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.
DNA; quadruplex; telomere; potassium; temperature-dependent denaturation; folding intermediate; circular dichroism; fluorescence; 2-aminopurine; FRET; triple helix
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.
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.
Thermodynamic studies of G-quadruplex stability are an essential complement to structures obtained by NMR or x-ray crystallography. An understanding of the energetics of quadruplex folding provides a necessary foundation for the physical interpretation of quadruplex formation and reactivity. While thermal denaturation methods are most commonly used to evaluate quadruplex stability, it is also possible to study folding using isothermal titration methods. G-quadruplex folding is tightly coupled to specific cation binding. We describe here protocols for monitoring the cation-driven quadruplex folding transition using circular dichroism or absorbance, and for determination of the distribution of free and bound cation using a fluorescence indicator. Together these approaches provide insight into quadruplex folding at constant temperature, and characterize the linkage between cation binding and folding.
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.
G-quadruplex; Nucleic acids; Drug discovery; Hydrodynamic; Bead Models; Sedimentation
A new bisintercalating anthracycline (WP762) has been designed, in which monomeric units of daunorubicin have been linked through their amino groups on the daunosamine moieties using an m-xylenyl linker. Differential scanning calorimetry and UV melting experiments were used to measure the ultratight binding of WP762 to DNA. The binding constant for the interaction of WP762 with herring sperm DNA was determined to be 7.3 (±0.2) × 1012 M−1 at 20°C. The large favorable binding free energy of −17.3 kcal mol−1 was found to result from a large negative enthalpic contribution of −33.8 kcal mol−1 and an opposing entropic term (−TΔS = +16.5 kcal mol−1). A comparative molecular modeling study rationalized the increased binding by the m-xylenyl linker of WP762 positioning in the DNA minor groove compared to the p-xylenyl linker found in WP631, the first bis-anthracycline of this type. The cytotoxicity of WP762 was compared to that of other anthracyclines in Jurkat T lymphocytes. These studies, together with an analysis of the cell-cycle traverse in the presence of WP762, suggest that in these cells the new drug is more cytotoxic than the structurally related WP631.
G-quadruplex formation in the sequences 5′-(TTAGGG)n and 5′(TTAGGG)nTT (n=4,8,12) was studied using circular dichroism, sedimentation velocity, differential scanning calorimetry and molecular dynamics simulations. Sequences containing 8 and 12 repeats formed higher-order structures with two and three contiguous quadruplexes, respectively. Plausible structures for these sequences were determined by molecular dynamics simulations followed by experimental testing of predicted hydrodynamic properties by sedimentation velocity. These structures featured folding of the strand into contiguous quadruplexes with mixed hybrid conformations. Thermodynamic studies showed the strands folded spontaneous to contain the maximum number contiguous quadruplexes. For the sequence 5′(TTAGGG)12TT, more than 90% of the strands contained completely folded structures with three quadruplexes. Statistical mechanical-based deconvolution of thermograms for three quadruplex structures showed that each quadruplex melted independently with unique thermodynamic parmameters. Thermodynamic analysis revealed further that quadruplexes in higher-ordered structures were destabilized relative to their monomeric counterparts, with unfavorable coupling free energies. Quadruplex stability thus depends critically on the sequence and structural context.
A key part of drug design and development is the optimization of molecular interactions between an engineered drug candidate and its binding target. Thermodynamic characterization provides information about the balance of energetic forces driving binding interactions and is essential for understanding and optimizing molecular interactions.
This review discusses the information that can be obtained from thermodynamic measurements and how this can be applied to the drug development process. Current approaches for the measurement and optimization of thermodynamic parameters are presented, specifically higher throughput and calorimetric methods. Relevant literature for this review was identified in part by bibliographic searches for the period 2004 – 2011 using the Science Citation Index and PUBMED and the keywords listed below.
The most effective drug design and development platform comes from an integrated process utilizing all available information from structural, thermodynamic and biological studies. Continuing evolution in our understanding of the energetic basis of molecular interactions and advances in thermodynamic methods for widespread application are essential to realize the goal of thermodynamically-driven drug design. Comprehensive thermodynamic evaluation is vital early in the drug development process to speed drug development towards an optimal energetic interaction profile while retaining good pharmacological properties. Practical thermodynamic approaches, such as enthalpic optimization, thermodynamic optimization plots and the enthalpic efficiency index, have now matured to provide proven utility in design process. Improved throughput in calorimetric methods remains essential for even greater integration of thermodynamics into drug design.
