Molecular crowding regulates the structural switch of the DNA G-quadruplex

Almost all biochemical reactions in vitro have been investigated through numerous experiments conducted in dilute solutions containing low concentrations of solutes. However, biomacromolecules such as nucleic acids, proteins, and polysaccharides are designed to function and/or form their native structures in a living cell containing high concentrations of biomacromolecules, substrates, cofactors, salts, and so on. In the present study, we have demonstrated quantitatively the effect of molecular crowding on structures and stabilities of the G-quadruplex of d(G(4)T(4)G(4)). Molecular crowding with poly(ethylene glycol) (PEG) induced a structural transition from the antiparallel to the parallel G-quadruplex of d(G(4)T(4)G(4)), while molecular crowding with polycations did not alter the structure of the antiparallel G-quadruplex. The binding constants of putrescine, one of the polycations, for d(G(4)T(4)G(4)) in the absence and presence of Na(+) are calculated to be 277 and 2.5 M(-)(1), respectively. This indicates that the polycations coordinate to d(G(4)T(4)G(4)) with electrostatic interactions. The thermodynamic parameters of the antiparallel G-quadruplex formation under the crowding and noncrowding conditions induced by putrescine were also estimated. The stability of the antiparallel G-quadruplex decreased (-DeltaG degrees (25) decreased from 28 to 22 kcal mol(-)(1)) with molecular crowding by putrescine. Also, enthalpy and entropy changes in the structural formation under crowding and noncrowding conditions clearly showed that destabilization was entropy-driven. These quantitative parameters indicated that both the volume excluded by PEG and chemical interactions such as electrostatic interaction with solute polycations are critical for determining how molecular crowding affects the structure and stability of highly ordered DNA structures.     

The triazatruxene derivative azatrux binds to the parallel form of the human telomeric G-quadruplex under molecular crowding conditions: biophysical and molecular modeling studies

The present study has employed a combination of spectroscopic, calorimetric and computational methods to explore the binding of the three side-chained triazatruxene derivative, termed azatrux, to a human telomeric G-quadruplex sequence, under conditions of molecular crowding. The binding of azatrux to the tetramolecular parallel [d(TGGGGT)]₄ quadruplex in the presence and absence of crowding conditions, was also characterized. The data indicate that azatrux binds in an end-stacking mode to the parallel G-quadruplex scaffold and highlights the key structural elements involved in the binding. The selectivity of azatrux for the human telomeric G-quadruplex relative to another biologically relevant G-quadruplex (c-Kit87up) and to duplex DNA was also investigated under molecular crowding conditions, showing that azatrux has good selectivity for the human telomeric G-quadruplex over the other investigated DNA structures.     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to [d(ACGTACGT)]2 and [d(TCGATCGA)]2

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.     

Synthesis and characterization of monomolecular DNA G-quadruplexes formed by tetra-end-linked oligonucleotides

Guanine-rich DNA sequences are widely dispersed in the eukaryotic genome and are abundant in regions with relevant biological significance. They can form quadruplex structures stabilized by guanine quartets. These structures differ for number and strand polarity, loop composition, and conformation. We report here the syntheses and the structural studies of a set of interconnected d(TG(4)T) fragments which are tethered, with different orientations, to a tetra-end-linker in an attempt to force the formation of specific four-stranded DNA quadruplex structures. Two synthetic strategies have been used to obtain oligodeoxyribonucleotide (ODN) strands linked with their 3'- or 5'-ends to each of the four arms of the linker. The first approach allowed the synthesis of tetra-end-linked ODN (TEL-ODN) containing the four ODN strands with a parallel orientation, while the latter synthetic pathway led to the synthesis of TEL-ODNs each containing antiparallel ODN pairs. The influence of the linker at 3'- or 5'-ODN, on the quadruplex typology and stability, in the presence of sodium or potassium ions, has been investigated by circular dichroism (CD), CD thermal denaturation, (1)H NMR experiments at variable temperature, and molecular modeling. All synthesized TEL-ODNs formed parallel G-quadruplex structures. Particularly, the TEL-ODN containing all parallel ODN tracts formed very stable parallel G-quadruplex complexes, whereas the TEL-ODNs containing antiparallel ODN pairs led to relatively less stable parallel G-quadruplexes. The molecular modeling data suggested that the above antiparallel TEL-ODNs can adopt parallel G-quadruplex structures thanks to a considerable folding of the tetra-end-linker around the whole quadruplex scaffold.     

