Molecular Dynamics Investigations of Hybrid Nanopores for Single Molecule DNA Sequencing

Abstract

High throughput methods accelerate the drug discovery, biomolecule detection and sequencing applications pipeline. Single molecule detection technique facilitates the implementation of high throughput methods by eliminating multi step evaluation. Label free high throughput biosensing methods pave way for studying nanoscale interactions of biological molecules in real-time. Nanopore based techniques have enabled the design of biosensors emerged as a new and rapidly developing label free real-time single molecule sensing technique.

Biological nanopores typically realised using pore forming proteins, such as MspA porin and alpha hemolysin, are generally inserted into a lipid bilayer. These are great for sensitivity, but the proteins are difficult to handle. To mimic this biological nanopore, we have taken help from top-down and bottom-up nanotechnology to fabricate a bilayer nanopore. Scaffolded DNA origami is also a novel and unique method to fabricate tailor-made nanostructures with wide range of applications. The major advantage of DNA origami is the accurate control over the shape of the structure at the nanometer level.

Combining DNA origami on the top layer with solid-state nanopore on the bottom layer, we aim to introduce great many possibilities for changing and enhancing pore properties and features. Computational models were built to detect and distinguish biological molecules. Statistical analysis of the data obtained from computations run using supercomputers were used to propose, predict, and aid the experimental designs.

Specifically, we investigate molecular dynamics simulation of proposed nanopores using graphene, integrated with DNA Origami for DNA detection. This enables us to achieve pore functionalisation enabling selective base control. The incorporation of bait-prey mechanism for selectivity is made possible by optimizing the bait configuration of the DNA bases near the nanopore in the DNA origami layer, resulting in base specific residence times. Such pore functionalization techniques can also be used to selectively trap certain proteins whilst allowing a free passage of others. Especially MD simulations thrombin binding aptamers attached to the pores formed using DNA origami were observed to hold thrombin for a longer time when compared to a larger streptavidin molecule. Such simulations paved way for a better design of DNA origami pores which can be realised using experiments.

Graphene, although a wonder material for sequencing, exhibits strong hydrophobic interactions with DNA resulting in non-uniform translocation, thereby reducing the effectiveness detection using ionic currents. Heterostructure nanopore system was proposed with aligned nanopores to overcome this issue. Vertically stacked graphene and hexagonal Boron Nitride nanopores revealed a very pivotal role in the ordering of heterostructure. This makes use of the absence of very strong adsorption of DNA bases on hexagonal boron nitride when compared to graphene. Simulations point to accomplishing distinct residence times and ionic currents for AT and CG base-pairs and simultaneously exploring the possibility of underside sticking of DNA to the nanopore membrane.

It should be noted that owing to the versatility, nanopore devices are marking their entry in the commercial marketplaces too, apart from academia. We believe that our results from the understanding of nanopores will enable experimentalists to design pipelines which result in rapid and precise detection of biomolecules.