Affinity; binding; calorimetry; drug design and development; DSC; energetics; enthalpy; entropy; free energy; ITC; ligand; molecular interactions; screening; thermal shift assay; thermodynamics
Formation of DNA quadruplexes requires monovalent cation binding. To characterize the cation binding stoichiometry and linkage between binding and folding, we carried out KCl titrations of Tel22 (d[A(GGGTTA)3]), a model of the human telomere sequence, using a fluorescent indicator to determine [K+]free and circular dichroism to assess the extent of folding. At [K+]free = 5 mM (sufficient for >95% folding), the apparent binding stoichiometry is 3 K+/Tel22; at [K+]free = 20 mM, it increased to 8–10 K+/Tel22. Thermodynamic analysis shows that at [K+]free = 5 mM, K+ binding contributes approximately −4.9 kcal/mol for folding Tel22. The overall folding free energy is −2.4 kcal/mol, indicating that there are energetically unfavorable contributions to folding. Thus, quadruplex folding is driven almost entirely by the energy of cation binding with little or no contribution from other weak molecular interactions.
human telomere; G-quadruplex; potassium binding; folding-cation linkage
Multidimensional “3D” melting curves for G-quadruplexes are obtained by recording whole spectra (absorbance, CD, fluorescence) as a function of temperature rather than the common approach of recording the spectral response to temperature at a single wavelength. 3D melting curves are richer in information, and can be used to enumerate the number of significant species and intermediates states required to properly analyze the thermal denaturation reaction to obtain thermodynamic information. This unit describes the application of the method of singular value decomposition to the analysis of 3D melting data obtained for G-quadruplex structures, and how the results of such an analysis can be used to provide a more complete characterization of the mechanism of quadruplex unfolding.
G-quadruplex; thermodynamics; spectroscopy; thermal melting; singular value decomposition
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.
drug discovery; in silico screening; SURFLEX-DOCK; DNA; G-quadruplex; high-throughput screening
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.
Structural knowledge of telomeric DNA is critical for understanding telomere biology and for the utilization of telomeric DNA as a therapeutic target. Very little is known about the structure of long human DNA sequences that may form more than one quadruplex unit. Here we report a combination of molecular dynamics simulations and experimental biophysical studies to explore the structural and dynamic properties of the human telomeric sequence (TTAGGG)8TT that folds into two contiguous quadruplexes. Five higher order quadruplex models were built combining known single human telomeric quadruplex structures as unique building blocks. The biophysical properties of this sequence in K+ solution were experimentally investigated by means of analytical ultracentrifugation and UV spectroscopy. Additionally, the environments of loop adenines were probed by fluorescence studies using systematic single-substitutions of 2-aminopurine for the adenine bases. The comparison of the experimentally determined properties with the corresponding quantities predicted from the models allowed us to test the validity of each of the structural models. One model emerged whose properties are most consistent with the predictions, and which therefore is the most probable structure in solution. This structure features contiguous quadruplex units in an alternating hybrid-1-hybrid-2 conformation with a highly-ordered interface composed of loop residues from both quadruplexes.
Thermodynamic and kinetic studies complement high-resolution structures of G-quadruplexes. Such studies are essential for a thorough understanding of the mechanisms that govern quadruplex folding and conformational changes in quadruplexes. This perspective article reviews representative thermodynamic and kinetic studies of the folding of human telomeric quadruplex structures. Published thermodynamic data vary widely and are inconsistent. Possible reasons for these inconsistencies are discussed. The key issue of whether or not such folding reactions are a simple two-state process is examined. A tentative energy balance for the folding of telomeric quadruplexes in Na+ and K+ solution, and for conformational transition between these forms will be presented.
