Label-free, atomic force microscopy-based mapping of DNA intrinsic curvature for the nanoscale comparative analysis of bent duplexes

We propose a method for the characterization of the local intrinsic curvature of adsorbed DNA molecules. It relies on a novel statistical chain descriptor, namely the ensemble averaged product of curvatures for two nanosized segments, symmetrically placed on the contour of atomic force microscopy imaged chains. We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature. The centrosymmetric character of the chain descriptor enables targetting strands with unknown orientation. This overcomes a remarkable limitation of the current experimental strategies that estimate curvature maps solely from the trajectories of end-labeled molecules or palindromes. As a consequence our approach paves the way for a reliable, unbiased, label-free comparative analysis of bent duplexes, aimed to detect local conformational changes of physical or biological relevance in large sample numbers. Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far. We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes

Autonomously designed free-form 2D DNA origami

Scaffolded DNA origami offers the unique ability to organize molecules in nearly arbitrary spatial patterns at the nanometer scale, with wireframe designs further enabling complex 2D and 3D geometries with irregular boundaries and internal structures. The sequence design of the DNA staple strands needed to fold the long scaffold strand to the target geometry is typically performed manually, limiting the broad application of this materials design paradigm. Here, we present a fully autonomous procedure to design all DNA staple sequences needed to fold any free-form 2D scaffolded DNA origami wireframe object. Our algorithm uses wireframe edges consisting of two parallel DNA duplexes and enables the full autonomy of scaffold routing and staple sequence design with arbitrary network edge lengths and vertex angles. The application of our procedure to geometries with both regular and irregular external boundaries and variable internal structures demonstrates its broad utility for nanoscale materials science and nanotechnology.National Science Foundation (U.S.) (Grant CCF-1564025)National Science Foundation (U.S.) (Grant CMMI-1334109)Office of Naval Research (Grant N000141210621

Surface and Interfacial Approaches for the Characterization of Biomolecular Interactions and to Optimize Desi-MS Performance

This work involves the surface and interfacial characterization of biomolecular interactions by atomic force microscopy and optimization of desorption electrospray ionization mass spectrometry. In the first part (part I), suitable surface of functionalized porous silicon are created and characterized to enhance Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) capability. DESI-MS is a powerful emerging analytical tool that finds applications in fundamental research and as a diagnostic tool. Enhancement of the performance of this technique will make it superior and improve the scope of its applications. The use of super hydrophobic porous silicon proved to enhance the desorption/ionization mechanism of DESI. It improves the ionization efficiency almost two fold when compared to the traditionally used glass slide and porous polytetraflouroethylene surfaces under the same conditions. The functionalized porous surfaces showed incredible stability, which is suitable for long time and high throughput analysis. We proposed a mechanism whereby the porous silicon acts as a barrier for the spray solvent, and creates a pool of analyte during desorption, leading to greater stability. On the other hand, the super hydrophobic functionality improves the ionization power of the technique by increasing analyte concentration over the area sampled and preventing filing of the pores. The functionalized porous surfaces are also suitable for DESI-imaging of biomolecules and tissue cells. In the second part (part II) of this thesis, Atomic Force Microscopy (AFM) was used to confirm site-specific protein-DNA interaction of the vancomycin resistance associated regulatory protein (VraR) from S. aureus. The protein stoichiometry at the binding site was confirmed as being mostly dimer for VraR, and as oligomers for phosphorylated VraR. In another project, AFM proved to be a very useful technique for the visualization and characterization of RNA. It enable us to visualized for the first time, the three dimensional structural architecture of genomic RNA of tomato bushy stunt virus. AFM allowed us to confirm the proposed long range-RNA-RNA interaction existing within the genome, leading to a more compact structure. Volume analyses enable to confirm the existence of compact structures as visualized in the AFM images. These results are consistent with expected conformations utilized by the TBSV virus for different viral processes. Sub-genomic RNA expressed by the TBSV virus, also exhibited compact structures with different degree of protrusions. All observed RNA and sub-genomic RNA structures from our AFM images were consistent with the selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) predicted structures

