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Engineering of protein nanopores for sequencing, chemical or protein sensing and disease diagnosis.

ABSTRACT: Biological systems contain highly-ordered structures performing diverse functions. The elegant structures of biomachines have inspired the development of nanopores as single molecule sensors. Over the years, the utility of nanopores for detecting a wide variety of analytes have rapidly emerged for sensing, sequencing and diagnostic applications. Several protein channels with diverse shapes and sizes, such as motor channels from bacteriophage Phi29, SPP1, T3, and T4, as well as ?-hemolysin, MspA, aerolysin, FluA, OmpF/G, CsgG, ClyA, have been continually investigated and developed as nanopores. Herein, we focus on advances in biological nanopores for single molecule sensing and DNA sequencing from a protein engineering standpoint for changing pore sizes, altering charge distributions, enhancing sensitivity, improving stability, and imparting new detection capabilities.

SUBMITTER: Wang S

PROVIDER: S-EPMC5994363 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Engineering of protein nanopores for sequencing, chemical or protein sensing and disease diagnosis.

Wang Shaoying S Zhao Zhengyi Z Haque Farzin F Guo Peixuan P

Current opinion in biotechnology 20171210

Biological systems contain highly-ordered structures performing diverse functions. The elegant structures of biomachines have inspired the development of nanopores as single molecule sensors. Over the years, the utility of nanopores for detecting a wide variety of analytes have rapidly emerged for sensing, sequencing and diagnostic applications. Several protein channels with diverse shapes and sizes, such as motor channels from bacteriophage Phi29, SPP1, T3, and T4, as well as α-hemolysin, MspA, ...[more]

PMID: 29232619

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Project description:Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5' phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5' end of the oligos and to leave a 3' adenosine overhang after binding to their target. The 3' adenosine overhang of the adapter and the terminal phosphate makes the 5' end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.

Dataset Information

Engineering of protein nanopores for sequencing, chemical or protein sensing and disease diagnosis.

Publications

Engineering of protein nanopores for sequencing, chemical or protein sensing and disease diagnosis.

Similar Datasets

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