Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering.

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.

Strategy for modeling higher-order G-quadruplex structures recalcitrant to NMR determination.

Guanine-rich nucleic acids can form intramolecularly folded four-stranded structures known as G-quadruplexes (G4s). Traditionally, G4 research has focused on short, highly modified DNA or RNA sequences that form well-defined homogeneous compact structures. However, the existence of longer sequences with multiple G4 repeats, from proto-oncogene promoters to telomeres, suggests the potential for more complex higher-order structures with multiple G4 units that might offer selective drug-targeting sites for therapeutic development.

Higher-order G-quadruplexes in promoters are untapped drug targets.

G-quadruplexes (G4s) are four-stranded nucleic acid secondary structures that form within guanine-rich regions of chromatin. G4 motifs are abundant in the genome, with a sizable proportion (∼40%) existing within gene promoter regions. G4s are proven epigenetic features that decorate the promoter landscape as binding centers for transcription factors.

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.

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 . Unimolecular G4 can potentially form in sequences with runs of guanines interspersed with what will become loops in the folded structure: 5'GLGLGLG, where is typically 2-4 and is highly variable.

Drug discovery of small molecules targeting the higher-order hTERT promoter G-quadruplex.

DNA G-quadruplexes (G4s) are now widely accepted as viable targets in the pursuit of anticancer therapeutics. To date, few small molecules have been identified that exhibit selectivity for G4s over alternative forms of DNA, such as the ubiquitous duplex. We posit that the lack of current ligand specificity arises for multiple reasons: G4 atomic models are often small, monomeric, single quadruplex structures with few or no druggable pockets; targeting G-tetrad faces frequently results in the enrichment of extended electron-deficient polyaromatic end-pasting scaffolds; and virtual drug discovery efforts often under-sample chemical search space.

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.

POT1 stability and binding measured by fluorescence thermal shift assays.

The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions.

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.

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.

Putting a New Spin of G-Quadruplex Structure and Binding by Analytical Ultracentrifugation.

Analytical ultracentrifugation is a powerful biophysical tool that provides information about G-quadruplex structure, stability, and binding reactivity. This chapter provides a simplified explanation of the method, along with examples of how it can be used to characterize G4 formation and to monitor small-molecule binding.

G-quadruplex virtual drug screening: A review.

Over the past two decades biologists and bioinformaticians have unearthed substantial evidence supporting a role for G-quadruplexes as important mediators of biological processes. This includes telomere damage signaling, transcriptional activity, and splicing. Both their structural heterogeneity and their abundance in oncogene promoters makes them ideal targets for drug discovery.