Robert Buscaglia

Hydration is a major determinant of the G-quadruplex stability and conformation of the human telomere 3' sequence of d(AG3(TTAG3)3).

The factors that determine the conformation and stability of G-quadruplex forming sequences remain poorly understood. Here we demonstrate the influence of cosolvents on the conformation and stability of the human telomeric sequence d(A(GGGTTA)3GGG)) in both K(+) and Na(+) containing solutions using a combination of circular dichroism, NMR, and thermodynamics. Molecular crowding arguments have previously been used to suggest that the parallel quadruplex form may be biologically relevant. However, the small cosolvents previously used, PEG 200 and 400, are actually dehydrating agents. We have used acetonitrile as a non-hydrogen-bonding dehydrating agent; similar conformational transitions were observed in K(+) solution. Moreover, NMR analysis shows that the resulting structure contains non-anti guanine glycosyl torsion angles suggesting that the conformation present in acetonitrile is not identical to the all-parallel crystal structure, despite the supposed parallel type CD spectrum.

Polyethylene glycol binding alters human telomere G-quadruplex structure by conformational selection

Polyethylene glycols (PEGs) are widely used to perturb the conformations of nucleic acids, including G-quadruplexes. The mechanism by which PEG alters G-quadruplex conformation is poorly understood. We describe here studies designed to determine how PEG and other co-solutes affect the conformation of the human telomeric quadruplex. Osmotic stress studies using acetonitrile and ethylene glycol show that conversion of the ‘hybrid’ conformation to an all-parallel ‘propeller’ conformation is accompanied by the release of about 17 water molecules per quadruplex and is energetically unfavorable in pure aqueous solutions. Sedimentation velocity experiments show that the propeller form is hydrodynamically larger than hybrid forms, ruling out a crowding mechanism for the conversion by PEG. PEGs do not alter water activity sufficiently to perturb quadruplex hydration by osmotic stress. PEG titration experiments are most consistent with a conformational selection mechanism in which PEG binds more strongly to the propeller conformation, and binding is coupled to the conformational transition between forms. Molecular dynamics simulations show that PEG binding to the propeller form is sterically feasible and energetically favorable. We conclude that PEG does not act by crowding and is a poor mimic of the intranuclear environment, keeping open the question of the physiologically relevant quadruplex conformation.

Modeling complex equilibria in isothermal titration calorimetry experiments: thermodynamic parameters estimation for a three-binding-site model.

Isothermal titration calorimetry (ITC) is a powerful technique that can be used to estimate a complete set of thermodynamic parameters (e.g., K(eq) (or ΔG), ΔH, ΔS, and n) for a ligand-binding interaction described by a thermodynamic model. Thermodynamic models are constructed by combining equilibrium constant, mass balance, and charge balance equations for the system under study. Commercial ITC instruments are supplied with software that includes a number of simple interaction models, for example, one binding site, two binding sites, sequential sites, and n-independent binding sites. More complex models, for example, three or more binding sites, one site with multiple binding mechanisms, linked equilibria, or equilibria involving macromolecular conformational selection through ligand binding, need to be developed on a case-by-case basis by the ITC user. In this paper we provide an algorithm (and a link to our MATLAB program) for the nonlinear regression analysis of a multiple-binding-site model with up to four overlapping binding equilibria. Error analysis demonstrates that fitting ITC data for multiple parameters (e.g., up to nine parameters in the three-binding-site model) yields thermodynamic parameters with acceptable accuracy.

Induction of G-quadruplex DNA structure by Zn(II) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin.

