Abstract 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.

The protein POT1 ( P rotection o f T elomeres 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 first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 – DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

With the long-term goal of developing an ultra-sensitive microcantilever-based biosensor for versatile biomarker detection, new controlled bioreceptor-analytes systems are being explored to overcome the disadvantages of conventional ones. Gold (Au) microwires have been used as a probe to overcome the tolerance problem that occurs in response to changes in environmental conditions. However, the cytotoxicity of Au microwires is still unclear. Here, we examined the cytotoxicity of Au microwires systems using both commercial and as-synthesised Au microwires. In vitro experiments show that commercial Au microwires with an average quoted length of 5.6 µm are highly toxic against Gram-negative Escherichia coli ( E. coli ) at 50 µg/mL. However, this toxicity is due to the presence of CTAB surfactant not by the microwires. Conversely, the as-synthesised Au microwires show non-cytotoxicity even at the maximum viable concentration (330 µg/mL). These findings may lead to the development of potentially life-saving cytotoxicity-free biosensors for an early diagnostic of potential diseases.

We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program automates identification and tracing of individual molecules in circular and linear conformations without user input. TopoStats was able to identify and trace a range of molecules within AFM images, finding, on average, 90% of all individual molecules and molecular assemblies within a wide field of view, and without the need for prior processing. DNA minicircles of varying size, DNA origami rings and pore forming proteins were identified and accurately traced with contour lengths of traces typically within 10 nm of the predicted contour length. TopoStats was also able to reliably identify and trace linear and enclosed circular molecules within a mixed population. The program is freely available via GitHub ( https://github.com/afm-spm/TopoStats ) and is intended to be modified and adapted for use if required.

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.

Super-resolution microscopy enables the study of vaccinia architecture at subviral resolution with molecular specificity. Here, we outline how to use structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM) to detect fluorescently tagged or immunolabeled viral proteins on purified virions. Tens to hundreds of individual virions can be imaged in a single field of view providing data for single-particle averaging or quantitative analysis of viral protein spatial organization.

The protein ALL1 fused from chromosome 1q (AF1q) is overexpressed in a variety of cancers and acts to activate several signaling pathways that lead to oncogenesis. For example, AF1q has been shown to interact with T-cell Factor 7 (TCF7; also known as TCF1) from the Wnt/beta-catenin pathway resulting in the transcriptional activation of the CD44 and the enhancement of breast cancer metastasis. Despite the importance of AF1q in facilitating oncogenesis and metastasis, the structural and biophysical properties of AF1q remain largely unexplored due to the absence of a viable method for producing recombinant protein. Here, we report the overexpression of AF1q in E. coli as a fusion to a N-terminal His e-tag, which forms inclusion bodies (IBs) during expression. The AF1q protein was purified from IBs under denaturing conditions by immobilized metal affinity chromatography followed by a successful one-step dialysis refolding. Refolded AF1q was further purified to homogeneity by gel filtration chromatography resulting in an overall yield of 35 mg/L culture. Our nuclear magnetic resonance (NMR) and analytical ultracentrifugation (AUC) measurements reveal AF1q interacts with TCF7, specifically with TCF7's high-mobility group (HMG) domain (residues 154-237), which is, to our knowledge, the first biophysical characterization of the AF1q and TCF7 interaction.

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins(1). For many viruses, the clustering of fusion proteins-and their distribution on virus particles-is crucial for fusion activity(2,3). Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion(4). However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity(5). Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

Circular dichroism and stopped-flow UV spectroscopies were used to investigate the thermodynamic stability and the folding pathway of d(TGAG3TG3TAG3TG3TA2) at 25C in solutions containing 25 mM KCl. Under these conditions the oligonucleotide adopts a thermally stable, all-parallel G-quadruplex topography containing three stacked quartets. K+-induced folding shows three resolved relaxation times, each with distinctive spectral changes. Folding is complete within 200 s. These data indicate a folding pathway that involves at least two populated intermediates, one of which seems to be an antiparallel structure that rearranges to the final all-parallel conformation. Molecular dynamics reveals a stereochemically plausible folding pathway that does not involve complete unfolding of the intermediate. The rate of unfolding was determined using complementary DNA to trap transiently unfolded states to form a stable duplex. As assessed by 1D-1H NMR and fluorescence spectroscopy, unfolding is extremely slow with only one observable rate-limiting relaxation time.

Mammalian and bird ribosomes are nearly twice the mass of prokaryotic ribosomes in part because of their extraordinarily long rRNA tentacles. Human rRNA tentacles are not fully observable in current three-dimensional structures and their conformations remain to be fully resolved. In previous work we identified sequences that favor G-quadruplexes in silico and in vitro in rRNA tentacles of the human large ribosomal subunit. We demonstrated by experiment that these sequences form G-quadruplexes in vitro. Here, using a more recent motif definition, we report additional G-quadruplex sequences on surfaces of both subunits of the human ribosome. The revised sequence definition reveals expansive arrays of potential G-quadruplex sequences on LSU tentacles. In addition, we demonstrate by a variety of experimental methods that fragments of the small subunit rRNA form G-quadruplexes in vitro. Prior to this report rRNA sequences that form G-quadruplexes were confined to the large ribosomal subunit. Our combined results indicate that the surface of the assembled human ribosome contains numerous sequences capable of forming G-quadruplexes on both ribosomal subunits. The data suggest conversion between duplexes and G-quadruplexes in response to association with proteins, ions, or other RNAs. In some systems it seems likely that the integrated population of RNA G-quadruplexes may be dominated by rRNA, which is the most abundant cellular RNA.

Super-resolution microscopy (SRM) has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for SRM designed to combine high performance and ease of use. We named it NanoJ—a reference to the popular ImageJ software it was developed for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image reconstruction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.

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.

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.

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.

The spatial distribution of binding and fusion proteins on most viruses and the functional relevance of this organization remains largely unexplored. Employing super-resolution microscopy we define the nanoscale membrane architecture of the prototypic poxvirus, vaccinia. We show that binding and entry fusion complex (EFC) proteins are organized into distinct functional domains with fusion proteins polarized to the tips of virions. Repression of individual EFC components disrupted fusion protein polarization, correlating with a loss of fusion activity. Repression of vaccinia A27, a non-EFC protein implicated in fusion, revealed that disruption of EFC localization impacts virus fusion pore formation. We propose that the polarized distribution of EFCs is essential for poxvirus fusion efficiency.

The Florida manatee (Trichechus manatus latirotris) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.

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 RFID assay, coupled with PCA and HCA, provides a useful tool for the discovery of ligands selective for particular nucleic acid structures.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm - VirusMapper - packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

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.

Circular dichroism (CD) is a phenomenon that arises from the differential absorption of left- and right-handed circularly polarized light, and may be seen with optically active molecules. CD spectroscopy provides useful spectral signatures for biological macromolecules in solution, and provides low-resolution structural information about macromolecular conformation. CD spectroscopy is particularly useful for monitoring conformational changes in macromolecules upon environmental perturbations. G-quadruplex structures show unique CD spectral signatures, and CD is an important tool for characterizing their formation and global structure. This protocol offers step-by-step methods for determining reliable and reproducible CD spectra of quadruplex structures and normalizing the spectra for presentation. CD spectra properly normalized with respect to quadruplex concentration and path length are required to facilitate accurate comparison of results among laboratories. The standard operating procedures proposed are recommended to make such comparison accurate and informative. © 2017 by John Wiley & Sons, Inc.