The objective of this renewal proposal is to bring nanopore sequencing technology with MspA to fruition. With funding through this program, our group has engineered the porin MspA specifically for nanopore sequencing. By combining MspA with a processive enzyme technology our team demonstrated the visionary goal of nanopore sequencing first conceived two decades ago. With continued funding, we will take the necessary steps to ready nanopore sequencing for industrial integration. We will also explore improvements and variations that make nanopore sequencing an even more powerful tool.
Our specific aims are: (1) develop the base calling algorithms necessary for our present nanopore sequencing process, (2) further refine the porin MspA through rational - and bold - genetic engineering in order to optimize its sequencing quality, (3) understand and improve DNA translocation dynamics by reducing its stochastic character and (4) demonstrate sequencing of long segments of native genomic DNA including identification of epigenetic modifications. Our team's success to date has enabled us to form partnerships and gain support from many excellent labs whose expertise has assisted us to move nanopore sequencing forward. We will work with our partner labs to complete the aims outlined in this proposal. It is our goal to improve the quality of human life by delivering a technique that can accurately sequence a human genome for well under $1000 and provide sequencing results within the short timeframe required for clinical practice.
The objective of this collaborative proposal is to bring nanopore sequencing to full fruition. With previous support we have engineered the porin MspA for nanopore sequencing and combined it with a processive enzyme to demonstrate nanopore sequencing. We propose to take the next steps necessary to ready the process for industrial integration, but we will also explore improvements and variations that make nanopore sequencing even more powerful.
| Craig, Jonathan M; Laszlo, Andrew H; Brinkerhoff, Henry et al. (2017) Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers. Proc Natl Acad Sci U S A 114:11932-11937 |
| Laszlo, A H; Derrrington, I M; Gundlach, J H (2017) Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT. Methods Enzymol 582:387-414 |
| Nova, Ian C; Derrington, Ian M; Craig, Jonathan M et al. (2017) Investigating asymmetric salt profiles for nanopore DNA sequencing with biological porin MspA. PLoS One 12:e0181599 |
| Pavlenok, Mikhail; Niederweis, Michael (2016) Hetero-oligomeric MspA pores in Mycobacterium smegmatis. FEMS Microbiol Lett 363: |
| Comer, Jeffrey; Aksimentiev, Aleksei (2016) DNA sequence-dependent ionic currents in ultra-small solid-state nanopores. Nanoscale 8:9600-13 |
| Laszlo, Andrew H; Derrington, Ian M; Gundlach, Jens H (2016) MspA nanopore as a single-molecule tool: From sequencing to SPRNT. Methods 105:75-89 |
| Bhattacharya, Swati; Yoo, Jejoong; Aksimentiev, Aleksei (2016) Water Mediates Recognition of DNA Sequence via Ionic Current Blockade in a Biological Nanopore. ACS Nano 10:4644-51 |
| Craig, Jonathan M; Laszlo, Andrew H; Derrington, Ian M et al. (2015) Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore. PLoS One 10:e0143253 |
| Derrington, Ian M; Craig, Jonathan M; Stava, Eric et al. (2015) Subangstrom single-molecule measurements of motor proteins using a nanopore. Nat Biotechnol 33:1073-5 |
| Morton, Danielle; Mortezaei, Shahab; Yemenicioglu, Sukru et al. (2015) Tailored Polymeric Membranes for Mycobacterium Smegmatis Porin A (MspA) Based Biosensors. J Mater Chem B 3:5080-5086 |
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