Structure of a 28.5 kDa duplex-embedded G-quadruplex system resolved to 7.4 Å resolution with cryo-EM.

Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are transcriptional regulators. Little structural information exists for these G4s embedded within duplexes, their presumed genomic environment. Here, we report the 7.4 Å resolution structure and dynamics of a 28.5 kDa duplex-G4-duplex (DGD) model system using cryo-EM, molecular dynamics, and small-angle X-ray scattering (SAXS) studies. The DGD cryo-EM refined model features a 53° bend induced by a stacked duplex-G4 interaction at the 5' G-tetrad interface with a persistently unstacked 3' duplex. The surrogate complement poly dT loop preferably stacks onto the 3' G-tetrad interface resulting in occlusion of both 5' and 3' tetrad interfaces. Structural analysis shows that the DGD model is quantifiably more druggable than the monomeric G4 structure alone and represents a new structural drug target. Our results illustrate how the integration of cryo-EM, MD, and SAXS can reveal complementary detailed static and dynamic structural information on DNA G4 systems.

Long promoter sequences form higher-order G-quadruplexes: an integrative structural biology study of c-Myc, k-Ras and c-Kit promoter sequences.

We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned. We report here five structures of long (34-70 nt) wild-type sequences selected from three cancer-related promoters: c-Myc, c-Kit and k-Ras. Each sequence studied has a unique structure. Three sequences form structures with two contiguous, stacked, G-quadruplex units. One longer sequence from c-Myc forms a structure with three contiguous stacked quadruplexes. A longer c-Kit sequence forms a quadruplex-hairpin structure. Each structure exhibits interfacial regions between stacked quadruplexes or novel loop geometries that are possible druggable targets. We also report methodological advances in our integrated structural biology approach, which now includes quantitative CD for counting stacked G-tetrads, DNaseI cleavage for hairpin detection and SAXS model refinement. Our results suggest that higher-order quadruplex assemblies may be a common feature within the genome, rather than simple single quadruplex structures.

G-quadruplex DNA: A Longer Story.

G-quadruplexes (G4s) are distinctive four-stranded DNA or RNA structures found within cells that are thought to play functional roles in gene regulation and transcription, translation, recombination, and DNA damage/repair. While G4 structures can be uni-, bi-, or tetramolecular with respect to strands, folded unimolecular conformations are most significant in vivo. Unimolecular G4 can potentially form in sequences with runs of guanines interspersed with what will become loops in the folded structure: 5'GxLyGxLyGxLyGx, where x is typically 2-4 and y is highly variable. Such sequences are highly conserved and specifically located in genomes. In the folded structure, guanines from each run combine to form planar tetrads with four hydrogen-bonded guanine bases; these tetrads stack on one another to produce four strand segments aligned in specific parallel or antiparallel orientations, connected by the loop sequences. Three types of loops (lateral, diagonal, or "propeller") have been identified. The stacked tetrads form a central cavity that features strong coordination sites for monovalent cations that stabilize the G4 structure, with potassium or sodium preferred. A single monomeric G4 typically forms from a sequence containing roughly 20-30 nucleotides. Such short sequences have been the primary focus of X-ray crystallographic or NMR studies that have produced high-resolution structures of a variety of monomeric G4 conformations. These structures are often used as the basis for drug design efforts to modulate G4 function.We believe that the focus on monomeric G4 structures formed by such short sequences is perhaps myopic. Such short sequences for structural studies are often arbitrarily selected and removed from their native genomic sequence context, and then are often changed from their native sequences by base substitutions or deletions intended to optimize the formation of a homogeneous G4 conformation. We believe instead that G-quadruplexes prefer company and that in a longer natural sequence context multiple adjacent G4 units can form to combine into more complex multimeric G4 structures with richer topographies than simple monomeric forms. Bioinformatic searches of the human genome show that longer sequences with the potential for forming multiple G4 units are common. Telomeric DNA, for example, has a single-stranded overhang of hundreds of nucleotides with the requisite repetitive sequence with the potential for formation of multiple G4s. Numerous extended promoter sequences have similar potentials for multimeric G4 formation. X-ray crystallography and NMR methods are challenged by these longer sequences (>30 nt), so other tools are needed to explore the possible multimeric G4 landscape. We have implemented an integrated structural biology approach to address this challenge. This approach integrates experimental biophysical results with atomic-level molecular modeling and molecular dynamics simulations that provide quantitatively testable model structures. In every long sequence we have studied so far, we found that multimeric G4 structures readily form, with a surprising diversity of structures dependent on the exact native sequence used. In some cases, stable hairpin duplexes form along with G4 units to provide an even richer landscape. This Account provides an overview of our approach and recent progress and provides a new perspective on the G-quadruplex folding landscape.

