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All-atom simulations have become increasingly popular to study conformational and dynamical properties of nucleic acids as they are accurate and provide high spatial and time resolutions. This high resolution, however, comes at a heavy computational cost, and, within the time scales of simulations, nucleic acids weakly fluctuate around their ideal structure exploring a limited set of conformations. We introduce the RBB-NA algorithm (available as a package in the Open Source Library PLUMED), which is capable of controlling rigid base parameters in all-atom simulations of nucleic acids. With suitable biasing potentials, this algorithm can “force” a DNA or RNA molecule to assume specific values of the six rotational (tilt, roll, twist, buckle, propeller, opening) and/or the six translational parameters (shift, slide, rise, shear, stretch, stagger). The algorithm enables the use of advanced sampling techniques to probe the structure and dynamics of locally strongly deformed nucleic acids. We illustrate its performance showing some examples in which DNA is strongly twisted, bent, or locally buckled. In these examples, RBB-NA reproduces well the unconstrained simulations data and other known features of DNA mechanics, but it also allows one to explore the anharmonic behavior characterizing the mechanics of nucleic acids in the high deformation regime.

We present a synthetic nanoscale piston that uses chemical energy to perform molecular transport against an applied bias. Such a device comprises a 13 by 5 nm protein cylinder, embedded in a biological membrane enclosing a single-stranded DNA (ssDNA) rod. Hybridization with DNA cargo rigidifies the rod, allowing for transport of a selected DNA molecule across the nanopore. A strand displacement reaction from ssDNA fuel on the other side of the membrane then liberates the DNA cargo back into solution and regenerates the initial configuration. The entropic penalty of ssDNA confinement inside the nanopore drives DNA transport regardless of the applied bias. Multiple automated and reciprocating cycles are observed, in which the DNA piston moves through the 10 nm length of the nanopore. In every cycle, a single DNA molecule is transported across the nanopore against an external bias force, which is the hallmark of biological transporters.

DNA surface-hybridization biosensors utilize the selective hybridization of target sequences in solution to surface-immobilized probes. In this process, the target is usually assumed to be in excess, so that its concentration does not significantly vary while hybridizing to the surface-bound probes. If the target is initially at low concentrations and/or if the number of probes is very large, and they have high affinity for the target, the DNA in solution may become depleted. In this paper we analyze the equilibrium and kinetics of hybridization of DNA biosensors in the case of strong target depletion, by extending the Langmuir adsorption model. We focus, in particular, on the detection of a small amount of a single-nucleotide “mutant” sequence (concentration c2) in a solution, which differs by one or more nucleotides from an abundant “wild-type” sequence (concentration c1≫ c2). We show that depletion can give rise to a strongly enhanced sensitivity of the biosensors. Using representative values of rate constants and hybridization free energies, we find that in the depletion regime one could detect relative concentrations c2/c1that are up to 3 orders of magnitude smaller than in the conventional approach. The kinetics is surprisingly rich and exhibits a nonmonotonic adsorption with no counterpart in the no-depletion case. Finally, we show that, alongside enhanced detection sensitivity, this approach offers the possibility of sample enrichment, by substantially increasing the relative amount of the mutant over the wild-type sequence.

Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited.