Intrastrand triplex DNA repeats in bacteria: a source of genomic instability.

Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at http://bioinformatics.uni-konstanz.de/utils/ITxF/. Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.

Structural characterization of quadruplex DNA with in-cell EPR approaches.

Guanosine-rich DNA sequences have the potential to adopt four-stranded conformations termed quadruplexes. The chromosomes of higher organisms are capped by so-called telomeres that are composed of repeats of the sequence TTAGGG. Up to 200 nucleotides of the G-rich strand form an overhang that is suspected to fold into intramolecular G-quadruplexes. Since induction of quadruplexes at the telomeres results in anti-proliferative effects, the intracellular structure of G-quadruplexes is of high interest as an anti-cancer drug target. Here we give a perspective on the elucidation of DNA sequence folds by electron paramagnetic resonance (EPR) distance measurements. The technique complements X-ray crystallography and NMR spectroscopy, as it can be applied in noncrystalline states, is not intrinsically limited by the size of the bio-macromolecular complex, and is able to analyze flexible structures or coexisting DNA conformation.

Shape Analysis of DNA-Au Hybrid Particles by Analytical Ultracentrifugation.

Current developments in nanotechnology have increased the demand for nanocrystal assemblies with well-defined shapes and tunable sizes. DNA is a particularly well-suited building block in nanoscale assemblies because of its scalable sizes, conformational variability, and convenient self-assembly capabilities via base pairing. In hybrid materials, gold nanoparticles (AuNPs) can be assembled into nanoparticle structures with programmable interparticle distances by applying appropriate DNA sequences. However, the development of stoichiometrically defined DNA/NP structures is still challenging since product mixtures are frequently obtained and their purification and characterization is the rate-limiting step in the development of DNA-NP hybrid assemblies. Improvements in nanostructure fractionation and characterization techniques offer great potential for nanotechnology applications in general. This study reports the application of analytical ultracentrifugation (AUC) for the characterization of anisotropic DNA-linked metal-crystal assemblies. On the basis of transmission electron microscopy data and the DNA primary sequence, hydrodynamic bead models are set up for the interpretation of the measured frictional ratios and sedimentation coefficients. We demonstrate that the presence of single DNA strands on particle surfaces as well as the shape factors of multiparticle structures in mixtures can be quantitatively described by AUC. This study will significantly broaden the possibilities to analyze mixtures of shape-anisotropic nanoparticle assemblies. By establishing insights into the analysis of nanostructure mixtures based on fundamental principles of sedimentation, a wide range of potential applications in basic research and industry become accessible.

Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd(3+)-DOTA.

Gd(3+) is increasingly used in EPR spectroscopy due to its increased intracellular stability and signal-to-noise ratios. Here we present the incorporation of Gd(3+)-DOTA into internal positions in DNA. Distance measurements via pulsed Electron Paramagnetic Resonance (EPR) spectroscopy in vitro and in cellula proved enhanced stability and efficiency compared to nitroxide labels.

A matter of location: influence of G-quadruplexes on Escherichia coli gene expression.

We provide important insights into secondary-structure-mediated regulation of gene expression in Escherichia coli. In a comprehensive survey, we show that the strand orientation and the exact position of a G-quadruplex sequence strongly influence its effect on transcription and translation. We generated a series of reporter gene constructs that contained systematically varied positions of quadruplexes and respective control sequences inserted into several positions within the promoter, 50-UTR, and 30-UTR regions. G-rich sequences at specific locations in the promoter and also in proximity to the ribosome-binding site (RBS) showed pronounced inhibitory effects. Additionally, we rationally designed a system where quadruplex formation showed a gene-activating behavior. Moreover, we characterized quadruplexes in proximity to the RBS that occur naturally in E. coli genes, demonstrating that some of these quadruplexes exert significant modulation of gene expression. Taken together, our data show strong position-dependent effects of quadruplex secondary structures on bacterial gene expression.