The solution structures of higher-order human telomere G-quadruplex multimers.

Human telomeres contain the repeat DNA sequence 5'-d(TTAGGG), with duplex regions that are several kilobases long terminating in a 3' single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate models proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5'-d(TTAGGG)n, with n = 8, 12 and 16, fold into multimeric structures containing the maximal number (2, 3 and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of ∼34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

The hTERT core promoter forms three parallel G-quadruplexes.

The structure of the 68 nt sequence with G-quadruplex forming potential within the hTERT promoter is disputed. One model features a structure with three stacked parallel G-quadruplex units, while another features an unusual duplex hairpin structure adjoined to two stacked parallel and antiparallel quadruplexes. We report here the results of an integrated structural biology study designed to distinguish between these possibilities. As part of our study, we designed a sequence with an optimized hairpin structure and show that its biophysical and biochemical properties are inconsistent with the structure formed by the hTERT wild-type sequence. By using circular dichroism, thermal denaturation, nuclear magnetic resonance spectroscopy, analytical ultracentrifugation, small-angle X-ray scattering, molecular dynamics simulations and a DNase I cleavage assay we found that the wild type hTERT core promoter folds into a stacked, three-parallel G-quadruplex structure. The hairpin structure is inconsistent with all of our experimental data obtained with the wild-type sequence. All-atom models for both structures were constructed using molecular dynamics simulations. These models accurately predicted the experimental hydrodynamic properties measured for each structure. We found with certainty that the wild-type hTERT promoter sequence does not form a hairpin structure in solution, but rather folds into a compact stacked three-G-quadruplex conformation.

Human POT1 unfolds G-quadruplexes by conformational selection.

The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (-10.1 ± 0.3 kcal mol-1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

A rapid fluorescent indicator displacement assay and principal component/cluster data analysis for determination of ligand-nucleic acid structural selectivity.

We describe a rapid fluorescence indicator displacement assay (R-FID) to evaluate the affinity and the selectivity of compounds binding to different DNA structures. We validated the assay using a library of 30 well-known nucleic acid binders containing a variety chemical scaffolds. We used a combination of principal component analysis and hierarchical clustering analysis to interpret the results obtained. This analysis classified compounds based on selectivity for AT-rich, GC-rich and G4 structures. We used the FID assay as a secondary screen to test the binding selectivity of an additional 20 compounds selected from the NCI Diversity Set III library that were identified as G4 binders using a thermal shift assay. The results showed G4 binding selectivity for only a few of the 20 compounds. Overall, we show that this R-FID assay, coupled with PCA and HCA, provides a useful tool for the discovery of ligands selective for particular nucleic acid structures.

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.

G-Quadruplex Secondary Structure Obtained from Circular Dichroism Spectroscopy.

A curated library of circular dichroism spectra of 23 G-quadruplexes of known structure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principal component analysis and singular value decomposition were used to characterize the reference spectral library. CD spectra were successfully fit to obtain estimates of the amounts of base steps in anti-anti, syn-anti or anti-syn conformations, in diagonal or lateral loops, or in other conformations. The results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra.

"Inside-Out" PEGylation of Bovine ß-Cross-Linked Hemoglobin.

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to ß-cross-linked (ßXL-Hb) hemoglobin (Hb) tetramers through the Cys ß93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three ßXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P50  = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P50  = 9.67 Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.

Clinical application of plasma thermograms. Utility, practical approaches and considerations.

