Improving the prospects of cleavage-based nanopore sequencing engines

Abstract

Recently proposed methods for DNA sequencing involve the use of cleavage-based enzymes attached to the opening of a nanopore. The idea is that DNA interacting with either an exonuclease or polymerase protein will lead to a small molecule being cleaved near the mouth of the nanopore, and subsequent entry into the pore will yield information about the DNA sequence. The prospects for this approach seem promising, but it has been shown that diffusion related effects impose a limit on the capture probability of molecules by the pore, which limits the efficacy of the technique. Here, we revisit the problem with the goal of optimizing the capture probability via a step decrease in the nucleotide diffusion coefficient between the pore and bulk solutions. It is shown through random walk simulations and a simplified analytical model that decreasing the molecule's diffusion coefficient in the bulk relative to its value in the pore increases the nucleotide capture probability. Specifically, we show that at sufficiently high applied transmembrane potentials (≥100 mV), increasing the potential by a factor f is equivalent to decreasing the diffusion coefficient ratio D(bulk)/D(pore) by the same factor f. This suggests a promising route toward implementation of cleavage-based sequencing protocols. We also discuss the feasibility of forming a step function in the diffusion coefficient across the pore-bulk interface.

Studies of RNA Sequence and Structure Using Nanopores

“…This results in bases being read out of sequence, as well as insertion and deletion errors. Several improvements to this method have been proposed to circumvent the diffusion problem, 18 but to date this remains an open problem.…”

Theory of diffusion-influenced reactions in complex geometries

“…Modern examples of emerging nanotechnologies that rely on controlled alterations of diffusion and reaction pathways in DIRs include different sorts of chemical and biochemical catalysis involving complex nano-reactors [12,13], nanopore-based sequencing engines [14] and morphology control and surface functionalization of inorganic-based delivery vehicles for controlled intracellular drug release [15,16].…”

“…In general, the presence of multiple reactive and non-reactive particles/boundaries cannot be neglected in the study of (bio)chemical reactions occurring in real milieux, where the geometrical compactness of the environment may have profound effects, such as first-passage times that are non-trivially influenced by the starting point [23]. Relevant complex media include the cell cytoplasm [8,[24][25][26], porous or other artificial confining media [23,[27][28][29][30][31][32], which be considered as offering important tunable features for technological applications [12,14,16].In this paper, we take a major step forward by solving the general problem of computing the steady-state reaction rate constant for a diffusion-influenced chemical reaction between a mobile ligand and an explicit arbitrary, static 3D configuration of spherical reactive boundaries of arbitrary sizes and intrinsic reactivities.To set the stage for the forthcoming discussion, let us first consider the simple problem of two molecules A and B of size R and a, respectively, diffusing in solution. Upon encountering, the two species can form a complex, which catalyzes the transformation of species B into some product P with rate constant k,…”

“…Diffusion-influenced reactions (DIR) are ubiquitous in many contexts in physics, chemistry and biology [1,2] and keep on sparking intense theoretical and computational activity in many fields [3][4][5][6][7][8][9][10][11]. Modern examples of emerging nanotechnologies that rely on controlled alterations of diffusion and reaction pathways in DIRs include different sorts of chemical and biochemical catalysis involving complex nano-reactors [12,13], nanopore-based sequencing engines [14] and morphology control and surface functionalization of inorganic-based delivery vehicles for controlled intracellular drug release [15,16].However, while the mathematical foundations for the description of such problems have been laid nearly a century ago [17], many present-day problems of the utmost importance at both the fundamental and applied level are still challenging. Notably, arduous difficulties arise in the quantification of the important role played by the environment's geometry (obstacles, compartmentalization) [18] and distributed reactivity (patterns of competitive reaction targets or traps) in coupling transport and reaction pathways in many natural and artificial (bio)chemical reactions [1,19,20].…”

“…Diffusion-influenced reactions (DIR) are ubiquitous in many contexts in physics, chemistry and biology [1,2] and keep on sparking intense theoretical and computational activity in many fields [3][4][5][6][7][8][9][10][11]. Modern examples of emerging nanotechnologies that rely on controlled alterations of diffusion and reaction pathways in DIRs include different sorts of chemical and biochemical catalysis involving complex nano-reactors [12,13], nanopore-based sequencing engines [14] and morphology control and surface functionalization of inorganic-based delivery vehicles for controlled intracellular drug release [15,16].…”