Observation of water molecules within the bimolecular d(G₃CT₄G₃C)₂G-quadruplex

G-Rich oligonucleotides with cytosine residues in their sequences can form G-quadruplexes where G-quartets are flanked by G·C Watson-Crick base pairs. In an attempt to probe the role of cations in stabilization of a structural element with two G·C base pairs stacked on a G-quartet, we utilized solution state nuclear magnetic resonance to study the folding of the d(G(3)CT(4)G(3)C) oligonucleotide into a G-quadruplex upon addition of (15)NH(4)(+) ions. Its bimolecular structure exhibits antiparallel strands with edge-type loops. Two G-quartets in the core of the structure are flanked by a couple of Watson-Crick G·C base pairs in a sheared arrangement. The topology is equivalent to the solution state structure of the same oligonucleotide in the presence of Na(+) and K(+) ions [Kettani, A., et al. (1998) J. Mol. Biol.282, 619, and Bouaziz, S., et al. (1998) J. Mol. Biol.282, 637). A single ammonium ion binding site was identified between adjacent G-quartets, but three sites were expected. The remaining potential cation binding sites between G-quartets and G·C base pairs are occupied by water molecules. This is the first observation of long-lived water molecules within a G-quadruplex structure. The flanking G·C base pairs adopt a coplanar arrangement and apparently do not require cations to neutralize unfavorable electrostatic interactions among proximal carbonyl groups. A relatively fast movement of ammonium ions from the inner binding site to bulk with the rate constants of 21 s(-1) was attributed to the lack of hydrogen bonds between adjacent G·C base pairs and the flexibility of the T(4) loops.     

Measured and calculated CD spectra of G-quartets stacked with the same or opposite polarities

Circular dichroism (CD) spectroscopy is widely used to characterize the structures of DNA G-quadruplexes. CD bands at 200-300 nm have been empirically related to G-quadruplexes having parallel or antiparallel sugar-phosphate backbones. We propose that a more fundamental interpretation of the origin of the CD bands is in the stacking interactions of neighboring G-quartets, which can have the same or opposing polarities of hydrogen bond acceptors and donors. From an empirical summation of CD spectra of the d(G)5 G-quadruplex and of the thrombin binding aptamer that have neighboring G-quartets with the same and opposite polarities, respectively, the spectra of aptamers selected by the Ff gene 5 protein (g5p) appear to arise from a combination of the two types of polarities of neighboring G-quartets. The aptamer CD spectra resemble the spectrum of d(G3T4G3), in which two adjacent quartets have the same and two have opposite polarities. Quantum-chemical spectral calculations were performed using a matrix method, based on guanine chromophores oriented as in d(G3T4G3). The calculations show that the two types of G-quartet stacks have CD spectra with features resembling experimental spectra of the corresponding types of G-quadruplexes.     

The solution structure of d(G(4)T(4)G(3))(2): a bimolecular G-quadruplex with a novel fold

The G-rich 11-mer oligonucleotide d(G(4)T(4)G(3)) forms a bimolecular G-quadruplex in the presence of sodium ions with a topology that is distinct from the folds of the closely related and well-characterized sequences d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)). The solution structure of d(G(4)T(4)G(3))(2) has been determined using a combination of NMR spectroscopy and restrained molecular dynamics calculations. d(G(4)T(4)G(3))(2) forms an asymmetric dimeric fold-back structure consisting of three stacked G-quartets. The two T(4) loops that span diagonally across the outer faces of the G-quartets assume different conformations. The glycosidic torsion angle conformations of the guanine bases are 5'-syn-anti-syn-anti-(T(4) loop)-anti-syn-anti in one strand and 5'-syn-anti-syn-anti-(T(4) loop)-syn-anti-syn in the other strand. The guanine bases of the two outer G-quartets exhibit a clockwise donor-acceptor hydrogen-bonding directionality, while those of the middle G-quartet exhibit the anti-clockwise directionality. The topology of this G-quadruplex, like other bimolecular fold-back structures with diagonal loops, places each strand of the G-quartet region next to a neighboring parallel and an anti-parallel strand. The two guanine residues not involved in G-quartet formation, G4 and G12 (i.e. the fourth guanine base of one strand and the first guanine base of the other strand), adopt distinct conformations. G4 is stacked on top of an adjacent G-quartet, and this base-stacking continues along with the bases of the loop residues T5 and T6. G12 is orientated away from the core of G-quartets; stacked on the T7 base and apparently involved in hydrogen-bonding interactions with the phosphodiester group of this same residue. The cation-dependent folding of the d(G(4)T(4)G(3))(2) quadruplex structure is distinct from that observed for similar sequences. While both d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)) form bimolecular, diagonally looped G-quadruplex structures in the presence of Na(+), K(+) and NH(4)(+), we have observed this folding to be favored for d(G(4)T(4)G(3)) in the presence of Na(+), but not in the presence of K(+) or NH(4)(+). The structure of d(G(4)T(4)G(3))(2) exhibits a "slipped-loop" element that is similar to what has been proposed for structural intermediates in the folding pathway of some G-quadruplexes, and therefore provides support for the feasibility of these proposed transient structures in G-quadruplex formation.     