Circular dichroism spectra demonstrate formation of the thrombin-binding DNA aptamer G-quadruplex under stabilizing-cation-deficient conditions

It is noteworthy that the formation of the DNA G-quadruplex is induced by factors other than stabilizing cations because this event probably occurs in living cells. Previous studies have shown that thrombin-binding DNA aptamer (TBA) forms a chair-type intramolecular G-quadruplex structure that binds with thrombin protein in the absence of stabilizing cations. Here, we used circular dichroism (CD) spectroscopy to confirm G-quadruplex formation in the presence of thrombin without stabilizing cations. We obtained characteristic CD spectra that demonstrated that TBA forms the distinctive G-quadruplex structure. Additionally, we investigated G-quadruplex formation induced by change of solvent environment: the influence of low-temperature conditions and molecular crowding.     

Structural polymorphism of telomeric DNA regulated by pH and divalent cation

DNA oligonucleotides can form multi-stranded structures such as a duplex, triplex, and quadruplex, while the double helical structure is generally considered as the canonical structure of DNA oligonucleotides. Guanine-rich or cytosine-rich oligonucleotides, which are observed in telomere, centromere, and other biologically important sequences in vivo, can form four-stranded G-quadruplex and I-motif structures in vitro. In this study, we have investigated the effects of pH and cation on the structures and their stabilities of d(G4T4G4) and d(C4A4C4). The CD spectra and thermal melting curves of DNAs at various pHs demonstrated that acidic conditions induced a stable I-motif structure of d(C4A4C4), while the pH value did not affect the G-quadruplex structure and stability of d(G4T4G4). The CD spectra of the 1:1 mixture of d(G4T4G4) and d(C4A4C4) indicated that the acidic conditions inhibit the duplex formation between d(G4T4G4) and d(C4A4C4). Isothermal titration calorimetry measurements of the duplex formation at various pHs also quantitatively indicated that the acidic conditions inhibit the duplex formation. On the other hand, the CD spectra and thermal melting curves of DNAs in the absence and presence of Ca2+ indicated that Ca2+ induces a parallel G-quadruplex structure of d(G4T4G4) and then inhibits the duplex formation. These results lead to the conclusion that both the pH and coexisting cation can induce and regulate the structural polymorphisms the oligonucleotides in which they form the G-quadruplex, I-motif, and duplex depending on the conditions. Thus, the results reported here indicate pivotal roles of pH and coexisting cations in biological processes by regulating the conformational switching between the duplex and quadruplexes structures of the guanine-rich or cytosine-rich oligonucleotides in vivo.     

A unified computational view of DNA duplex,triplex, quadruplex and their donor–acceptor interactions

DNA can assume various structures as a result of interactions at atomic and molecular levels (e.g., hydrogen bonds, π–π stacking interactions, and electrostatic potentials), so understanding of the consequences of these interactions could guide development of ways to produce elaborate programmable DNA for applications in bio- and nanotechnology. We conducted advanced ab initio calculations to investigate nucleobase model structures by componentizing their donor-acceptor interactions. By unifying computational conditions, we compared the independent interactions of DNA duplexes, triplexes, and quadruplexes, which led us to evaluate a stability trend among Watson–Crick and Hoogsteen base pairing, stacking, and even ion binding. For a realistic solution-like environment, the influence of water molecules was carefully considered, and the potassium-ion preference of G-quadruplex was first analyzed at an ab initio level by considering both base-base and ion-water interactions. We devised new structure factors including hydrogen bond length, glycosidic vector angle, and twist angle, which were highly effective for comparison between computationally-predicted and experimentally-determined structures; we clarified the function of phosphate backbone during nucleobase ordering. The simulated tendency of net interaction energies agreed well with that of real world, and this agreement validates the potential of ab initio study to guide programming of complicated DNA constructs.     

