz between F2′ and H6/H8 have previously been observed for free 2′fluoro-arabinonucleosides but also in a 2′F-ANA/RNA hybrid duplex with compelling evidence pointing to the formation of a C8−H8···F2′ pseudohydrogen bond rather than to a five-bond coupling.20−23 The influence of fluorosugars on the structure of nucleic acids has been extensively studied by Damha and Gonzáles in recent years. Based on the geometry of calculated solution structures, a preferential formation of sequential Fi··· CHi+1 hydrogen bonds of energetic importance was proposed for Fara substitutions in a hybrid duplex but also in quadruplexes.24−26 In contrast, the here-presented experimental observation of 19F−1H scalar couplings in the FaraG-substituted quadruplex with flipped 5′-tetrad unequivocally demonstrates that intraresidual CH···F hydrogen bonds of partial covalent character are formed and likely are responsible for the superimposed C8 deshielding effects (Figure 3).27 Accordingly, the attenuation of shielding effects due to the conformational
Figure 2. Populations of quadruplexes with inverted 5′-tetrad (top) and change in melting temperature ΔTm (bottom) as a function of different FaraG and FrG substitution patterns.
fraction of rearranged G4 structures is plotted together with changes in melting temperature for all FaraG-modified sequences. Reported values for corresponding FrG substitutions are included for comparison.12 On the basis of the CD analysis, at least two substitution sites are required for an efficient tetrad polarity switch in both FaraG and FrG analogs. The larger impact of FrG incorporated at position 1 was attributed to unfavorable fluorine interactions within the G4 narrow groove, enforcing a north pucker with its high propensity for an anti conformational transition.12,18 In contrast to FrG, single substitutions with FaraG analogs at position 1 are least influential in affecting a tetrad flip yet seem to strongly promote a polarity reversal in disubstituted quadruplexes (Figure 2). Conspicuously, all single modifications at the three nonmatching positions exert a destabilizing effect and thermal stabilities further decrease with each additional FrG surrogate but tend to increase again in the case of FaraG analogs. Thus, whereas trisubstituted quadruplexes suggested to entirely undergo a tetrad flip are destabilized by 13 °C with FrG modifications, stability is hardly compromised in the case of FaraG incorporation. For a more detailed structural analysis 1D and 2D NMR experiments were performed on the FaraG trisubstituted sequence. The observation of 12 guanine imino resonances confirms the formation of a single quadruplex with three Gtetrads (Figure S2). Additional assignments are based on standard strategies and supported by characteristic H1′-F2′ and 5149
DOI: 10.1021/acs.jpclett.7b02428 J. Phys. Chem. Lett. 2017, 8, 5148−5152
Letter
The Journal of Physical Chemistry Letters
favorable impact of noncanonical CH···F hydrogen bonds in anti nucleotides. NMR-derived distances were used as restraints to determine the three-dimensional structure of the FaraG-trisubstituted quadruplex through molecular dynamics calculations in explicit water. On the basis of extracted scalar couplings and strong H1′−H4′ crosspeaks, all FaraG nucleotides were set to adopt a sugar pucker between O4′-endo and C2′-endo in line with previously reported data (Figure S5).23,29 To ensure formation of the experimentally observed CH···F pseudohydrogen bonds, additional soft restraints for intraresidual F2′−H8 distances and C8−H8···F2′ angles of 90° were implemented.30 The FaraG-modified quadruplex is well-defined with an rmsd of 0.8 Å for the G-core residues and an overall rmsd of 2.0 Å (Figure 5). Its global fold closely resembles the parent unmodified G4 (Figure S6a). Small structural deviations include the lateral loop with stacking of A18 instead of G17. Owing to their common flipped 5′-tetrad, FaraG and FrG substituted quadruplexes are largely superimposable (Figure S6b). Structural details on the CH···F hydrogen bond in the F araG nucleotides are shown in Figure 5 for a representative structure. In general, higher coupling constants J(H8,F2′) are correlated with shorter F2′−H8 distances and larger C−H···F angles as expected (Figure S7). Noticeably, observed angles for the C−H···F hydrogen bridge