Elongational-flow-induced scission of DNA nanotubes in laminar flow

The length distributions of polymer fragments subjected to an elongational-flow-induced scission are profoundly affected by the fluid flow and the polymer bond strengths. In this paper, laminar elongational flow was used to induce chain scission of a series of circumference-programmed DNA nanotubes. The DNA nanotubes served as a model system for semiflexible polymers with tunable bond strength and cross-sectional geometry. The expected length distribution of fragmented DNA nanotubes was calculated from first principles by modeling the interplay between continuum hydrodynamic elongational flow and the molecular forces required to overstretch multiple DNA double helices. Our model has no-free parameters; the only inferred parameter is obtained from DNA mechanics literature, namely, the critical tension required to break a DNA duplex into two single-stranded DNA strands via the overstretching B-S DNA transition. The nanotube fragments were assayed with fluorescence microscopy at the single-molecule level and their lengths are in agreement with the scission theory

How round is a protein? Exploring protein structures for globularity using conformal mapping.

We present a new algorithm that automatically computes a measure of the geometric difference between the surface of a protein and a round sphere. The algorithm takes as input two triangulated genus zero surfaces representing the protein and the round sphere, respectively, and constructs a discrete conformal map f between these surfaces. The conformal map is chosen to minimize a symmetric elastic energy E S (f) that measures the distance of f from an isometry. We illustrate our approach on a set of basic sample problems and then on a dataset of diverse protein structures. We show first that E S (f) is able to quantify the roundness of the Platonic solids and that for these surfaces it replicates well traditional measures of roundness such as the sphericity. We then demonstrate that the symmetric elastic energy E S (f) captures both global and local differences between two surfaces, showing that our method identifies the presence of protruding regions in protein structures and quantifies how these regions make the shape of a protein deviate from globularity. Based on these results, we show that E S (f) serves as a probe of the limits of the application of conformal mapping to parametrize protein shapes. We identify limitations of the method and discuss its extension to achieving automatic registration of protein structures based on their surface geometry

A Multiscale Model for Virus Capsid Dynamics

Viruses are infectious agents that can cause epidemics and pandemics. The understanding of virus formation, evolution, stability, and interaction with host cells is of great importance to the scientific community and public health. Typically, a virus complex in association with its aquatic environment poses a fabulous challenge to theoretical description and prediction. In this work, we propose a differential geometry-based multiscale paradigm to model complex biomolecule systems. In our approach, the differential geometry theory of surfaces and geometric measure theory are employed as a natural means to couple the macroscopic continuum domain of the fluid mechanical description of the aquatic environment from the microscopic discrete domain of the atomistic description of the biomolecule. A multiscale action functional is constructed as a unified framework to derive the governing equations for the dynamics of different scales. We show that the classical Navier-Stokes equation for the fluid dynamics and Newton's equation for the molecular dynamics can be derived from the least action principle. These equations are coupled through the continuum-discrete interface whose dynamics is governed by potential driven geometric flows

Sizing single nanoscale objects from polarization forces

Sizing natural or engineered single nanoscale objects is fundamental in many areas of science and technology. To achieve it several advanced microscopic techniques have been developed, mostly based on electron and scanning probe microscopies. Still for soft and poorly adhered samples the existing techniques face important challenges. Here, we propose an alternative method to size single nanoscale objects based on the measurement of its electric polarization. The method is based on Electrostatic Force Microscopy measurements combined with a specifically designed multiparameter quantification algorithm, which gives the physical dimensions (height and width) of the nanoscale object. The proposed method is validated with ~50 nm diameter silver nanowires, and successfully applied to ~10 nm diameter bacterial polar flagella, an example of soft and poorly adhered nanoscale object. We show that an accuracy comparable to AFM topographic imaging can be achieved. The main advantage of the proposed method is that, being based on the measurement of long-range polarization forces, it can be applied without contacting the sample, what is key when considering poorly adhered and soft nanoscale objects. Potential applications of the proposed method to a wide range of nanoscale objects relevant in Material, Life Sciences and Nanomedicine is envisaged