Effect of G-tract length on the topology and stability of intramolecular DNA quadruplexes

G-Rich sequences are known to form four-stranded structures that are based on stacks of G-quartets, and sequences with the potential to adopt these structures are common in eukaryotic genomes. However, there are few rules for predicting the relative stability of folded complexes that are adopted by sequences with different-length G-tracts or variable-length linkers between them. We have used thermal melting, circular dichroism, and gel electrophoresis to examine the topology and stability of intramolecular G-quadruplexes that are formed by sequences of the type d(GnT)4 and d(GnT2)4 (n = 3-7) in the presence of varying concentrations of sodium and potassium. In the presence of potassium or sodium, d(GnT)4 sequences form intramolecular parallel complexes with the following order of stability: n = 3 > n = 7 > n = 6 > n = 5 > n = 4. d(G3T)4 is anomalously stable. In contrast, the stability of d(GnT2)4 increases with the length of the G-tract (n = 7 > n = 6 > n = 5 > n = 4 > n = 3). The CD spectra for d(GnT)4 in the presence of potassium exhibit positive peaks around 260 nm, consistent with the formation of parallel topologies. These peaks are retained in sodium-containing buffers, but when n = 4, 5, or 6, CD maxima are observed around 290 nm, suggesting that these sequences [especially d(G5T)4] have some antiparallel characteristics. d(G3T2)4 adopts a parallel conformation in the presence of both sodium and potassium, while all the other d(GnT2)4 complexes exhibit predominantly antiparallel features. The properties of these complexes are also affected by the rate of annealing, and faster rates favor parallel complexes.     

Crystal structure of d(gcGXGAgc) with X=G: a mutation at X is possible to occur in a base-intercalated duplex for multiplex formation

DNA fragments with the sequences d(gcGX[Y]n Agc) (n=1, X=A, and Y=A, T, or G)form base-intercalated duplexes, which is a basic unit for formation of multiplexes such as octaplex and hexaplex. To examine the stability of multiplexes, a DNA with X=Y=G and n=1 was crystallized under conditions different from those of the previously determined sequences, and its crystal structure has been determined. The two strands are coupled in an anti-parallel fashion to form a base-intercalated duplex, in which the first and second residues form Watson-Crick type G:C pairs and the third and sixth residues form a sheared G:A pairs at both ends of the duplex. The G4 and G5 bases are stacked alternatively on those of the counter strand to form a long G column of G3-G4-G5*-G5-G4*-G3*, the central four Gs being protruded. In addition, the three duplexes are associated to form a hexaplex around a mixture of calcium and sodium cations on the crystallographic threefold axis. These structural features are similar to those of the previous crystals, though slightly different in detail. The present study indicates that mutation at the 4th position is possible to occur in a base-intercalated duplex for multiplex formations, suggesting that DNA fragments with any sequence sandwiched between the two triplets gcG and Agc can form a multiplex.     

Role of loop residues and cations on the formation and stability of dimeric DNA G-quadruplexes