quadruplex DNA; thermodynamics; kinetics; free energy; enthalpy
Human telomeric DNA consists of tandem repeats of the DNA sequence d(GGGTTA). Oligodeoxynucletotide telomere models such as d[A(GGGTTA)3GGG] (Tel22) fold in a cation-dependent manner into quadruplex structures consisting of stacked G-quartets linked by d(TTA) loops. NMR has shown that in Na+ solutions Tel22 forms a ‘basket’ topology of four antiparallel strands; in contrast, Tel22 in K+ solutions consists of a mixture of unknown topologies. Our previous studies on the mechanism of folding of Tel22 and similar telomere analogs utilized changes in UV absorption between 270 and 325 nm that report primarily on G-quartet formation and stacking showed that quadruplex formation occurs within milliseconds upon mixing with an appropriate cation. In the current study, we assessed the dynamics and equilibria of folding of specific loops by using Tel22 derivatives in which the dA residues were serially substituted with the fluorescent reporter base, 2-aminopurine (2-AP). Tel22 folding induced by Na+ or K+ assessed by changes in 2-AP fluorescence consists of at least three kinetic steps with time constants spanning a range of ms to several hundred seconds. Na+-dependent equilibrium titrations of Tel22 folding could be approximated as a cooperative two-state process. In contrast, K+-dependent folding curves were biphasic, revealing that different conformational ensembles are present in 1 mM and 30 mM K+. This conclusion was confirmed by 1H NMR. Molecular dynamics simulations revealed a K+ binding pocket in Tel22 located near dA1 that is specific for the so-called hybrid-1 conformation in which strand 1 is in a parallel arrangement. The possible presence of this topologically specific binding site suggests that K+ may play an allosteric role in regulating telomere conformation and function by modulating quadruplex tertiary structure.
DNA·RNA hybrid duplexes are functionally important structures in gene expression that are underutilized as potential drug targets. Several tools are described here for the discovery and characterization of small molecules capable of the selective recognition of DNA·RNA hybrid structures. Competition dialysis and thermal denaturation of mixtures of polynucleotide structures can be used to identify small molecules that bind selectively to DNA·RNA hybrids. An assay that measures small molecule inhibition of RNase H can be used to measure a functional response to these ligands.
DNA·RNA hybrid; RNase H; dialysis; thermal denaturation; enzyme inhibition; UV spectrophotometry
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.
quadruplex DNA; telomere model; polynucleotide folding; loop structure; fluorescence; fluorescence quenching; quantum yield; fluorescence lifetime; 2-aminopurine; solvent accessibility
Analytical ultracentrifugation (AUC) is a powerful technique for the characterization of hydrodynamic and thermodynamic properties. The intent of this article is to demonstrate the utility of sedimentation velocity (SV) studies to obtain hydrodynamic information for G-quadruplex systems and to provide insights into one part of this process, namely, data analysis of existing SV data. An array of data analysis software is available, mostly written and continually developed by established researchers in the AUC field, with particularly rapid advances in the analysis of SV data. Each program has its own learning curve and this article is intended as a resource in the data analysis process for beginning researchers in the field. We discuss the application of three of the most commonly used data analysis programs, DCDT+, Sedfit and SedAnal, to the interpretation of SV data obtained in our laboratory on two G-quadruplex systems.
analytical ultracentrifugation; sedimentation velocity; sedimentation coefficient; frictional ratio; hydrodynamic; solution conformation; G-quadruplex DNA; data analysis; DCDT+; Sedfit; SedAnal
Circular dichroism and differential scanning calorimetry were used to determine the energetics of the conformational switch of the human telomere quadruplex formed by the sequence d[AGGG(TTAGGG)3] between the sodium basket form and the potassium hybrid form. The energy barrier separating the two conformations was found to be modest, only 1.4−2.4 kcal mol−1. The kinetics of exchange of bound K+ for Na+ cations and the concomitant conformational switch was assessed by measuring time-dependent changes in the circular dichroism spectrum accompanying the cation exchange reaction. The time course of these changes was found to consist of three distinct kinetic processes: a rapid phase that was complete in less than 5 ms followed by two slower phases with relaxation times of 40−50 s and 600−800 s at 25 °C and pH 7.0. We interpret these kinetics in terms of a model in which the bound Na+ cations are rapidly replaced by K+ followed by relatively slow structural rearrangements to generate the final K+-bound product(s). Circular dichroism studies showed that addition of the porphyrin TmPyP4 promoted conversion of the basket to the hybrid form. The kinetics of the TmPyP4-induced conformational change were the same as those observed for the cation exchange reaction.
The structure of a higher-order G-quadruplex structure for human telomeric DNA is presented. The structure was determined by a novel integrated approach in which molecular dynamics simulations were used to produce a stable structure, from which specific experimentally accessible properties were predicted. These properties were tested by sedimentation velocity and steady-state fluorescence measurements. The structure that emerges is a dimeric structure with two quadruplex units, each with a different structure. The interface between the quadruplex units is stabilized by specific stacking interactions of loop nucleotides. The interface is a unique structure and a unique target for drug design.
