Structural characterization of G-Quadruplexes within duplex DNA

The G-Quadruplex (GQ) is a repetitive, guanine rich DNA sequence that occurs throughout the genome. This specific DNA motif is capable of forming an alternative DNA secondary structure similar to a rectangular prism. The most common GQ motif consists of four sets of three guanine bases separated by varying intervening loops, typically comprised of 1 to 9 bases in length. The four sets of guanine triplets stabilize the GQ structure through Hoogsteen base pairing with the assistance of monovalent cations. Two predominant folding motifs of these structures have been identified within a single-stranded (ss) DNA context: parallel and antiparallel. Further work is needed to establish if these structural folding trends are consistent in the double-stranded (ds) DNA context. Limited studies in this area have occurred due to the lack of available methodologies for characterizing these GQ structures within a duplex DNA context (i.e. a GQ structure with its complementary sequence embedded within a dsDNA context). The biological importance of these structures can be traced to GQ involvement in regulation of replication and transcription, genome rearrangements, translation and telomere processing. In support of experimental findings, computational studies have revealed a 230-fold enrichment of GQ sequences in upstream of promoters over the genomic average, amounting to nearly half of all human promoters containing putative GQ sequences. Moreover, GQ sequences are highly likely to be found in oncogenes and regulatory genes. Auxiliary to these observations are findings that GQ sequences are less likely to be located within the template strand, coding regions, tumor suppressor genes, and housekeeping genes. The highly selective positions of GQ imply that GQs may regulate particular set of biological processes and suggest that the stabilization of the structure may serve as a novel pharmaceutical target. Despite the plethora of reports on telomeric DNA, relatively few studies have looked into GQs located within genomic regions. Furthermore, most studies focused on several well-characterized sequences such as c-Myc, TERT and BCL2 formed in the context of ssDNA. Although ssDNA may be relevant for studying the telomeric overhang (naturally single-stranded), it cannot be an appropriate platform for investigating ~800,000 putative GQ-forming sequences in dsDNA found throughout the human genome. Previously accepted methodologies in GQ structural investigations have proven to be cumbersome, at best, when utilized within the dsDNA context. Additionally, current, widely-available GQ investigational tools provide only qualitative insight into GQ formation and structure. Circular dichroism, the standard methodology for identifying folding properties of putative GQ sequences, fails to provide characteristic signals for GQs when probing dsDNA. The limitations of current techniques provide an opportunity for the development of more quantitative, biologically applicable analysis tools. We have developed a bulk-phase induced fluorescence-based assay that can distinguish between folded and unfolded GQs, as well as identify predominant folding motifs (parallel or antiparallel). Our central objective is to elucidate the rules governing GQ folding within dsDNA and identify potential GQs that can affect crucial biological processes such as replication, transcription and translation. This thesis describes four key findings. First, GQ formation is much less robust within the duplex setting as compared to single-stranded contexts. Second, stable GQ folding within a dsDNA context is driven by both sequence composition and loop length. Third, strong GQ folding within genomic DNA is underrepresented near genetic regulatory elements. Fourth, GQ folding imposes barrier effects in gene expression in e. coli. Based on these findings, a comprehensive GQ folding atlas was developed which highlights potentially important GQ structures and their function in gene expression

G-quadruplex DNA and the Regulation of Human Telomere Accessibility

Human telomeres are nucleoprotein structures that cap the ends of chromosomes, preventing them from degradation. In normal cells, they are shortened due to the end replication problem. However, most cancer cells overcome this inherent growth limitation by upregulating telomerase, an enzyme that elongates telomeres to yield an immortal phenotype. The remaining cancerous cells immortalize by activating the alternative telomerelengthening pathway (ALT). Thus, treatments that directly target the telomere could disrupt both mechanisms the cancer cell employs for unlimited proliferation. The telomere overhang is G-rich DNA that spontaneously folds into a G-quadruplex. In vitro, the G-quadruplex structure has been shown to inhibit telomere extension by telomerase, making it an attractive potential therapeutic target. However, G-quadruplex capping properties and regulatory roles are still poorly understood. In this study, we used a FRET-based assay to probe the stability and accessibility of the G-quadruplex. We examined the folding behavior of variable telomeric repeat lengths (4- 8). Accessibility of the G-quadruplex was measured by the rate of quadruplex unfolding after the addition of complementary DNA, ALT pathway proteins, and telomerase. These measurements revealed a distinct periodic pattern of overhang accessibility where DNA and protein binding is limited for multiples of four TTAGGG repeats, whereas five to seven repeats are more accessible (7>6>5). Contrastingly, POT1, a telomere-specific binding protein, showed accessibility independent of the number of repeats. These results demonstrate the role of G-quadruplexes as physical impedances to the binding of telomere associated proteins and as critical regulatory structures for theOpe

Predicting G-quadruplex Formation

Guanine-rich regions of genomic DNA can spontaneously fold into secondary structures called G-quadruplexes (GQs). Akin to tiny switches, GQs regulate genetic processes through their folding and unfolding. Their interest to basic science, as well as their potential as therapeutic targets for human diseases, has motivated the creation of computational tools for their prediction. Currently, GQ folding predictors are based on results from studies of GQs formed in single-stranded DNA. As a result, existing tools perform poorly when applied to the prediction of GQ formation in double-stranded (ds) DNA, the native context within which genomic GQs are found. Here, we present a probabilistic model of GQ formation, which is learned from large-scale human genomic pull-down experiments and applied to the analysis of gene ontological data. Advances in the characterization of GQs in dsDNA have enabled us to integrate results from small-molecule binding assays and singlemolecule FRET microscopy into our model. In order to obtain training sets of sequences, we identified nearly 700,000 unique, potential GQs and categorized them according to pulldown experiment outcomes. Model parameters learned from these training sets agree with experimental evidence and, when asked to predict the folding of dsDNA GQ sequences, outperformed existing models of GQ folding. This tool can be applied to genomic sequences to locate the most strongly forming GQs, revealing valuable information for the design of GQ-targeting therapies, and represents the next step toward the practical, widespread use of GQs in medicine and technology.Ope