Proton NMR study of the [d(ACGTATACGT)]2-2echinomycin complex: conformational changes between echinomycin binding sites.

The interactions of echinomycin and the DNA decamer [d(ACGTATACGT)]2 were studied by proton NMR. Echinomycin binds cooperatively as a bisintercalator at the CpG steps. The terminal A.T base pairs are Hoogsteen base paired, but none of the four central A.T base pairs are Hoogsteen base paired. However, binding of the drug induces unwinding of the DNA which is propagated to the central ApT step. All four central A.T base pairs are destabilized relative to those in the free DNA. Furthermore, based on these and other results from our laboratory, we conclude that the formation of stable Hoogsteen base pairs may not be the relevant structural change in vivo. The structural changes propagated between adjacent ACGT binding sites are the unwinding of the duplex and destabilization of the base pairing between binding sites.     

The four repeat Giardia lamblia telomere forms tetramolecular G-quadruplex with antiparallel topology

Abstract

Guanine rich DNA sequences of regulatory genomic regions form secondary structures known as G-quadruplexes usually stabilized by tetrads of Hoogsteen hydrogen bonded guanines. The in vivo existence of G-quadruplexes ascertains their biological roles. Human telomeric repeats are the most studied G-rich sequences. The four repeat Giardia telomeric sequence (TAGGG)₄ differs from its human counterpart (TTAGGG)_4, by deletion of one T at the G-tract intervening site of each repeat. We show here that whilst the two repeat Giardia telomeric sequence (TAGGG)₂ forms parallel and antiparallel quadruplexes with tetramolecular topology exclusively, the four repeat version (TAGGG)₄ forms a tetramolecular (antiparallel) and unimolecular (parallel) quadruplexes in Na⁺. The tetramolecular (antiparallel) G-quadruplex formed by four repeats of Giardia telomeric sequence is stabilized by the additional Watson-Crick bonding between its intervening TA bases aligned in antiparallel fashion. Four stranded antiparallel quadruplex for four repeats of any telomeric sequence have not been characterized till date. We hypothesize that telomeric association in antiparallel fashion, (via G-overhangs to form tetramolecular quadruplex) could be a biologically relevant molecular event. Further, coexistence of Hoogsteen as well as Watson-Crick base pairing might give insight for recognition of conformationally diverse DNA structures by ligands.

Communicated by Ramaswamy H. Sarma     

Effects of deficient of the Hoogsteen base-pairs on the G-quadruplex stabilization and binding mode of a cationic porphyrin

BackgroundIn stabilization of the G-quadruplex, formation of a Hoogsteen base-pair between the guanine (G) bases is essential. However, the contribution of each Hoogsteen base-pair at different positions to whole stability of the G-quadruplex has not been known. In this study, the effect of a deficiency of the Hoogsteen type hydrogen bond in the G-quadruplex stability was investigated. Spectral properties of meso-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) associated with various G-quadruplexes were also examined.MethodsThe thermal stability of the thrombin-binding DNA aptamer 5′G₁G₂TTG₅G₆TG₈TG₁₀G₁₁TTG₁₄G₁₅ G-quadruplex, in which the guanine (G) base at 1, 2, 5, 6 and 8th positions was replaced with an inosine (I) base, one at a time, was investigated by circular dichroism (CD). The absorption, CD and fluorescence decay curve for the G-quadruplex associated TMPyP were also measured.ResultsThe transition from the G-quadruplex to a single stranded form was endothermic and induced by an increase in entropy. The order in stability was 0>8>6>2>5>1, where the numbers denote the position of the replacement and 0 represents no replacements of the G base, suggesting the significant contribution of the G₁ base in the stability of the G-quadruplex. Alteration in the spectral property of TMPyP briefly followed the order in thermal stability.ConclusionsReplacement of a G base with an I base resulted in destabilization of the G-quadruplex. The missing hydrogen bond at position 1 destabilized the G-quadruplex most efficiently. TMPyP binds near the I base-replaced location namely, the side of the G-quadruplex.General significanceThe Hoogsteen base-pairing is confirmed to be essential in stabilization of G-quadruplex. When G is replaced with I, the latter base is mobile to interact with cationic porphyrin.     

