Sequence- and structural-selective nucleic acid binding revealed by the melting of mixtures

A simple method for the detection of sequence- and structural-selective ligand binding to nucleic acids is described. The method is based on the commonly used thermal denaturation method in which ligand binding is registered as an elevation in the nucleic acid melting temperature (T(m)). The method can be extended to yield a new, higher -throughput, assay by the simple expediency of melting designed mixtures of polynucleotides (or oligonucleotides) with different sequences or structures of interest. Upon addition of ligand to such mixtures at low molar ratios, the T(m) is shifted only for the nucleic acid containing the preferred sequence or structure. Proof of principle of the assay is provided using first a mixture of polynucleotides with different sequences and, second, with a mixture containing DNA, RNA and two types of DNA:RNA hybrid structures. Netropsin, ethidium, daunorubicin and actinomycin, ligands with known sequence preferences, were used to illustrate the method. The applicability of the approach to oligonucleotide systems is illustrated by the use of simple ternary and binary mixtures of defined sequence deoxyoligonucleotides challenged by the bisanthracycline WP631. The simple mixtures described here provide proof of principle of the assay and pave the way for the development of more sophisticated mixtures for rapidly screening the selectivity of new nucleic acid binding compounds

Kinetics and mechanism of K+- and Na+-induced folding of models of human telomeric DNA into G-quadruplex structures

Cation-induced folding into quadruplex structures for three model human telomeric oligonucleotides, d[AGGG(TTAGGG)3], d[TTGGG(TTAGGG)3A] and d[TTGGG(TTAGGG)3], was characterized by equilibrium titrations with KCl and NaCl and by multiwavelength stopped flow kinetics. Cation binding was cooperative with Hill coefficients of 1.5–2.2 in K+ and 2.4–2.9 in Na+ with half-saturation concentrations of 0.5–1 mM for K+ and 4–13 mM for Na+ depending on the oligonucleotide sequence. Oligonucleotide folding in 50 mM KCl at 25°C consisted of single exponential processes with relaxation times τ of 20–60 ms depending on the sequence. In contrast, folding in100 mM NaCl consisted of three exponentials with τ-values of 40–85 ms, 250–950 ms and 1.5–10.5 s. The folding rate constants approached limiting values with increasing cation concentration; in addition, the rates of folding decreased with increasing temperature over the range 15–45°C. Taken together, these results suggest that folding of G-rich oligonucleotides into quadruplex structures proceeds via kinetically significant intermediates. These intermediates may consist of antiparallel hairpins in rapid equilibrium with less ordered structures. The hairpins may subsequently form nascent G-quartets stabilized by H-bonding and cation binding followed by relatively slow strand rearrangements to form the final completely folded topologies. Fewer kinetic intermediates were evident with K+ than Na+, suggesting a simpler folding pathway in K+ solutions

Conformational profiling of a G-rich sequence within the c-KIT promoter

G-quadruplexes (G4) within oncogene promoters are considered to be promising anticancer targets. However, often they undergo complex structural rearrangements that preclude a precise description of the optimal target. Moreover, even when solved structures are available, they refer to the thermodynamically stable forms but little or no information is supplied about their complex multistep folding pathway. To shed light on this issue, we systematically followed the kinetic behavior of a G-rich sequence located within the c-KIT proximal promoter (kit2) in the presence of monovalent cations K + and Na + . A very short-lived intermediate was observed to start the G4 folding process in both salt conditions. Subsequently, the two pathways diverge to produce distinct thermodynamically stable species (parallel and antiparallel G-quadruplex in K + and Na + , respectively). Remarkably, in K + -containing solution a branched pathway is required to drive the wild type sequence to distribute between a monomeric and dimeric G-quadruplex. Our approach has allowed us to identify transient forms whose relative abundance is regulated by the environment; some of them were characterized by a half-life within the timescale of physiological DNA processing events and thus may represent possible unexpected targets for ligands recognition

Energetics and kinetics of a conformational switch in G-quadruplex DNA

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

G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots

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

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

Thermal difference spectra: a specific signature for nucleic acid structures

We show that nucleic acid structures may be conveniently and inexpensively characterized by their UV thermal difference spectra. A thermal difference spectrum (TDS) is obtained for a nucleic acid by simply recording the ultraviolet absorbance spectra of the unfolded and folded states at temperatures above and below its melting temperature (T(m)). The difference between these two spectra is the TDS. The TDS has a specific shape that is unique for each type of nucleic acid structure, a conclusion that is based on a comparison of >900 spectra from 200 different sequences. The shape of the TDS reflects the subtleties of base stacking interactions that occur uniquely within each type of nucleic acid structure. TDS provides a simple, inexpensive and rapid method to obtain structural insight into nucleic acid structures, which is applicable to both DNA and RNA from short oligomers to polynucleotides. TDS complements circular dichroism as a tool for the structural characterization of nucleic acids in solution

Not so crystal clear: the structure of the human telomere G-quadruplex in solution differs from that present in a crystal

The structure of human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The sequence [5′-AGGG(TTAGGG)(3)] has been used as a model for telomere DNA in both NMR and X-ray crystallographic studies, the results of which show dramatically different structures. In Na(+) solution, NMR revealed an antiparallel G-quadruplex structure that featured both diagonal and lateral TTA loops. Crystallographic studies in the presence of K(+) revealed a flattened, propeller-shaped structure featuring a parallel-stranded G-quadruplex with symmetrical external TTA loops. We report the results of biophysical experiments in solution and computational studies that are inconsistent with the reported crystal structure, indicating that a different structure exists in K(+) solutions. Sedimentation coefficients were determined experimentally in both Na(+) and K(+) solutions and were compared with values calculated using bead models for the reported NMR and crystal structures. Although the solution NMR structure accurately predicted the observed S-value in Na(+) solution, the crystal structure predicted an S-value that differed dramatically from that experimentally observed in K(+) solution. The environments of loop adenines were probed by quantitative fluorescence studies using strategic and systematic single-substitutions of 2-aminopurine for adenine bases. Both fluorescence intensity and quenching experiments in K(+) yielded results at odds with quantitative predictions from the reported crystal structure. Circular dichroism and fluorescence quenching studies in the presence of the crowding agent polyethylene glycol showed dramatic changes in the quadruplex structure in K(+) solutions, but not in Na(+) solutions, suggesting that the crystal environment may have selected for a particular conformational form. Molecular dynamics simulations were performed to yield model structures for the K(+) quadruplex form that are consistent with our biophysical results and with previously reported chemical modification studies. These models suggest that the biologically relevant structure of the human telomere quadruplex in K(+) solution is not the one determined in the published crystalline state

Discovery of novel triple helical DNA intercalators by an integrated virtual and actual screening platform

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

Rational selection of small molecules that increase transcription through the GAA repeats found in Friedreich’s ataxia

AbstractFriedreich’s ataxia (FRDA) is an autosomal recessive trinucleotide repeat disease with no effective therapy. Expanded GAA repeats in the first intron of the FRDA gene are thought to form unusual non-B DNA conformations that decrease transcription and subsequently reduce levels of the encoded protein, frataxin. Frataxin plays a crucial role in iron metabolism and detoxification. To discover small molecules that increase transcription through the GAA repeat region in FRDA, we have made stable cell lines containing a portion of expanded intron 1 fused to a GFP reporter. Small molecules identified using the competition dialysis method were found to increase FRDA-intron 1-reporter gene expression. One of these compounds, pentamidine, increases frataxin levels in patient cells. Thus our approach can be used to detect small molecules of potential therapeutic value in FRDA