Formation of guanine-quadruplexes by four DNA oligonucleotides with common sequence dG4-loop-dG4 has been studied by a combination of NMR and UV spectroscopy. The loops consisted of 1',2'-dideoxyribose, propanediol, hexaethylene glycol, and thymine residues. The comparison of data on modified and parent oligonucleotides gave insight into the role of loop residues on formation and stability of dimeric G-quadruplexes. All modified oligonucleotides fold into dimeric fold-back G-quadruplexes in the presence of sodium ions. Multiple structures form in the presence of potassium and ammonium ions, which is in contrast to the parent oligonucleotide with dT4 loop. 15N-filtered 1H NMR spectra demonstrate that all studied G-quadruplexes exhibit three 15NH4(+) ion binding sites. Topology of intermolecular G-quadruplexes was evaluated by NMR measurements and diffusion experiments. The spherical, prolate-ellipsoid and symmetric cylinder models were used to interpret experimental translational diffusion constants in terms of diameters and lengths of unfolded oligonucleotides and their respective G-quadruplexes. UV melting and annealing curves show that oligonucleotides with non-nucleosidic loop residues fold faster, exhibit no hysteresis, and are less stable than dimeric d(G4T4G4)2 which can be attributed to the absence of H-bonds, stacking between loop residues and the outer G-quartets as well as cation-pi interactions. Oligonucleotide consisting of hexaethylene glycol linkage with only two phosphate groups in the loop exhibits higher melting temperature and more negative deltaH(o) and deltaG(o) values than oligonucleotides with four 1',2'-dideoxyribose or propanediol residues.     

G-quadruplex induced stabilization by 2'-deoxy-2'-fluoro-D-arabinonucleic acids (2'F-ANA)

The impact of 2'-deoxy-2'-fluoroarabinonucleotide residues (2'F-araN) on different G-quadruplexes derived from a thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), an anti-HIV phosphorothioate aptamer PS-d(T2G4T2) and a DNA telomeric sequence d(G4T4G4) via UV thermal melting (T(m)) and circular dichroism (CD) experiments has been investigated. Generally, replacement of deoxyguanosines that adopt the anti conformation (anti-guanines) with 2'F-araG can stabilize G-quartets and maintain the quadruplex conformation, while replacement of syn-guanines with 2'F-araG is not favored and results in a dramatic switch to an alternative quadruplex conformation. It was found that incorporation of 2'F-araG or T residues into a thrombin-binding DNA G-quadruplex stabilizes the complex (DeltaT(m) up to approximately +3 degrees C/2'F-araN modification); 2'F-araN units also increased the half-life in 10% fetal bovine serum (FBS) up to 48-fold. Two modified thrombin-binding aptamers (PG13 and PG14) show an approximately 4-fold increase in binding affinity to thrombin, as assessed via a nitrocellulose filter binding assay, both with increased thermal stability (approximately 1 degrees C/2'F-ANA modification increase in T(m)) and nuclease resistance (4-7-fold) as well. Therefore, the 2'-deoxy-2'-fluoro-d-arabinonucleic acid (2'F-ANA) modification is well suited to tune (and improve) the physicochemical and biological properties of naturally occurring DNA G-quartets.     

d(G3T4G4) forms unusual dimeric G-quadruplex structure with the same general fold in the presence of K+, Na+ or NH4+ ions

We have recently communicated that DNA oligonucleotide d(G(3)T(4)G(4)) forms a dimeric G-quadruplex in the presence of K(+) ions [J. Am. Chem. Soc.2003, 125, 7866-7871]. The high-resolution NMR structure of d(G(3)T(4)G(4))(2) G-quadruplex exhibits G-quadruplex core consisting of three stacked G-quartets. The two overhanging G3 and G11 residues are located at the opposite sides of the end G-quartets and are not involved in G-quartet formation. d(G(3)T(4)G(4))(2) G-quadruplex represents the first bimolecular G-quadruplex where end G-quartets are spanned by diagonal (T4-T7) as well as edge-type loops (T15-T18). Three of the G-rich strands are parallel while one is anti-parallel. The G12-G22 strand demonstrates a sharp reversal in strand direction between residues G19 and G20 that is accommodated with the leap over the middle G-quartet. The reversal in strand direction is achieved without any extra intervening residues. Here we furthermore examined the influence of different monovalent cations on the folding of d(G(3)T(4)G(4)). The resolved imino and aromatic proton resonances as well as (sequential) NOE connectivity patterns showed only minor differences in key intra- and interquartet NOE intensities in the presence of K(+), Na(+) and NH(4)(+) ions, which were consistent with subtle structural differences while retaining the same folding topology of d(G(3)T(4)G(4))(2) G-quadruplex.     

