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1.  Modeling Complex Equilibria in ITC Experiments: Thermodynamic Parameters Estimation for a Three Binding Site Model 
Analytical biochemistry  2012;434(2):233-241.
Isothermal Titration Calorimetry, ITC, is a powerful technique that can be used to estimate a complete set of thermodynamic parameters (e.g. Keq (or ΔG), ΔH, ΔS, and n) for a ligand binding interaction described by a thermodynamic model. Thermodynamic models are constructed by combination of 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 non-linear 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.
PMCID: PMC3572830  PMID: 23262283
ITC; calorimetry; fitting; analysis; non-linear regression; three binding site model; multiple binding sites
2.  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 meso-Tetra(N-methyl-4-pyridyl)porphine 
Organic & biomolecular chemistry  2012;10(47):9393-9404.
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-III1 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 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.
PMCID: PMC3501587  PMID: 23108607
3.  Populated Intermediates in the Thermal Unfolding of the Human Telomeric Quadruplex 
Journal of the American Chemical Society  2012;134(40):16834-16844.
Thermal denaturation profiles of several model oligonucleotides of the human telomere DNA sequence including d[A(GGGTTA)3GGG] (Tel22) were determined using circular dichroism (CD), fluorescence of adenine → 2-aminopurine analogs and fluorescence resonance energy transfer (FRET) to monitor the unfolding process at specific locations within the quadruplex. The resulting optical spectra vs. temperature data matrices were analyzed by singular value decomposition (SVD) to ascertain the minimum number of species required to reproduce the unfolding spectral profiles. Global non-linear least squares fitting of the SVD amplitude vectors was used to estimate thermodynamic parameters and optical spectra of all species for a series of unfolding mechanisms that included one-, two-, and three-step sequential pathways F ⇌ In ⇌ U, n = 0, 1 or 2) as well as two mechanisms with spectroscopically distinct starting structures (F1 and F2). The CD and FRET data for Tel22 unfolding between 4 and 94 °C in 25 mM KCl were best described by a sequential unfolding model with two intermediates, while the 2-aminopurine analogs required one intermediate. The higher melting intermediate I2 had a transition midpoint temperature (Tm) of 61 °C and a CD spectrum with a maximum at ~265 nm and a minimum at ~245 nm. The fluorescence emission spectra of the 2-aminopurine and FRET derivatives suggest greater solvent exposure of the 5′-AGGGTTA- segment in the intermediate compared to the folded state. The spectroscopic properties of the 61 °C intermediate suggest that it may be a triple helical structure.
PMCID: PMC3477705  PMID: 22989179
DNA; quadruplex; telomere; potassium; temperature-dependent denaturation; folding intermediate; circular dichroism; fluorescence; 2-aminopurine; FRET; triple helix
4.  Polyethylene glycol binding alters human telomere G-quadruplex structure by conformational selection 
Nucleic Acids Research  2013;41(16):7934-7946.
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.
PMCID: PMC3763525  PMID: 23804761
5.  Calculation of Hydrodynamic Properties for G-Quadruplex Nucleic Acid Structures from in silico Bead Models 
Topics in current chemistry  2013;330:179-210.
Nucleic acids enriched in guanine bases can adopt unique quadruple helical tertiary structures known as G-quadruplexes. G-quadruplexes have emerged as attractive drug targets as many G-quadruplex-forming sequences have been discovered in functionally critical sites within the human genome, including the telomere, oncogene promoters, and mRNA processing sites. A single G-quadruplex-forming sequence can adopt one of multiple folding topologies often resulting in a lack of a single definitive atomic-level resolution structure for many of these sequences and a major challenge to the discovery of G-quadruplex-selective small molecule drugs. Low-resolution techniques employed to study G-quadruplex structures (e.g. CD spectroscopy) are often unable to discern between G-quadruplex structural ensembles while high-resolution techniques (e.g. NMR spectroscopy) can be overwhelmed by a highly polymorphic system. Hydrodynamic bead modeling is an approach to studying G-quadruplex structures that could bridge the gap between low-resolution techniques and high-resolution molecular models. Here, we present a discussion of hydrodynamic bead modeling in the context of studying G-quadruplex structures, highlighting recent successes and limitations to this approach, as well as an example featuring a G-quadruplex structure formed from the human telomere. This example can easily be adapted to the investigation of any other G-quadruplex-forming sequences.
PMCID: PMC3580009  PMID: 22886555
G-quadruplex; Nucleic acids; Drug discovery; Hydrodynamic; Bead Models; Sedimentation
6.  A Discovery Funnel for Nucleic Acid Binding Drug Candidates 
Drug development research  2011;72(2):178-186.
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. While in silico screens targeting proteins has proven successful, other classes of targets, in particular DNA, remain largely unexplored using virtual screening methods. With the realization of the functional importance of many non-cannonical DNA structures such as G-quadruplexes, increased efforts are underway to discover new small molecules that can bind selectively to DNA structures. Here, 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 ten 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.
PMCID: PMC3090163  PMID: 21566705
drug discovery; in silico screening; SURFLEX-DOCK; DNA; G-quadruplex; high-throughput screening
7.  G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots 
Nucleic Acids Research  2012;40(9):4203-4215.
The use of time-resolved fluorescence measurements in studies of telomeric G-quadruplex folding and stability has been hampered by the complexity of fluorescence lifetime distributions in solution. The application of phasor diagrams to the analysis of time-resolved fluorescence measurements, collected from either frequency-domain or time-domain instrumentation, allows for rapid characterization of complex lifetime distributions. Phasor diagrams are model-free graphical representations of transformed time-resolved fluorescence results. Simplification of complex fluorescent decays by phasor diagrams is demonstrated here using a 2-aminopurine substituted telomeric G-quadruplex sequence. The application of phasor diagrams to complex systems is discussed with comparisons to traditional non-linear regression model fitting. Phasor diagrams allow for the folding and stability of the telomeric G-quadruplex to be monitored in the presence of either sodium or potassium. Fluorescence lifetime measurements revealed multiple transitions upon folding of the telomeric G-quadruplex through the addition of potassium. Enzymatic digestion of the telomeric G-quadruplex structure, fluorescence quenching and Förster resonance energy transfer were also monitored through phasor diagrams. This work demonstrates the sensitivity of time-resolved methods for monitoring changes to the telomeric G-quadruplex and outlines the phasor diagram approach for analysis of complex time-resolved results that can be extended to other G-quadruplex and nucleic acid systems.
PMCID: PMC3351194  PMID: 22241767
8.  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 non-destructive, 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.
PMCID: PMC3008626  PMID: 20012419
quadruplex DNA; telomere model; polynucleotide folding; loop structure; fluorescence; fluorescence quenching; quantum yield; fluorescence lifetime; 2-aminopurine; solvent accessibility

Results 1-8 (8)