Hypertension 66 (20.3%) 24 (24.2%) 30 (16.3%) NS Diabetes 20 (6.2%) 7 (7.1%) 10 (5.4%) NS Excess weight 78 (24%) 27 (27.3%) 44 (23.9%) NS Smokers 64 (19.7%) 17 (17.2%) 35 (19.0%) NS Age >50 years 137 (42.2%) 54 (54.5%) 67 (36.4%) <0.02 Kidney disease 7 (2.2%) 1 (1%) 5 (2.7%) NS Family history, DM 102 (31.4%) 28 (28.3%) 66 (35.9%) NS

Conflict of interest statement. None declared.

In addition to the canonical double helix, DNA can fold into various other inter- and intramolecular secondary structures. Although many such structures were long thought to be in vitro artefacts, bioinformatics demonstrates that DNA sequences capable of forming these structures are conserved throughout evolution, suggesting the existence of non-B-form DNA in vivo. In addition, genes whose products promote formation or resolution of these structures are found in diverse organisms, and a growing body of work suggests that the resolution of DNA secondary structures is critical for genome integrity. This Review focuses on emerging evidence relating to the characteristics of G-quadruplex structures and the possible influence of such structures on genomic stability and cellular processes, such as transcription.

DNA secondary structures: stability and function of G-quadruplex structures

Detection of analytes by means of field-effect transistors bearing ligand-specific receptors is fundamentally limited by the shielding created by the electrical double layer (the "Debye length" limitation). We detected small molecules under physiological high-ionic strength conditions by modifying printed ultrathin metal-oxide field-effect transistor arrays with deoxyribonucleotide aptamers selected to bind their targets adaptively. Target-induced conformational changes of negatively charged aptamer phosphodiester backbones in close proximity to semiconductor channels gated conductance in physiological buffers, resulting in highly sensitive detection. Sensing of charged and electroneutral targets (serotonin, dopamine, glucose, and sphingosine-1-phosphate) was enabled by specifically isolated aptameric stem-loop receptors.

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Aptamer–field-effect transistors overcome Debye length limitations for small-molecule sensing

Quadruplex-forming sequences are widely prevalent in human and other genomes, including bacterial ones. These sequences are over-represented in eukaryotic telomeres, promoters, and 5′ untranslated regions. They can form quadruplex structures, which may be transient in many situations in normal cells since they can be effectively resolved by helicase action. Mutated helicases in cancer cells are unable to unwind quadruplexes, which are impediments to transcription, translation, or replication, depending on their location within a particular gene. Small molecules that can stabilize quadruplex structures augment these effects and produce cell and proliferation growth inhibition. This article surveys the chemical biology of quadruplexes. It critically examines the major classes of quadruplex-binding small molecules that have been developed to date and the various approaches to discovering selective agents. The challenges of requiring (and achieving) small-molecule targeted selectivity for a particular quadrup...

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Quadruplex Nucleic Acids as Novel Therapeutic Targets.

INTRODUCTION Many cellular RNAs contain regions that fold into stable structures required for function. Both Watson−Crick and noncanonical interactions can play important roles in forming these structures. An intriguing noncanonical structure is the RNA G-quadruplex (RG4), a four-stranded structure containing two or more layers of G-quartets, in which the Watson–Crick face of each of four G residues pairs to the Hoogsteen face of the neighboring G residues. RG4 regions can be very stable in vitro, particularly in the presence of K + , and thus they are generally assumed to be predominantly folded within cells, which have ample K + . Indeed, these structures have been implicated in mRNA processing and translation, with recently proposed roles in cancer and other human diseases. However, the number of cellular RNAs that can fold into RG4 structures has been unclear, as has been the extent to which these RG4 regions are folded in cells. RATIONALE Enzymes and chemicals that act on RNA with structure-dependent preferences provide valuable tools for detecting and monitoring RNA folding. For example, dimethyl sulfate (DMS) treatment of RNA, either in vitro or in cells, coupled with high-throughput sequencing of abortive primer-extension products can monitor the folding states of many RNAs in one experiment. Analogous high-throughput methods use cell-permeable variants of SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) reagents. These methods reveal important differences between RNA structures formed in vivo and those formed in vitro. However, they are designed to detect Watson−Crick pairing and thus do not identify RG4 structures or provide information on their folding states. After recognizing that RG4 regions can block reverse transcriptase, we reasoned that this property, together with the known ability of RG4s to protect the N7 of participating G nucleotides from DMS modification, could be used to develop a suite of high-throughput methods to both identify endogenous RNAs that can fold into RG4s in vitro and determine whether these regions also fold in cells. RESULTS We first developed a high-throughput method that identifies RG4 regions on the basis of their propensity to stall reverse transcriptase in a K + -dependent manner. Applying this method to RNA from mammalian cell lines and yeast, we identified >10,000 endogenous regions that form RG4s in vitro, thereby expanding by a factor of >100 the catalog of endogenous regions with experimentally supported propensity to fold into RG4 structures. To infer the folding state of these RG4 regions in vitro and in cells, DMS treatment was performed before profiling of reverse-transcriptase stops. These analyses showed that, in contrast to previous assumptions, regions that folded into RG4 structures in vitro were overwhelmingly unfolded in vivo, as indicated by their accessibility to DMS modification in cells. A complementary probing strategy using a SHAPE reagent confirmed the unfolded state of most RG4 regions in eukaryotic cells. Moreover, RG4 regions remained unfolded both in cells depleted of adenosine 5′-triphosphate and in cells lacking a helicase known to unfold RG4 regions in vitro. Applying our probing methods to bacteria revealed a different behavior, in that model RG4 regions that were unfolded in eukaryotic cells were folded when expressed in Escherichia coli . However, these ectopically expressed quadruplexes impaired mRNA translation and cell growth, which helps explain why very few endogenous sequences that could fold into RG4s were detected in the transcriptomes of E. coli and the two other eubacteria analyzed. CONCLUSION In mammals, thousands of endogenous RNA sequences have regions that can fold into RG4s in vitro, but these regions are globally unfolded in eukaryotic cells, presumably by robust and effective machinery that remains to be fully characterized. In contrast, RG4 regions are permitted to fold in E. coli cells, but E. coli and other bacteria have undergone evolutionary depletion of endogenous RG4-forming sequences.

RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria

Here we review studies that provided important information about conformational properties of DNA using circular dichroic (CD) spectroscopy. The conformational properties include the B-family of structures, A-form, Z-form, guanine quadruplexes, cytosine quadruplexes, triplexes and other less characterized structures. CD spectroscopy is extremely sensitive and relatively inexpensive. This fast and simple method can be used at low- as well as high-DNA concentrations and with short- as well as long-DNA molecules. The samples can easily be titrated with various agents to cause conformational isomerizations of DNA. The course of detected CD spectral changes makes possible to distinguish between gradual changes within a single DNA conformation and cooperative isomerizations between discrete structural states. It enables measuring kinetics of the appearance of particular conformers and determination of their thermodynamic parameters. In careful hands, CD spectroscopy is a valuable tool for mapping conformational properties of particular DNA molecules. Due to its numerous advantages, CD spectroscopy significantly participated in all basic conformational findings on DNA.

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Circular dichroism and conformational polymorphism of DNA

Circular dichroism and guanine quadruplexes

Circular dichroism spectroscopy of DNA: from duplexes to quadruplexes.

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G(3)(TTAG(3))(3) forms an antiparallel quadruplex of the same basket type in solution containing either K(+) or Na(+) ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K(+)-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G(3)(TTAG(3))(3) motif. Both G(3)(TTAG(3))(3) and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K(+)-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K(+)-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG(3)(TTAG(3))(3) by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K(+) ions.

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions

Hydroxymethylcytosine (5-hmC) was recently identified as a relatively frequent base in eukaryotic genomes. Its physiological function is still unclear, but it is supposed to serve as an intermediate in DNA de novo demethylation. Using X-ray diffraction, we solved five structures of four variants of the d(CGCG AATTCGCG) dodecamer, containing either 5-hmC or 5-methylcytosine (5-mC) at position 3 or at position 9. The observed resolutions were between 1.42 and 1.99 A ˚ . Cytosine modification in all cases influences neither the whole B-DNA double helix structure nor the modified base pair geometry. The additional hydroxyl group of 5-hmC with rotational freedom along the C5-C5A bond is preferentially oriented in the 3 0 direction. A comparison of thermodynamic properties of the dodecamers shows no effect of 5- mC modification and a sequence-dependent only slight destabilizing effect of 5-hmC modification. Also taking into account the results of a previous functional study (Mun zel et al. (2011) (Improved synthesis and mutagenicity of oligonucleotides containing 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Chem. Eur. J., 17, 13782� 13788)), we conclude that the 5 position of cytosine is an ideal place to encode epigenetic information. Like this, neither the helical structure nor the thermodynamics are changed, and polymerases cannot distinguish 5- hmC and 5-mC from unmodified cytosine, all these effects are making the former ones non-mutagenic.

Daniel Renčiuk

Papers

Circular dichroism and conformational polymorphism of DNA

Circular dichroism and guanine quadruplexes

Circular dichroism spectroscopy of DNA: from duplexes to quadruplexes.

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions

Crystal structures of B-DNA dodecamer containing the epigenetic modifications 5-hydroxymethylcytosine or 5-methylcytosine.