Stability of telomeric G-quadruplexes

In most eukaryotes, telomeric DNA consists of repeats of a short motif that includes consecutive guanines and may hence fold into G-quadruplexes. Budding yeasts have telomeres composed of longer repeats and show variation in the degree of repeat homogeneity. Although telomeric sequences from several organisms have been shown to fold into G-quadruplexes in vitro, surprisingly, no study has been dedicated to the comparison of G-quadruplex folding and stability of known telomeric sequences. Furthermore, to our knowledge, folding of yeast telomeric sequences into intramolecular G-quadruplexes has never been investigated. Using biophysical and biochemical methods, we studied sequences mimicking about four repetitions of telomeric motifs from a variety of organisms, including yeasts, with the aim of comparing the G-quadruplex folding potential of telomeric sequences among eukaryotes. G-quadruplex folding did not appear to be a conserved feature among yeast telomeric sequences. By contrast, all known telomeric sequences from eukaryotes other than yeasts folded into G-quadruplexes. Nevertheless, while G(3)T(1-4)A repeats (found in a variety of organisms) and G(4)T(2,4) repeats (found in ciliates) folded into stable G-quadruplexes, G-quadruplexes formed by repetitions of G(2)T(2)A and G(2)CT(2)A motifs (found in many insects and in nematodes, respectively) appeared to be in equilibrium with non-G-quadruplex structures (likely hairpin-duplexes).     

Mitochondria and G-quadruplex evolution: an intertwined relationship

G-quadruplexes (G4s) are non-canonical structures formed in guanine (G)-rich sequences through stacked G tetrads by Hoogsteen hydrogen bonding. Several studies have demonstrated the existence of G4s in the genome of various organisms, including humans, and have proposed that G4s have a regulatory role in various cellular functions. However, little is known regarding the dissemination of G4s in mitochondria. In this review, we report the observation that the number of potential G4-forming sequences in the mitochondrial genome increases with the evolutionary complexity of different species, suggesting that G4s have a beneficial role in higher-order organisms. We also discuss the possible function of G4s in mitochondrial (mt)DNA and long noncoding (lnc)RNA and their role in various biological processes.     

A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple.

The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher-order DNA architectures. Further, these two small unimolecular G-quadruplexes, which are distinct from each other and from parallel-stranded G-quadruplexes, provide novel targets for ligand recognition. Our results demonstrate that the double chain reversal loop connectivity identified previously by our laboratory within the Tetrahymena telomere d(T2G4)4 quadruplex, is a robust folding topology, since it has now also been observed within the d(G2T4G2CAG2GT4G2T) quadruplex. The identification of a G-(T-T) triad and a T-T-T triple, expands on the available recognition alignments for base triads and triples.     

End-stacking of copper cationic porphyrins on parallel-stranded guanine quadruplexes

Nucleic acids that contain multiple sequential guanines assemble into guanine quadruplexes (G-quadruplexes). Drugs that induce or stabilize G-quadruplexes are of interest because of their potential use as therapeutics. Previously, we reported on the interaction of the Cu(2+) derivative of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine (CuTMpyP4), with the parallel-stranded G-quadruplexes formed by d(T(4)G( n )T(4)) (n = 4 or 8) (Keating and Szalai in Biochemistry 43:15891-15900, 2004). Here we present further characterization of this system using a series of guanine-rich oligonucleotides: d(T(4)G( n )T(4)) (n = 5-10). Absorption titrations of CuTMpyP4 with all d(T(4)G( n )G(4)) quadruplexes produce approximately the same bathochromicity (8.3 +/- 2 nm) and hypochromicity (46.2-48.6%) of the porphyrin Soret band. Induced emission spectra of CuTMpyP4 with d(T(4)G( n )T(4))(4) quadruplexes indicate that the porphyrin is protected from solvent. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry revealed a maximum porphyrin to quadruplex stoichiometry of 2:1 for the shortest (n = 4) and longest (n = 10) quadruplexes. Electron paramagnetic resonance spectroscopy shows that bound CuTMpyP4 occupies magnetically noninteracting sites on the quadruplexes. Consistent with our previous model for d(T(4)G(4)T(4)), we propose that two CuTMpyP4 molecules are externally stacked at each end of the run of guanines in all d(T(4)G( n )T(4)) (n = 4-10) quadruplexes.     

