Osmosis-Driven Motion-Type Modulation of Biological Nanopores for Parallel Optical Nucleic Acid Sensing

Abstract

Recent developments in nanopore sequencing have inspired new concepts in precision medicine but limited in throughput. By optically encoding calcium flux from an array of nanopores, parallel measurements from hundreds of nanopores were reported, while lateral drifts of biological nanopores set obstacles for signal processing. In this paper, optical single-channel recording (oSCR) serves to track nanopores with high precision and a general principle of nanopore motion kinetics is quantitatively investigated. By finely adjusting the osmosis-oriented interactions between the lipid/substrate interfaces, motions of nanopores could be controllably restricted. Improved signal-to-noise ratio is observed from motion-restricted nanopores, which is experimentally demonstrated. To systematically evaluate oSCR with asymmetric salt concentrations, a finite element method simulation is established. oSCR with an array of immobilized nanopores suggests new strategies for sequencing DNA by microscopic imaging in high throughput and is widely applicable to the investigation of other transmembrane proteins.

Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O6‐Carboxymethyl Guanine and Reveals Its Behavior in Replication

“…NPS was carried out using the enzymatic ratcheting strategy with a mutant MspA NP (see SI) inserted in a self‐assembled 1,2‐diphytanoyl‐sn‐glycero‐3‐phosphocholine (DPhPC) lipid bilayer, which separates the cis and the trans side of a custom‐made electrophysiology chamber . Wildtype Phi29 DNAP was used in the cis side of the chamber to promote the DNA copying for NPS (fully described in the SI, Figure S4).…”

Electrode-free nanopore sensing by DiffusiOptoPhysiology

“…Here, the positive direction of the osmotic pressure is defined to be from trans to cis. This osmotic pressure subsequently drives an oriented flow of water, ions, and analytes through a biological nanopore, which is inserted in the membrane ( 27 ). As a result, an enhanced translocation efficiency for analyte should be achieved with this introduced asymmetry.…”

“…The protein nanopores used in this paper were α-HL WT and ClyA-RR (fig. S11), which were expressed in E. coli and purified on the basis of published protocols ( 27 , 44 ).…”

Single molecule observation of hard–soft-acid–base (HSAB) interaction in engineeredMycobacterium smegmatisporin A (MspA) nanopores

“…Three engineered MspA mutants were prepared and characterized as previously reported. 18 For simplicity, these mutants were respectively named MspA-D (D93N/D90N/D118R/D134R/E139K), MspA-H (D93N/ D91H/D90N/D118R/D134R/E139K) and MspA-C (D93N/D91C/ D90N/D118R/D134R/E139K) (ESI Methods, Fig. S1-S3 †) throughout the paper.…”

“…In this paper, three MspA mutants, which have histidine, cysteine or aspartic acid placed at site 91 in a monomeric subunit, were prepared as previously reported. 18 When compatible analyte ions were applied, MspA mutants report noticeable events during single channel recordings. The recorded event from binding of a single ion has reached $10 pA in amplitude, which is considerably greater than that measured with a-HL mutants.…”