Utkur Mirsaidov

Nanopore Sequencing: Electrical Measurements of the Code of Life

Sequencing a single molecule of deoxyribonucleic acid (DNA) using a nanopore is a revolutionary concept because it combines the potential for long read lengths (>5 kbp) with high speed (1 bp/10 ns), while obviating the need for costly amplification procedures due to the exquisite single molecule sensitivity. The prospects for implementing this concept seem bright. The cost savings from the removal of required reagents, coupled with the speed of nanopore sequencing places the

Slowing the translocation of double-stranded DNA using a nanopore smaller than the double helix

1000 genome within grasp. However, challenges remain: high fidelity reads demand stringent control over both the molecular configuration in the pore and the translocation kinetics. The molecular configuration determines how the ions passing through the pore come into contact with the nucleotides, while the translocation kinetics affect the time interval in which the same nucleotides are held in the constriction as the data is acquired. Proteins like ¿-hemolysin and its mutants offer exquisitely precise self-assembled nanopores and have demonstrated the facility for discriminating individual nucleotides, but it is currently difficult to design protein structure ab initio, which frustrates tailoring a pore for sequencing genomic DNA. Nanopores in solid-state membranes have been proposed as an alternative because of the flexibility in fabrication and ease of integration into a sequencing platform. Preliminary results have shown that with careful control of the dimensions of the pore and the shape of the electric field, control of DNA translocation through the pore is possible. Furthermore, discrimination between different base pairs of DNA may be feasible. Thus, a nanopore promises inexpensive, reliable, high-throughput sequencing, which could thrust genomic science into personal medicine.

Direct observation of stick-slip movements of water nanodroplets induced by an electron beam

It is now possible to slow and trap a single molecule of double-stranded DNA (dsDNA), by stretching it using a nanopore, smaller in diameter than the double helix, in a solid-state membrane. By applying an electric force larger than the threshold for stretching, dsDNA can be impelled through the pore. Once a current blockade associated with a translocating molecule is detected, the electric field in the pore is switched in an interval less than the translocation time to a value below the threshold for stretching. According to molecular dynamics (MD) simulations, this leaves the dsDNA stretched in the pore constriction with the base-pairs tilted, while the B-form canonical structure is preserved outside the pore. In this configuration, the translocation velocity is substantially reduced from 1 bp/10 ns to approximately 1 bp/2 ms in the extreme, potentially facilitating high fidelity reads for sequencing, precise sorting, and high resolution (force) spectroscopy.

Nanopores in solid-state membranes engineered for single molecule detection

Dynamics of the first few nanometers of water at the interface are encountered in a wide range of physical, chemical, and biological phenomena. A simple but critical question is whether interfacial forces at these nanoscale dimensions affect an externally induced movement of a water droplet on a surface. At the bulk-scale water droplets spread on a hydrophilic surface and slip on a nonwetting, hydrophobic surface. Here we report the experimental description of the electron beam-induced dynamics of nanoscale water droplets by direct imaging the translocation of 10- to 80-nm-diameter water nanodroplets by transmission electron microscopy. These nanodroplets move on a hydrophilic surface not by a smooth flow but by a series of stick-slip steps. We observe that each step is preceded by a unique characteristic deformation of the nanodroplet into a toroidal shape induced by the electron beam. We propose that this beam-induced change in shape increases the surface free energy of the nanodroplet that drives its transition from stick to slip state.

Nanoelectromechanics of methylated DNA in a synthetic nanopore.

A nanopore is an analytical tool with single molecule sensitivity. For detection, a nanopore relies on the electrical signal that develops when a molecule translocates through it. However, the detection sensitivity can be adversely affected by noise and the frequency response. Here, we report measurements of the frequency and noise performance of nanopores </=8 nm in diameter in membranes compatible with semiconductor processing. We find that both the high frequency and noise performance are compromised by parasitic capacitances. From the frequency response we extract the parameters of lumped element models motivated by the physical structure that elucidates the parasitics, and then we explore four strategies for improving the electrical performance. We reduce the parasitic membrane capacitances using: (1) thick Si(3)N(4) membranes; (2) miniaturized composite membranes consisting of Si(3)N(4) and polyimide; (3) miniaturized membranes formed from metal-oxide-semiconductor (MOS) capacitors; and (4) capacitance compensation through external circuitry, which has been used successfully for patch clamping. While capacitance compensation provides a vast improvement in the high frequency performance, mitigation of the parasitic capacitance through miniaturization offers the most promising route to high fidelity electrical discrimination of single molecules.