G-quadruplexes (GQ) are formed by the association of guanine-rich stretches of DNA. Certain small molecules can influence kinetics and thermodynamics of this association. Understanding the mechanism of ligand-assisted GQ folding is necessary for the design of more efficient cancer therapeutics. The oligonucleotide d(TAGGG)(2) forms parallel bimolecular GQ in the presence of ≥66 mM K(+); GQs are not formed under Na(+), Li(+) or low K(+) conditions. The thermodynamic parameters for GQ folding at 60 μM oligonucleotide and 100 mM KCl are ΔH = -35 ± 2 kcal mol(-1) and ΔG(310) = -1.4 kcal mol(-1). Quadruplex [d(TAGGG)(2)](2) binds 2-3 K(+) ions with K(d) of 0.5 ± 0.2 mM. Our work addresses the question of whether metal free 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) and its Zn(II), Cu(II), and Pt(II) derivatives are capable of facilitating GQ folding of d(TAGGG)(2) from single stranded, or binding to preformed GQ, using UV-vis and circular dichroism (CD) spectroscopies. ZnTMPyP4 is unique among other porphyrins in its ability to induce GQ structure of d(TAGGG)(2), which also requires at least a low amount of potassium. ZnTMPyP4 binds with 2:1 stoichiometry possibly in an end-stacking mode with a ~10(6) M(-1) binding constant, determined through UV-vis and ITC titrations. This process is entropically driven and has ΔG(298) of -8.0 kcal mol(-1). TMPyP4 binds with 3:1 stoichiometry and K(a) of ~10(6) M(-1). ZnTMPyP4 and TMPyP4 are efficient stabilizers of [d(TAGGG)(2)](2) displaying ΔT(1/2) of 13.5 and 13.8 °C, respectively, at 1:2 GQ to porphyrin ratio; CuTMPyP4 shows a much weaker effect (ΔT(1/2) = 4.7 °C) and PtTMPyP4 is weakly destabilizing (ΔT(1/2) = -2.9 °C). The selectivity of ZnTMPyP4 for GQ versus dsDNA is comparable to that of TMPyP4. The ability of ZnTMPyP4 to bind and stabilize GQ, to induce GQ formation, and speed up its folding may suggest an important biological activity for this molecule.

2-Aminopurine as a Probe for Quadruplex Loop Structures

Fluorescent reporter groups have served for many years as sensitive probes of macromolecular structure. Such probes can be especially useful in comparative studies such as detection of conformational changes and discrimination among structural models. Spectroscopic methods such as fluorescence are attractive because they are rapid, require small amounts of material, are nondestructive, can be carried out with commonly available equipment, and are relatively inexpensive. In addition, there is a rich library of theoretical and practical materials available to aid in data interpretation.The intrinsic fluorescence of most nucleic acids is too low to be useful in structural studies. Thus, it is necessary to incorporate a suitable reporter group to utilize fluorescence methods involving polynucleotide structure. A highly fluorescent adenine analog, 2-aminopurine, has long served in this capacity. The present article describes our use of 2-aminopurine as a probe of loop structures in quadruplex DNA. In particular, we show how knowledge of the relative intensity of 2-aminopurine emission as well as its sensitivity to exogenous quenching molecules such as acrylamide can aid in comparing crystal and solution structures of an oligonucleotide model of the human telomere and in discrimination among models containing tandem repeats of the telomeric quadruplex.

Not all G-quadruplexes are created equally: an investigation of the structural polymorphism of the c-Myc G-quadruplex-forming sequence and its interaction with the porphyrin TMPyP4.