Structure of the DNA Duplex d(ATTAAT)2 with Hoogsteen Hydrogen Bonds

The traditional Watson-Crick base pairs in DNA may occasionally adopt a Hoogsteen conformation, with a different organization of hydrogen bonds. Previous crystal structures have shown that the Hoogsteen conformation is favored in alternating AT sequences of DNA. Here we present new data for a different sequence, d(ATTAAT)₂, which is also found in the Hoogsteen conformation. Thus we demonstrate that other all-AT sequences of DNA with a different sequence may be found in the Hoogsteen conformation. We conclude that any all-AT sequence might acquire this conformation under appropriate conditions. We also compare the detailed features of DNA in either the Hoogsteen or Watson-Crick conformations.     

NMR investigation of Hoogsteen base pairing in quinoxaline antibiotic--DNA complexes: comparison of 2:1 echinomycin, triostin A and [N-MeCys3,N-MeCys7] TANDEM complexes with DNA oligonucleotides.

Hoogsteen base pairs have been demonstrated to occur in base pairs adjacent to the CpG binding sites in complexes of triostin A and echinomycin with a variety of DNA oligonucleotides. To understand the relationship of these unusual base pairs to the sequence specificity of these quinoxaline antibiotics, the conformation of the base pairs flanking the YpR binding sites of the 2:1 drug-DNA complexes of triostin A with [d(ACGTACGT)]2 and of the TpA specific [N-MeCys3, N-MeCys7] TANDEM with [d(ATACGTAT)]2 have been studied by 1H NMR spectroscopy. In both the 2:1 triostin A-DNA complex and the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the terminal A.T base pairs are Hoogsteen base paired with the 5' adenine in the syn conformation. This indicates that both TpA specific and CpG specific quinoxaline antibiotics are capable of inducing Hoogsteen base pairs in DNA. However, in both 2:1 complexes, Hoogsteen base pairing is limited to the terminal base pairs. In the 2:1 triostin A complex, the internal adenines are anti and in the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the internal guanines are anti regardless of pH, which indicates that the central base pairs of both complexes form Watson-Crick base pairs. This indicates that the sequence dependent nature of Hoogsteen base pairing is the same in TpA specific and CpG specific quinoxaline antibiotic-DNA complexes. We have calculated a low resolution three-dimensional structure of the 2triostin A-[d(ACGTACGT)]2 complex and compared it with other CpG specific quinoxaline antibiotic-DNA complexes. The role of stacking in the formation of Hoogsteen base pairs in these complexes is discussed.     

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background

G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging.

Methods

Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA–drug interactions.

Results

We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines.

Conclusions

Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed.

General significance

The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties.     

Stabilizing parallel G-quadruplex DNA by a new class of ligands: Two non-planar alkaloids through interaction in lateral grooves

Human DNA sequences consisting of tandem guanine (G) nucleotides can fold into a four-stranded structure named G-quadruplex via Hoogsteen hydrogen bonding. As the sequences forming G-quadruplex exist in essential regions of eukaryotic chromosomes and are involved in many important biological processes, the study of their biological functions has currently become a hotspot. Compounds selectively binding and stabilizing G-quadruplex structures have the potential to inhibit telomerase activity or alter oncogene expression levels and thus may act as antitumor agents. Most of reported G-quadruplex ligands generally have planar structures which stabilize G-quadruplex by π–π stacking. However, based on a pharmacophore-based virtual screening two non-planar G-quadruplex ligands were found. These two ligands exhibit good capability for G-quadruplex stabilization and prefer binding to paralleled G-quadruplex rather than to duplex DNA. The binding of these ligands to G-quadruplex may result from groove binding at a 2:1 stoichiometry. These results have shown that planar structures are not essential for G-quadruplex stabilizers, which may represent a new class of G-quadruplex-targeted agents as potential antitumor drugs.