Circular dichroism and conformational polymorphism of DNA

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

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoRSl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoRSl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoRSl-DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoRSl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control. © 2013 The Author(s)

Control of bacterial nitrate assimilation by stabilization of G-quadruplex DNA

Here we present a chemical-biology study in the model soil bacterium Paracoccus denitrificans, where we show ligand-specific control of nitrate assimilation. Stabilization of a G-quadruplex in the promoter region of the nas genes, encoding the assimilatory nitrate/nitrite reductase system, is achieved using known quadruplex ligands and results in attenuation of gene transcription

Rudimentary G-Quadruplex-Based Telomere Capping In Saccharomyces Cerevisiae

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3\u27 overhang inhibits 5\u27-\u3e 3\u27 resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo

Cancer-selective antiproliferative activity is a general property of some G-rich oligodeoxynucleotides

Oligodeoxynucleotide libraries containing randomly incorporated bases are used to generate DNA aptamers by systematic evolution of ligands by exponential enrichment (SELEX). We predicted that combinatorial libraries with alternative base compositions might have innate properties different from the standard library containing equimolar A + C + G + T bases. In particular, we hypothesized that G-rich libraries would contain a higher proportion of quadruplex-forming sequences, which may impart desirable qualities, such as increased nuclease resistance and enhanced cellular uptake. Here, we report on 11 synthetic oligodeoxynucleotide libraries of various base combinations and lengths, with regard to their circular dichroism, stability in serum-containing medium, cellular uptake, protein binding and antiproliferative activity. Unexpectedly, we found that some G-rich libraries (composed of G + T or G + C nucleotides) strongly inhibited cancer cell growth while sparing non-malignant cells. These libraries had spectral features consistent with G-quadruplex formation, were significantly more stable in serum than inactive libraries and showed enhanced cellular uptake. Active libraries generally had strong protein binding, while the pattern of protein binding suggested that G/T and G/C libraries have distinct mechanisms of action. In conclusion, cancer-selective antiproliferative activity may be a general feature of certain G-rich oligodeoxynucleotides and is associated with quadruplex formation, nuclease resistance, efficient cellular uptake and protein binding

Redox-dependent control of i-Motif DNA structure using copper cations

Previous computational studies have shown that Cu+ can act as a substitute for H+ to support formation of cytosine (C) dimers with similar conformation to the hemi-protonated base pair found in i-motif DNA. Through a range of biophysical methods, we provide experimental evidence to support the hypothesis that Cu+ can mediate C–C base pairing in i-motif DNA and preserve i-motif structure. These effects can be reversed using a metal chelator, or exposure to ambient oxygen in the air that drives oxidation of Cu+ to Cu2+, a comparatively weak ligand. Herein, we present a dynamic and redox-sensitive system for conformational control of an i-motif forming DNA sequence in response to copper cations

Investigating the Antiproliferative Activity of High Affinity DNA Aptamer on Cancer Cells

10.1371/journal.pone.0050964PLoS ONE81

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich ‘structurally poor’ RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5–100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using ‘structurally poor’ RNA domains in regulating biological process

Solution structures of all parallel-stranded monomeric and dimeric G-quadruplex scaffolds of the human c-kit2 promoter

Previous studies have demonstrated that nuclease hypersensitivity regions of several proto-oncogenic DNA promoters, situated upstream of transcription start sites, contain guanine-rich tracts that form intramolecular G-quadruplexes stabilized by stacked G•G•G•G tetrads in monovalent cation solution. The human c-kit oncogenic promoter, an important target in the treatment of gastrointestinal tumors, contains two such stretches of guanine-rich tracts, designated c-kit1 and c-kit2. Our previous nuclear magnetic resonance (NMR)-based studies reported on the novel G-quadruplex scaffold of the c-kit1 promoter in K+-containing solution, where we showed for the first time that even an isolated guanine was involved in G-tetrad formation. These NMR-based studies are now extended to the c-kit2 promoter, which adopts two distinct all-parallel-stranded conformations in slow exchange, one of which forms a monomeric G-quadruplex (form-I) in 20 mM K+-containing solution and the other a novel dimeric G-quadruplex (form-II) in 100 mM K+-containing solution. The c-kit2 promoter dimeric form-II G-quadruplex adopts an unprecedented all-parallel-stranded topology where individual c-kit2 promoter strands span a pair of three-G-tetrad-layer-containing all-parallel-stranded G-quadruplexes aligned in a 3′ to 5′-end orientation, with stacking continuity between G-quadruplexes mediated by a sandwiched A•A non-canonical pair. We propose that strand exchange during recombination events within guanine-rich segments, could potentially be mediated by a synapsis intermediate involving an intergenic parallel-stranded dimeric G-quadruplex

The Formation and Stabilization of a Novel G-Quadruplex in the 5′-Flanking Region of the Relaxin Gene

It has been reported that binding of STAT3 protein to the 5′-flanking region of the relaxin gene may result in downregulation of the relaxin expression. There is a Guanine(G)-rich segment located in about 3.8 Kb upstream of the relaxin gene and very close to the STAT3's binding site. In our study, NMR spectroscopy revealed the formation of G-quadruplex by this G-rich strand, and the result was confirmed by ESI mass spectrometry and CD spectroscopy. The theoretical structure of RLX G-quadruplex was constructed and refined by molecular modeling. When this relaxin G-quadruplex was stabilized by berberine(ΔTm = 10°C), a natural alkaloid from a Chinese herb, the gene expression could be up-regulated in a dose-dependent manner which was proved by luciferase assay. This result is different from the general G-quadruplex function that inhibiting the telomere replication or down-regulating many oncogenes expression. Therefore, our study reported a novel G-quadruplex in the relaxin gene and complemented the regulation mechanism about gene expression by G-quadruplexes