A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange.

Background: Information was not available about prototoxin LYNX1 amino acid residues involved in binding to muscle and neuronal nicotinic receptors. Results: A series of water-soluble LYNX1 (ws-LYNX1) mutants was obtained and their interaction with nicotinic receptors was analyzed. Conclusion: There are both common and selective ws-LYNX1 residues recognizing distinct receptor types. Significance: For the first time, several functionally important residues in ws-LYNX1 are identified.

The thrombin-binding aptamer (15-TBA) is a 15-mer DNA oligonucleotide with sequence d(GGTTGGTGTGGTTGG). 15-TBA folds into a quadruplex DNA (G-DNA) structure with two planar G-quartets connected by three single-stranded loops. The arrangement of the 15-TBA-thrombin complex is unclear, particularly with respect to the precise 15-TBA residues that interact with the thrombin structure. Our present understanding suggests either the 15-TBA single stranded loops containing sequential thymidines (TT) or alternatively a single-stranded loop, containing a guanine flanked by 2 thymidines (TGT), physically associates with thrombin protein. In the present study, the explicit solvent molecular dynamics (MD) simulation method was utilized to further analyze the 15-TBA-thrombin three-dimensional structure. Functional annotation of the loop residues was made with long simulations in the parmbsc0 force field. In total, the elapsed time of simulations carried out in this study exceeds 12 microseconds, substantially surpassing previous G-DNA simulation reports. Our simulations suggest that the TGT-loop function is to stabilize the structure of the aptamer, while the TT-loops participate in direct binding to thrombin. The findings of the present report advance our understanding of the molecular structure of the 15-TBA-thrombin structure further enabling the construction of biosensors for aptamer bases and the development of anticoagulant agents.

The present work is devoted to the analysis of the G-quadruplex DNA structure using the bioinformatics method. The interest towards quadruplex DNAs is determined by their involvement in the functioning of telomeres and onco-promoters as well as by the possibility to create on their basis aptamers and nanostructures. Here, we present an algorithm for a general analysis of the polymorphism of the G-quadruplex structure from the data bank PDB using original parameters. 74 structures were grouped according to the following parameters: the number of DNA strands, the number of G-quartets, and the location and orientation of the connecting loops. Two quantitative parameters were used to describe the quadruplex structure: the twist angle between two adjacent quartets (analogous to that for the complementary pair in the duplex DNA) and the quartet planarity (an original parameter). The distribution patterns of these values are specific for each group of quadruplex structures and are dependent upon the type of connecting loops used (diagonal, lateral or propeller). The tetramolecular loopless parallel quadruplex was used as a comparison template. The lateral loops introduce the strongest distortion into the structure of quadruplexes: the values of the twist angles are the lowest and are not typical for the other quadruplex groups. The loops of the diagonal type introduce much weaker deformation into quadruplexes; the structures with propeller loops are characterized by the optimum geometry of G-quartets. Hence, the correlation between the twist angle and the tension in the structure of quadruplex DNA is revealed.

Thrombin-binding aptamers are promising anticoagulants. HD1 is a monomolecular antiparallel G-quadruplex with two G-quartets linked by three loops. Aptamer-thrombin interactions are mediated with two TT-loops that bind thrombin exosite I. Several cations were shown to be coordinated inside the G-quadruplex, including K, Na, NH, Ba, and Sr; on the contrary, Mn was coordinated in the grooves, outside the G-quadruplex. K or Na coordination provides aptamer functional activity. The effect of other cations on aptamer functional activity has not yet been described, because of a lack of relevant tests. Interactions between aptamer HD1 and a series of cations were studied. A previously developed enzymatic method was applied to evaluate aptamer inhibitory activity. The structure-function correlation was studied using the characterization of G-quadruplex conformation by circular dichroism spectroscopy. K coordination provided the well-known high inhibitory activity of the aptamer, whereas Na coordination supported low activity. Although NH coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba and Sr coordination. Mn coordination destabilized the G-quadruplex that drastically diminished aptamer inhibitory activity. Therefore, G-quadruplex existence per se is insufficient for aptamer inhibitory activity. To elicit the nature of these effects, we thoroughly analyzed nuclear magnetic resonance (NMR) and X-ray data on the structure of the HD1 G-quadruplex with various cations. The most reasonable explanation is that cation coordination changes the conformation of TT-loops, affecting thrombin binding and inhibition. HD1 counterparts, aptamers 31-TBA and NU172, behaved similarly with some distinctions. In 31-TBA, an additional duplex module stabilized antiparallel G-quadruplex conformation at high concentrations of divalent cations; whereas in NU172, a different sequence of loops in the G-quadruplex module provided an equilibrium of antiparallel and parallel G-quadruplexes that shifted with cation binding. In conclusion, structures of G-quadruplex aptamers are flexible enough and are fine-tuned with different cation coordination.

Modeling tools provide a valuable support for DNA origami design. However, current solutions have limited application for conformational analysis of the designs. In this work we present a tool for a thorough study of DNA origami structure and dynamics. The tool is based on a novel coarse-grained model dedicated to geometry optimization and conformational analysis of DNA origami. We explored the ability of the model to predict dynamic behavior, global shapes, and fine details of two single-layer systems designed in hexagonal and square lattices using atomic force microscopy, Förster resonance energy transfer spectroscopy, and all-atom molecular dynamic simulations for validation of the results. We also examined the performance of the model for multilayer systems by simulation of DNA origami with published cryo-electron microscopy and atomic force microscopy structures. A good agreement between the simulated and experimental data makes the model suitable for conformational analysis of DNA origami objects. The tool is available at http://vsb.fbb.msu.ru/cosm as a web-service and as a standalone version.

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Aptamers based on nucleic acids are a promising alternative to antibodies in therapy and diagnostics. Several DNA aptamers against human thrombin have been developed by selection from random libraries: a 15-mer and its derivatives, a 29-mer, and a 31-mer. Some of them are patented and already under clinical trial. The 15-mer structure was determined by X-ray and NMR and turned out to be a monomolecular antiparallel G-quadruplex. The other aptamers mentioned above have higher inhibitory activity than the initial 15-mer, but there are not yet structural data explaining this phenomenon. Here, the initial 15-mer, 31-mer, and novel RA-36 aptamers are compared to establish the structure-function correlation, providing a solid ground for further rational aptameric drug design. For the molecular dynamic simulation of aptamers, the force field parmbsc0 was ported onto GROMACS, and the main stabilizing parameters were revealed, leading to the novel DNA aptamer RA-36. The functional properties of the DNA aptamers were studied by conventional coagulation tests, which do not directly elucidate the mechanism of thrombin inhibition by aptamers. Improved turbidimetric measurements provided data to develop detailed kinetics showing that the 31-mer and RA-36, in contrast to the 15-mer, are competitive inhibitors. These data revealed RA-36 to be an efficient thrombin inhibitor with a dose-dependent effect. Animal tests of the studied DNA aptamers suggested an unexpected species-specificity of the novel RA-36.