CRISPR-guided DNA polymerase enabling diversification of all nucleotides in a tunable window

The capacity to diversify genetic codes advances our understanding and engineering of biological systems. A method to continuously diversify user-defined regions of a genome without requiring the integration of nucleic acid libraries would enable forward genetic approaches in systems not amenable to high efficiency homologydirected integration, rapid evolution of biotechnologically useful activity through accelerated and parallelized rounds of mutagenesis and selection, and cell lineage tracking. Here we developed EvolvR, the first system that can continuously diversify all nucleotides within a tunable window length at user-defined loci. Our results demonstrate that EvolvR enables multiplexed and continuous diversification of user-defined genomic loci that will be useful for a broad range of basic and biotechnological applications

Molecular flexibility in ab initio drug docking to DNA: binding-site and binding-mode transitions in all-atom Monte Carlo simulations

The dynamics of biological processes depend on the structure and flexibility of the interacting molecules. In particular, the conformational diversity of DNA allows for large deformations upon binding. Drug–DNA interactions are of high pharmaceutical interest since the mode of action of anticancer, antiviral, antibacterial and other drugs is directly associated with their binding to DNA. A reliable prediction of drug–DNA binding at the atomic level by molecular docking methods provides the basis for the design of new drug compounds. Here, we propose a novel Monte Carlo (MC) algorithm for drug–DNA docking that accounts for the molecular flexibility of both constituents and samples the docking geometry without any prior binding-site selection. The binding of the antimalarial drug methylene blue at the DNA minor groove with a preference of binding to AT-rich over GC-rich base sequences is obtained in MC simulations in accordance with experimental data. In addition, the transition between two drug–DNA-binding modes, intercalation and minor-groove binding, has been achieved in dependence on the DNA base sequence. The reliable ab initio prediction of drug–DNA binding achieved by our new MC docking algorithm is an important step towards a realistic description of the structure and dynamics of molecular recognition in biological systems

Extensive hydrogen-bonding network and an unusual cation conformation in [tris­(hydroxy­meth­yl)methyl]­ammonium tetra­oxidorhenate(VII)

The title compound, (C4H12NO3)[ReO4], contains two cations and two anions in the asymmetric unit, related by a non-crystallographic centre of symmetry. The crystal structure is stabilized by an extensive hydrogen-bonding network with the formation of puckered layers perpendicular to [001]. In the tris­(hydroxy­meth­yl)ammonium cations, intra­molecular O—H⋯O hydrogen bonds are present with the formation of an S 1 1(6) graph-set motif. The crystal structure is further consolid­ated by N—H⋯O hydrogen bonds

Role of sequence encoded κB DNA geometry in gene regulation by Dorsal

Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the ‘activator/repressor’ functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the ‘sequence-encoded structure’ of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the ‘A-tract’ core. The sixth nucleotide in the nonameric κB-motif, ‘A’ (A6) in the repressor motifs and ‘T’ (T6) in the activator motifs, is critical to confer this functional specificity as A6 → T6 mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that ‘sequence encoded κB DNA-geometry’ regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network

A flexible integrative approach based on random forest improves prediction of transcription factor binding sites

Transcription factor binding sites (TFBSs) are DNA sequences of 6-15 base pairs. Interaction of these TFBSs with transcription factors (TFs) is largely responsible for most spatiotemporal gene expression patterns. Here, we evaluate to what extent sequence-based prediction of TFBSs can be improved by taking into account the positional dependencies of nucleotides (NPDs) and the nucleotide sequence-dependent structure of DNA. We make use of the random forest algorithm to flexibly exploit both types of information. Results in this study show that both the structural method and the NPD method can be valuable for the prediction of TFBSs. Moreover, their predictive values seem to be complementary, even to the widely used position weight matrix (PWM) method. This led us to combine all three methods. Results obtained for five eukaryotic TFs with different DNA-binding domains show that our method improves classification accuracy for all five eukaryotic TFs compared with other approaches. Additionally, we contrast the results of seven smaller prokaryotic sets with high-quality data and show that with the use of high-quality data we can significantly improve prediction performance. Models developed in this study can be of great use for gaining insight into the mechanisms of TF binding

Geometric properties of nucleic acids with potential for autobuilding

Algorithms and geometrical properties are described for the automated building of nucleic acids in experimental electron density

Nucleic-acid recognition interfaces: How the greater ability of RNA duplexes to bend towards the surface influences electrochemical sensor performance

The influence of RNA versus DNA on the performance of electrochemical biosensors where redox-labelled nucleic acid duplexes bend towards the electrode surface has been assessed. Faster electron transfer was observed for duplexes containing RNA, suggesting duplexes with RNA are more flexible. These data are of particular importance for microRNA biosensors