G-quadruplexes, DNA tertiary structures highly localized to functionally important sites within the human genome, have emerged as important new drug targets. The putative G-quadruplex-forming sequence (Pu27) in the NHE-III(1) promoter region of the c-Myc gene is of particular interest as stabilization of this G-quadruplex with TMPyP4 has been shown to repress c-Myc transcription. In this study, we examine the Pu27 G-quadruplex-forming sequence and its interaction with TMPyP4. We report that the Pu27 sequence exists as a heterogeneous mixture of monomeric and higher-order G-quadruplex species in vitro and that this mixture can be partially resolved by size exclusion chromatography (SEC) separation. Within this ensemble of configurations, the equilibrium can be altered by modifying the buffer composition, annealing procedure, and dialysis protocol thereby affecting the distribution of G-quadruplex species formed. TMPyP4 was found to bind preferentially to higher-order G-quadruplex species suggesting the possibility of stabilization of the junctions of the c-Myc G-quadruplex multimers by porphyrin end-stacking. We also examined four modified c-Myc sequences that have been previously reported and found a narrower distribution of G-quadruplex configurations compared to the parent Pu27 sequence. We could not definitively conclude whether these G-quadruplex structures were selected from the original ensemble or if they are new G-quadruplex structures. Since these sequences differ considerably from the wild-type promoter sequence, it is unclear whether their structures have any actual biological relevance. Additional studies are needed to examine how the polymorphic nature of G-quadruplexes affects the interpretation of in vitro data for c-Myc and other G-quadruplexes. The findings reported here demonstrate that experimental conditions contribute significantly to G-quadruplex formation and should be carefully considered, controlled, and reported in detail.

DSC Deconvolution of the Structural Complexity of c-MYC P1 Promoter G-Quadruplexes

We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P(1) promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K(+)] and 130 mM [K(+)]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).

An Improved Model for the hTERT Promoter Quadruplex

Mutations occur at four specific sites in the hTERT promoter in >75% of glioblastomas and melanomas, but the mechanism by which the mutations affect gene expression remains unexplained. We report biophysical computational studies that show that the hTERT promoter sequence forms a novel G-quadruplex structure consisting of three contiguous, stacked parallel quadruplexes. The reported hTERT mutations map to the central quadruplex within this structure, and lead to an alteration of its hydrodynamic properties and stability.

An investigation of G-quadruplex structural polymorphism in the human telomere using a combined approach of hydrodynamic bead modeling and molecular dynamics simulation.

Guanine-rich oligonucleotides can adopt noncanonical tertiary structures known as G-quadruplexes, which can exist in different forms depending on experimental conditions. High-resolution structural methods, such as X-ray crystallography and NMR spectroscopy, have been of limited usefulness in resolving the inherent structural polymorphism associated with G-quadruplex formation. The lack of, or the ambiguous nature of, currently available high-resolution structural data, in turn, has severely hindered investigations into the nature of these structures and their interactions with small-molecule inhibitors. We have used molecular dynamics in conjunction with hydrodynamic bead modeling to study the structures of the human telomeric G-quadruplex-forming sequences at the atomic level. We demonstrated that molecular dynamics can reproduce experimental hydrodynamic measurements and thus can be a powerful tool in the structural study of existing G-quadruplex sequences or in the prediction of new G-quadruplex structures.

A discovery funnel for nucleic acid binding drug candidates

Computational approaches are becoming increasingly popular for the discovery of drug candidates against a target of interest. Proteins have historically been the primary targets of many virtual screening efforts. Although in silico screens targeting proteins have proved successful, other classes of targets, in particular DNA, remain largely unexplored using virtual screening methods. With the realization of the functional importance of many noncanonical DNA structures such as G‐quadruplexes, increased efforts are under way to discover new small molecules that can bind selectively to DNA structures. In the present work, we describe efforts to build an integrated in silico and in vitro platform for discovering compounds that may bind to a chosen DNA target. Millions of compounds are initially screened in silico for selective binding to a particular structure and ranked to identify several hundred best hits. An important element of our strategy is the inclusion of an array of possible competing structures in the in silico screen. The best hundred or so hits are validated experimentally for binding to the actual target structure by a high‐throughput 96‐well thermal denaturation assay to yield the top 10 candidates. Finally, these most promising candidates are thoroughly characterized for binding to their DNA target by rigorous biophysical methods, including isothermal titration calorimetry, differential scanning calorimetry, spectroscopy, and competition dialysis. This platform was validated using quadruplex DNA as a target and a newly discovered quadruplex binding compound with possible anti‐cancer activity was discovered. Some considerations when embarking on virtual screening and in silico experiments are also discussed. Drug Dev Res 72: 178–186, 2011.  © 2010 Wiley‐Liss, Inc.