Design Principles of DNA-Barcodes for Nanopore-FET Readout, Based on Molecular Dynamics and TCAD Simulations.

Nanopore field-effect transistor (NP-FET) devices hold great promise as sensitive single-molecule sensors, which provide CMOS-based on-chip readout and are also highly amenable to parallelization. A plethora of applications will therefore benefit from NP-FET technology, such as large-scale molecular analysis (e.g., proteomics). Due to its potential for parallelization, the NP-FET looks particularly well-suited for the high-throughput readout of DNA-based barcodes. However, to date, no study exists that unravels the bit-rate capabilities of NP-FET devices. In this paper, we design DNA-based barcodes by labeling a piece of double-stranded DNA with dumbbell-like DNA structures. We explore the impact of both the size of the dumbbells and their spacing on achievable bit-rates. The conformational fluctuations of this DNA-origami, as observed by molecular dynamics (MD) simulation, are accounted for when selecting label sizes. An experimentally informed 3D continuum nanofluidic-nanoelectronic device model subsequently predicts both the ionic current and FET signals. We present a barcode design for a conceptually generic NP-FET, with a 14 nm diameter pore, operating in conditions corresponding to experiments. By adjusting the spacing between the labels to half the length of the pore, we show that a bit-rate of 78 kbit·s-1 is achievable. This lies well beyond the state-of-the-art of ≈40 kbit·s-1, with significant headroom for further optimizations. We also highlight the advantages of NP-FET readout based on the larger signal size and sinusoidal signal shape.

Insights into elastic properties of coarse-grained DNA models: q-stiffness of cgDNA vs cgDNA.

Coarse-grained models have emerged as valuable tools to simulate long DNA molecules while maintaining computational efficiency. These models aim at preserving interactions among coarse-grained variables in a manner that mirrors the underlying atomistic description. We explore here a method for testing coarse-grained vs all-atom models using stiffness matrices in Fourier space (q-stiffnesses), which are particularly suited to probe DNA elasticity at different length scales. We focus on a class of coarse-grained rigid base DNA models known as cgDNA and its most recent version, cgDNA+. Our analysis shows that while cgDNA+ closely follows the q-stiffnesses of the all-atom model, the original cgDNA shows some deviations for twist and bending variables, which are rather strong in the q → 0 (long length scale) limit. The consequence is that while both cgDNA and cgDNA+ give a suitable description of local elastic behavior, the former misses some effects that manifest themselves at longer length scales. In particular, cgDNA performs poorly on twist stiffness, with a value much lower than expected for long DNA molecules. Conversely, the all-atom and cgDNA+ twist are strongly length scale dependent: DNA is torsionally soft at a few base pair distances but becomes more rigid at distances of a few dozen base pairs. Our analysis shows that the bending persistence length in all-atom and cgDNA+ is somewhat overestimated.

Mechanisms of DNA-Mediated Allostery.

Proteins often regulate their activities via allostery-or action at a distance-in which the binding of a ligand at one binding site influences the affinity for another ligand at a distal site. Although less studied than in proteins, allosteric effects have been observed in experiments with DNA as well. In these experiments two or more proteins bind at distinct DNA sites and interact indirectly with each other, via a mechanism mediated by the linker DNA molecule. We develop a mechanical model of DNA/protein interactions which predicts three distinct mechanisms of allostery. Two of these involve an enthalpy-mediated allostery, while a third mechanism is entropy driven. We analyze experiments of DNA allostery and highlight the distinctive signatures allowing one to identify which of the proposed mechanisms best fits the data.

Rigid Base Biasing in Molecular Dynamics Enables Enhanced Sampling of DNA Conformations.

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.

Mechanical properties of nucleic acids and the non-local twistable wormlike chain model.

Mechanical properties of nucleic acids play an important role in many biological processes that often involve physical deformations of these molecules. At sufficiently long length scales (say, above ∼20-30 base pairs), the mechanics of DNA and RNA double helices is described by a homogeneous Twistable Wormlike Chain (TWLC), a semiflexible polymer model characterized by twist and bending stiffnesses. At shorter scales, this model breaks down for two reasons: the elastic properties become sequence-dependent and the mechanical deformations at distal sites get coupled. We discuss in this paper the origin of the latter effect using the framework of a non-local Twistable Wormlike Chain (nlTWLC). We show, by comparing all-atom simulations data for DNA and RNA double helices, that the non-local couplings are of very similar nature in these two molecules: couplings between distal sites are strong for tilt and twist degrees of freedom and weak for roll. We introduce and analyze a simple double-stranded polymer model that clarifies the origin of this universal distal couplings behavior. In this model, referred to as the ladder model, a nlTWLC description emerges from the coarsening of local (atomic) degrees of freedom into angular variables that describe the twist and bending of the molecule. Different from its local counterpart, the nlTWLC is characterized by a length-scale-dependent elasticity. Our analysis predicts that nucleic acids are mechanically softer at the scale of a few base pairs and are asymptotically stiffer at longer length scales, a behavior that matches experimental data.

PlyAB Nanopores Detect Single Amino Acid Differences in Folded Haemoglobin from Blood.

The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult haemoglobin (HbA) and sickle cell anaemia haemoglobin (HbS), which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades. Foetal Hb, which shows a larger sequence variation, was distinguished with near 100 % accuracy. Continuum and Brownian dynamics simulations revealed that Hb occupies two energy minima, one near the inner constriction and one at the trans entry of the nanopore. Thermal fluctuations, the charge of the protein, and the external bias influence the dynamics of Hb within the nanopore, which in turn generates the unique ionic current signal in the Hb variants. Finally, Hb was counted from blood samples, demonstrating that direct discrimination and quantification of Hb from blood using nanopores, is feasible.

Length-scale-dependent elasticity in DNA from coarse-grained and all-atom models.

We investigate the influence of nonlocal couplings on the torsional and bending elasticities of DNA. Such couplings have been observed in the past by several simulation studies. Here, we use a description of DNA conformations based on the variables tilt, roll, and twist. Our analysis of both coarse-grained (oxDNA) and all-atom models indicates that these share strikingly similar features: there are strong off-site couplings for tilt-tilt and twist-twist, while they are much weaker in the roll-roll case. By developing an analytical framework to estimate bending and torsional persistence lengths in nonlocal DNA models, we show how off-site interactions generate a length-scale-dependent elasticity. Based on the simulation-generated elasticity data, the theory predicts a significant length-scale-dependent effect on torsional fluctuations but only a modest effect on bending fluctuations. These results are in agreement with experiments probing DNA mechanics from single base pair to kilobase pair scales.