Multistep nucleation of nanocrystals in aqueous solution

Methylation of cytosine is a covalent modification of DNA that can be used to silence genes, orchestrating a myriad of biological processes including cancer. We have discovered that a synthetic nanopore in a membrane comparable in thickness to a protein binding site can be used to detect methylation. We observe a voltage threshold for permeation of methylated DNA through a <2 nm diameter pore, which we attribute to the stretching transition; this can differ by >1 V/20 nm depending on the methylation level, but not the DNA sequence.

Nanoparticle Dynamics in a Nanodroplet

The nucleation and growth of solids from solutions impacts many natural processes and is fundamental to applications in materials engineering and medicine. For a crystalline solid, the nucleus is a nanoscale cluster of ordered atoms that forms through mechanisms still poorly understood. In particular, it is unclear whether a nucleus forms spontaneously from solution via a single- or multiple-step process. Here, using in situ electron microscopy, we show how gold and silver nanocrystals nucleate from supersaturated aqueous solutions in three distinct steps: spinodal decomposition into solute-rich and solute-poor liquid phases, nucleation of amorphous nanoclusters within the metal-rich liquid phase, followed by crystallization of these amorphous clusters. Our ab initio calculations on gold nucleation suggest that these steps might be associated with strong gold-gold atom coupling and water-mediated metastable gold complexes. The understanding of intermediate steps in nuclei formation has important implications for the formation and growth of both crystalline and amorphous materials.

Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore

We describe the dynamics of 3-10 nm gold nanoparticles encapsulated by ∼30 nm liquid nanodroplets on a flat solid substrate and find that the diffusive motion of these nanoparticles is damped due to strong interactions with the substrate. Such damped dynamics enabled us to obtain time-resolved observations of encapsulated nanoparticles coalescing into larger particles. Techniques described here serve as a platform to study chemical and physical dynamics under highly confined conditions.

Hydration Layer-Mediated Pairwise Interaction of Nanoparticles

Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence—GAATTC—in the absence of a Mg2+ ion cofactor. We have previously shown that there is a voltage threshold for permeation of DNA bound to restriction enzymes through a nanopore that is associated with a nanonewton force required to rupture the complex. By introducing mutations in the DNA, we now show that this threshold depends on the recognition sequence and scales linearly with the dissociation energy, independent of the pore geometry. To predict the effect of mutation in a base pair on the free energy of dissociation, MD is used to qualitatively rank the stability of bonds in the EcoRI–DNA complex. We find that the second base in the target sequence exhibits the strongest binding to the protein, followed by the third and first bases, with even the flanking sequence affecting the binding, corroborating our experiments.

Molecular diagnostics for personal medicine using a nanopore.

When any two surfaces in a solution come within a distance the size of a few solvent molecules, they experience a solvation force or a hydration force when the solvent is water. Although the range and magnitude of hydration forces are easy to characterize, the effects of these forces on the transient steps of interaction dynamics between nanoscale bodies in solution are poorly understood. Here, using in situ transmission electron microscopy, we show that when two gold nanoparticles in water approach each other at a distance within two water molecules (∼5 Å), which is the combined thickness of the hydration shell of each nanoparticle, they form a sterically stabilized transient nanoparticle dimer. The interacting surfaces of the nanoparticles come in contact and undergo coalescence only after these surfaces are fully dehydrated. Our observations of transient steps in nanoparticle interactions, which reveal the formation of hydration layer mediated metastable nanoparticle pairs in solution, have significant implications for many natural and industrial processes.