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Recent advances in biological nanopores for nanopore sequencing, sensing and comparison of functional variations in MspA mutants.

ABSTRACT: Biological nanopores are revolutionizing human health by the great myriad of detection and diagnostic skills. Their nano-confined area and ingenious shape are suitable to investigate a diverse range of molecules that were difficult to identify with the previous techniques. Additionally, high throughput and label-free detection of target analytes instigated the exploration of new bacterial channel proteins such as Fragaceatoxin C (FraC), Cytolysin A (ClyA), Ferric hydroxamate uptake component A (FhuA) and Curli specific gene G (CsgG) along with the former ones, like α-hemolysin (αHL), Mycobacterium smegmatis porin A (MspA), aerolysin, bacteriophage phi 29 and Outer membrane porin G (OmpG). Herein, we discuss some well-known biological nanopores but emphasize on MspA and compare the effects of site-directed mutagenesis on the detection ability of its mutants in view of the surface charge distribution, voltage threshold and pore-analyte interaction. We also discuss illustrious and latest advances in biological nanopores for past 2-3 years due to limited space. Last but not the least, we elucidate our perspective for selecting a biological nanopore and propose some future directions to design a customized nanopore that would be suitable for DNA sequencing and sensing of other nontrivial molecules in question.

SUBMITTER: Bhatti H

PROVIDER: S-EPMC9038099 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Recent advances in biological nanopores for nanopore sequencing, sensing and comparison of functional variations in MspA mutants.

Bhatti Huma H Jawed Rohil R Ali Irshad I Iqbal Khurshid K Han Yan Y Lu Zuhong Z Liu Quanjun Q

RSC advances 20210831 46

Biological nanopores are revolutionizing human health by the great myriad of detection and diagnostic skills. Their nano-confined area and ingenious shape are suitable to investigate a diverse range of molecules that were difficult to identify with the previous techniques. Additionally, high throughput and label-free detection of target analytes instigated the exploration of new bacterial channel proteins such as Fragaceatoxin C (FraC), Cytolysin A (ClyA), Ferric hydroxamate uptake component A ( ...[more]

PMID: 35478559

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Project description:Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA.

Dataset Information

Recent advances in biological nanopores for nanopore sequencing, sensing and comparison of functional variations in MspA mutants.

Publications

Recent advances in biological nanopores for nanopore sequencing, sensing and comparison of functional variations in MspA mutants.

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