Molecular dynamics simulations reveal the balance of forces governing the formation of a guanine tetrad—a common structural unit of G-quadruplex DNA

G-quadruplexes (G4) are nucleic acid conformations of guanine-rich sequences, in which guanines are arranged in the square-planar G-tetrads, stacked on one another. G4 motifs form in vivo and are implicated in regulation of such processes as gene expression and chromosome maintenance. The structure and stability of various G4 topologies were determined experimentally; however, the driving forces for their formation are not fully understood at the molecular level. Here, we used all-atom molecular dynamics to probe the microscopic origin of the G4 motif stability. By computing the free energy profiles governing the dissociation of the 3′-terminal G-tetrad in the telomeric parallel-stranded G4, we examined the thermodynamic and kinetic stability of a single G-tetrad, as a common structural unit of G4 DNA. Our results indicate that the energetics of guanine association alone does not explain the overall stability of the G-tetrad and that interactions involving sugar–phosphate backbone, in particular, the constrained minimization of the phosphate–phosphate repulsion energy, are crucial in providing the observed enthalpic stabilization. This enthalpic gain is largely compensated by the unfavorable entropy change due to guanine association and optimization of the backbone topology.     

Kinetics of double-chain reversals bridging contiguous quartets in tetramolecular quadruplexes

Repetitive 5'GGXGG DNA segments abound in, or near, regulatory regions of the genome and may form unusual structures called G-quadruplexes. Using NMR spectroscopy, we demonstrate that a family of 5'GCGGXGGY sequences adopts a folding topology containing double-chain reversals. The topology is composed of two bistranded quadruplex monomeric units linked by formation of G:C:G:C tetrads. We provide a complete thermodynamic and kinetic analysis of 13 different sequences using absorbance spectroscopy and DSC, and compare their kinetics with a canonical tetrameric parallel-stranded quadruplex formed by TG4T. We demonstrate large differences (up to 10(5)-fold) in the association constants of these quadruplexes depending on primary sequence; the fastest samples exhibiting association rate equal or higher than the canonical TG4T quadruplex. In contrast, all sequences studied here unfold at a lower temperature than this quadruplex. Some sequences have thermodynamic stability comparable to the canonical TG4T tetramolecular quadruplex, but with faster association and dissociation. Sequence effects on the dissociation processes are discussed in light of structural data.     

A spectroscopic study on the interactions of porphyrin with G-quadruplex DNAs

Free-base porphyrin (5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine) (H(2)TMPyP4) has been shown to be an effective telomerase inhibitor by an in vitro assay. Here, we examined the interactions of the H(2)TMPyP4 with three distinct G-quadruplex DNAs, the parallel-stranded (TG(4)T)4, dimer-hairpin-folded (G(4)T(4)G(4))2, and monomer-folded AG(3)(T(2)AG(3))(3), by ultraviolet resonance Raman spectroscopy (UVRR), UV-vis absorption spectroscopy, fluorescence spectroscopy, and surface-enhanced Raman spectroscopy (SERS). The data obtained by the continuous variation titration method show that the binding stoichiometry of H(2)TMPyP4/G-quadruplex is 2:1 for (TG(4)T)4 and 4:1 for (G(4)T(4)G(4))2 or AG(3)(T(2)AG(3))(3). The results of SERS spectra, UV-vis absorption titration, and fluorescence emission spectra together with the binding stoichiometries reveal that two H(2)TMPyP4 molecules are externally stacked at two ends of the parallel (TG(4)T)4 G-quadruplex, whereas H(2)TMPyP4 molecules can intercalate within their diagonal or lateral loop regions and intervals between two G-tetrads for (G(4)T(4)G(4))2 and AG(3)(T(2)AG(3))(3) G-quadruplexes. The binding of H(2)TMPyP4 to (TG(4)T)4 G-quadruplex results in the hypochromicity of the UV Raman signal of (TG(4)T)4, indicating that the stacking effects between H(2)TMPyP4 and DNA bases are significant. The Raman hyperchromicities and shifts are observed after the binding of H(2)TMPyP4 to both (G(4)T(4)G(4))2 and AG(3)(T(2)AG(3))(3) G-quadruplexes. This indicates that the intercalative H(2)TMPyP4 can lengthen the vertical distance between adjacent G-tetrads of (G(4)T(4)G(4))2 and AG(3)(T(2)AG(3))(3) and change their conformations. The present study provides new insights into the effect of H(2)TMPyP4 binding on the structures of G-quadruplexes and also demonstrates that Raman spectroscopy is an ideal method for examining the interaction between drugs and G-quadruplexes.     

Intramolecular Telomeric G-Quadruplexes Dramatically Inhibit DNA Synthesis by Replicative and Translesion Polymerases,Revealing their Potential to Lead to Genetic Change

Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.