Telomeric Overhang Length Determines Structural Dynamics and Accessibility to Telomerase and ALT-Associated Proteins

SummaryThe G-rich single-stranded DNA at the 3′ end of human telomeres can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase or the recombination-based alternative lengthening of telomere (ALT) mechanism requires protein loading on the overhang. Using single-molecule fluorescence spectroscopy, we discovered that lengthening the telomeric overhang also increased the rate of dynamic exchanges between structural conformations. Overhangs with five to seven TTAGGG repeats, compared with four repeats, showed much greater dynamics and accessibility to telomerase binding and activity and loading of the ALT-associated proteins RAD51, WRN, and BLM. Although the eight repeats are highly dynamic, they can fold into two GQs, which limited protein accessibility. In contrast, the telomere-specific protein POT1 is unique in that it binds independently of repeat number. Our results suggest that the telomeric overhang length and dynamics may contribute to the regulation of telomere extension via telomerase action and the ALT mechanism

Walk well:a randomised controlled trial of a walking intervention for adults with intellectual disabilities: study protocol

Background - Walking interventions have been shown to have a positive impact on physical activity (PA) levels, health and wellbeing for adult and older adult populations. There has been very little work carried out to explore the effectiveness of walking interventions for adults with intellectual disabilities. This paper will provide details of the Walk Well intervention, designed for adults with intellectual disabilities, and a randomised controlled trial (RCT) to test its effectiveness. Methods/design - This study will adopt a RCT design, with participants allocated to the walking intervention group or a waiting list control group. The intervention consists of three PA consultations (baseline, six weeks and 12 weeks) and an individualised 12 week walking programme. A range of measures will be completed by participants at baseline, post intervention (three months from baseline) and at follow up (three months post intervention and six months from baseline). All outcome measures will be collected by a researcher who will be blinded to the study groups. The primary outcome will be steps walked per day, measured using accelerometers. Secondary outcome measures will include time spent in PA per day (across various intensity levels), time spent in sedentary behaviour per day, quality of life, self-efficacy and anthropometric measures to monitor weight change. Discussion - Since there are currently no published RCTs of walking interventions for adults with intellectual disabilities, this RCT will examine if a walking intervention can successfully increase PA, health and wellbeing of adults with intellectual disabilities

Cardiovascular risk among Aboriginal and non-Aboriginal smoking male prisoners: inequalities compared to the wider community

<p>Abstract</p> <p>Background</p> <p>Cardiovascular risk factors (CVRF) were collected as part of a randomised controlled trial of a multi-component intervention to reduce smoking among male prisoners. Cross-sectional baseline data on CVRF were compared among smoking male prisoners and males of similar age in the general population.</p> <p>Methods</p> <p>425 smoking prisoners were recruited (n = 407 in New South Wales; 18 in Queensland), including 15% of Aboriginal descent (mean age 33 years; median sentence length 3.6 years). We measured CVRF such as smoking, physical activity, blood pressure, risky alcohol use, symptoms of depression, and low socioeconomic status.</p> <p>Results</p> <p>We found that 39% of prisoners had 3+ CVRF, compared to 10% in a general community sample of most disadvantaged men of a similar age. Significantly more Aboriginal prisoners had 3+ CVRF than non-Aboriginal prisoners (55% vs 36%, p < 0.01) and were twice as likely to have 4+ CVRF (27% vs 12%). In addition to all prisoners in this study being a current smoker (with 70% smoking 20+ cigarettes per day), the prevalence of other CVRF was very high: insufficient physical activity (23%); hypertension (4%), risky drinking (52%), symptoms of depression (14%) and low socioeconomic status (SES) (44%). Aboriginal prisoners had higher levels of risky alcohol use, symptoms of depression, and were more likely to be of low SES.</p> <p>Conclusion</p> <p>Prisoners are at high risk for developing cardiovascular disease compared to even the most disadvantaged in their community and should be the focus of specific public health interventions.</p> <p>Trial Registration</p> <p>This trial is registered with the Australian New Zealand Clinical Trials Registry <a href="http://www.anzctr.org.au/ACTRN12606000229572.aspx">ACTRN#12606000229572</a>.</p

Characterization of inter-genomic and telomeric G-quadruplex structures through atomic force microscopy

The G-quadruplex DNA structure, a secondary folding motif of DNA, is found throughout the genome. The dynamics of these structures has been seen to affect processes such as telomerase elongation and regulation of gene transcription. Typical investigation techniques require these structures to be present in very high concentrations and in non-biological buffers. Adding to the issues with current methods of investigation, this structure currently relies on bulk measurements, which limit the accuracy with which it can be measured. In this report, we present newly developed atomic force microscopy-based assays for quantifying G-quadruplex folding patterns with sub nanometer accuracy. The assays are based upon a duplex DNA handle ranging from 18 to 525 base pairs in length containing an overhang sequence utilized to attach the DNA to a mica surface. The overhang sequence pairs with a possible G-quadruplex forming sequence and allows for the retention and identification of the folded structure. Using a 12 base DNA probe, we demonstrate that the location of G-quadruplex folding on long strands of DNA are not preferred to occur at the distal end, contrary to DMS foot printing studies. We demonstrate that this method enables detection of the two major folding motifs and is able to distinguish height differences between them. The high resolution and low concentration requirements, combined with the ability to image hundreds of single molecules per study, allow this method to provide quick and accurate characterization of G-quadruplex folding