Water is an integral part of DNA and the conserved water molecules at the binding sites can modulate drug binding to DNA or protein. We report here that anthracycline antitumor antibiotics, adriamycin (AM) and daunomycin (DM) binding to DNA is accompanied by different hydration changes, with AM binding resulting in the uptake of about twice as many water molecules as DM. These results indicate that water is playing an important role in drug binding to DNA.
The molecular docking tools Autodock and Surflex accurately reproduce the crystallographic structures of a collection of small molecule ligands that have been shown to bind nucleic acids. Docking studies were performed with the intercalators daunorubicin and ellipticine and the minor groove binders distamycin and pentamidine. Autodock and Surflex dock daunorubicin and distamycin to their nucleic acid targets within a resolution of approximately 2 Å, which is similar to the limit of the crystal structure resolution. However, for the top ranked poses, Autodock and Surflex both dock ellipticine into the correct site but in a different orientation compared to the crystal structure. This appears not only to be partly related to the symmetry of the target nucleic acid, as ellipticine is able to dock from either side of the intercalation site, but also due to the shape of the ligand and docking accuracy. Surflex docks pentamidine in a symmetrically equivalent orientation relative to the crystal structure, while Autodock was able to dock this molecule in the original orientation. In the case of the Surflex docking of pentamidine, the initial rmsd is misleading, given the symmetrical structure of pentamidine. Importantly, the ranking functions of both of these programs are able to return a top pose within approximately 2 Å rmsd for daunorubicin, distamycin, and pentamidine and approximately 3 Å rmsd for ellipticine compared to their respective crystal structures. Some docking challenges and potential pitfalls are explored, such as the importance of hydrogen treatment on ligands as well as the scoring functions of Autodock and Surflex. Overall for this set of complexes, Surflex is preferred over Autodock for virtual screening, as although the results are comparable, Surflex has significantly faster performance and ease of use under the optimal software conditions tested. These experiments show that molecular docking techniques can be successfully extended to include nucleic acid targets, a finding which has important implications for virtual screening applications and in the design of new small molecules to target therapeutically relevant morphologies of nucleic acids.
The plasma proteome is a complex mixture of over 3000 proteins that has been routinely exploited by physicians for clinical diagnostic assays. More recently, the low abundance region of the proteome has been examined for potential biomarkers of disease. A novel calorimetric assay has been developed that exploits a new physical basis with which to interrogate the plasma proteome. This section provides a brief overview of the use of the plasma proteome in clinical diagnosis and biomarker discovery and then introduces the new calorimetric assay. Some initial results are reported that indicate the potential clinical utility of the assay.
Plasma proteome; thermal stability; differential scanning calorimetry; clinical diagnosis; biomarker
Virtual Screening is an increasingly attractive way to discover new small molecules with potential medicinal value. We introduce a novel strategy that integrates use of the molecular docking software Surflex with experimental validation by the method of competition dialysis. This integrated approach was used to identify ligands that selectively bind to the triplex DNA poly(dA)-[poly(dT)]2. A library containing ∼2 million ligands was virtually screened to identify compounds with chemical and structural similarity to a known triplex intercalator, the napthylquinoline MHQ-12. Further molecular docking studies using compounds with high structural similarity resulted in two compounds that were then demonstrated by competition dialysis to have a superior affinity and selectivity for the triplex nucleic acid than MHQ-12. One of the compounds has a different chemical backbone than MHQ-12, which demonstrates the ability of this strategy to ‘scaffold hop’ and to identify small molecules with novel binding properties. Biophysical characterization of these compounds by circular dichroism and thermal denaturation studies confirmed their binding mode and selectivity. These studies provide a proof-of-principle for our integrated screening strategy, and suggest that this platform may be extended to discover new compounds that target therapeutically relevant nucleic acid morphologies.
G-quadrplex DNA can exist in a rich variety of structural forms, ranging from unimolecular folded structures containing diverse types of loops and strand oreintations, to bimolecular dimeric structures, and finally to tetramolecular parallel-stranded structures. These diverse structures present numerous potential small molecule binding sites with distinctive properties. There is mounting evidence for important functional roles for G-quadruplex structures in biology. G-quadruplexes may participate in the maintainance of telomeres, in transcriptional regulation and, in mRNA, may act to modulate translation. G-quadruplexes thus represent an attractive target for new small-molecule therapeutic agents. Competition dialysis provides a useful tool for the discovery of small molecules that selectively recognize the unique structural features of G-quadruplexes. The principles and practice of the competition dialysis experiment are described here.
G-quadruplexDNA; binding; dialysis; drug discovery; structural-selectivity