Differential scanning calorimetry (DSC) studies of blood plasma are part of an emerging area of the clinical application of DSC to biofluid analysis. DSC analysis of plasma from healthy individuals and patients with various diseases has revealed changes in the thermal profiles of the major plasma proteins associated with the clinical status of the patient. The sensitivity of DSC to the concentration of proteins, their interactions with other proteins or ligands, or their covalent modification underlies the potential utility of DSC analysis. A growing body of literature has demonstrated the versatility and performance of clinical DSC analysis across a range of biofluids and in a number of disease settings. The principles, practice and challenges of DSC analysis of plasma are described in this article.

A small molecule--DNA binding landscape.

This brief account traces the development of a "competition dialysis" method used to characterize the structural and sequence selectivity of DNA binding compounds. The method was inspired by a simple "differential dialysis" method pioneered by Don Crothers to explore base-selective intercalator binding. Results from compiled competition dialysis studies provide a small-molecule DNA binding landscape that shows a rich diversity of interactions and molecular recognition.

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.

Calorimetric analysis of the plasma proteome: identification of type 1 diabetes patients with early renal function decline.

BACKGROUND: Microalbuminuria (MA) has been questioned as a predictor of progressive renal dysfunction in patients with type 1 diabetes (T1D). Consequently, new clinical end points are needed that identify or predict patients that are at risk for early renal function decline (ERFD). The potential clinical utility of differential scanning calorimetry (DSC) analysis of blood plasma and other biofluids has recently been reported. This method provides an alternate physical basis with which to study disease-associated changes in the bulk plasma proteome. METHODS: DSC analysis of blood plasma was applied to identify unique signatures of ERFD in subjects enrolled in the 1st Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a prospective cohort study of T1D patients. Recent data suggests that differences in the plasma peptidome of these patients correlate with longitudinal measures of renal function. Differences in DSC profile (thermogram) features were evaluated between T1D MA individuals exhibiting ERFD (n=15) and matched control subjects (n=14). RESULTS: The average control group thermogram resembled a previously defined healthy thermogram. Differences were evident between ERFD and control individuals. Heat capacity values of the main two transitions were found to be significant discriminators of patient status. CONCLUSIONS: Results from this pilot study suggest the potential utility of DSC proteome analysis to prognostic indicators of renal disease in T1D. GENERAL SIGNIFICANCE: DSC shows sensitivity to changes in the bulk plasma proteome that correlate with clinical status in T1D providing additional support for the utility of DSC profiling in clinical diagnostics.

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.

Supramolecular DNA-based catalysis in organic solvents.

The distinct folding accompanied by its polymorphic character renders DNA G-quadruplexes promising biomolecular building blocks to construct novel DNA-based and supramolecular assemblies. However, the highly polar nature of DNA limits the use of G-quadruplexes to water as a solvent. In addition, the archetypical G-quadruplex fold needs to be stabilized by metal-cations, which is usually a potassium ion. Here, we show that a noncovalent PEGylation process enabled by electrostatic interactions allows the first metal-free G-quadruplexes in organic solvents. Strikingly, incorporation of an iron-containing porphyrin renders the self-assembled metal-free G-quadruplex catalytically active in organic solvents. Hence, these "supraG4zymes" enable DNA-based catalysis in organic media. The results will allow the broad utilization of DNA G-quadruplexes in nonaqueous environments.

Calculation of hydrodynamic properties for G-quadruplex nucleic acid structures from in silico bead models.

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 many 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.

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.

Isothermal folding of G-quadruplexes.

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.

Populated intermediates in the thermal unfolding of the human telomeric quadruplex.

Thermal denaturation profiles of several model oligonucleotides of the human telomere DNA sequence including d[A(GGGTTA)(3)GGG] (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 nonlinear 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 ⇌ I(n) ⇌ U, n = 0, 1, or 2) as well as two mechanisms with spectroscopically distinct starting structures (F(1) and F(2)). 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 I(2) had a transition midpoint temperature (T(m)) of 61 °C and a CD spectrum with a maximum and minimum at ~265 and ~245 nm, respectively. 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.