MCMs: a novel nanopore
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
Nanopore sequencers utilise protein pores and motors to rapidly sequence individual DNA molecules. With the availability of Oxford Nanopore Technologies' (ONT) MinION platform, nanopore sequencing is rapidly becoming a mainstream platform for high throughput sequencing. This project will perform a detailed investigation of the molecular mechanisms governing the helicase action of the homohexameric minichromosome maintenance (MCM) complex found in a range of archaeal species. We will collaborate with Oxford Nanopore Technologies (ONT) to determine:
1) if the flow cells from MinION DNA sequencers can be used as highly parallel, single-molecule scientific tools for assaying DNA helicase activity.
2) whether a MCM variant can be engineered to function as a combined DNA motor and nanopore in a MinION DNA sequencer
The novel application of ONT's recently-available MinION technology will allow us for the first time to determine at single molecule resolution the precise molecular characteristics of the MCM helicase while it is unwinding DNA. Measurements will include the speed of DNA translocation and unwinding by the helicase, the step size for unwinding, the ATP hydrolysis rates of helicase activity and the regularity with which these functions occur. MinION technology allows highly parallel data on single molecule measurements at nucleotide resolution to be collected quickly and easily, facilitating rapid progress on this project.
To facilitate our understanding of the process at a molecular scale, the student will utilise a range of biophysical techniques including state-of-the-art NMR approaches, EM and cryo-EM reconstructions to study the structure and function of these large protein assemblies in solution. These techniques will be used to map at atomic resolution the path taken by nucleic acids through the MCM complex and the conformational changes that the protein must undergo in order for DNA unwinding to be realized.
Our project builds on an initial collaborative study with ONT that supports the concept that MCM motor function can be measured on MinION devices. Support for the project provided by ONT, including training in sample preparation, nanopore assays and data analysis at ONT's site in Oxford will generate data that will demonstrate the efficacy of the ONT flow cell system as a single molecule research tool.
1) if the flow cells from MinION DNA sequencers can be used as highly parallel, single-molecule scientific tools for assaying DNA helicase activity.
2) whether a MCM variant can be engineered to function as a combined DNA motor and nanopore in a MinION DNA sequencer
The novel application of ONT's recently-available MinION technology will allow us for the first time to determine at single molecule resolution the precise molecular characteristics of the MCM helicase while it is unwinding DNA. Measurements will include the speed of DNA translocation and unwinding by the helicase, the step size for unwinding, the ATP hydrolysis rates of helicase activity and the regularity with which these functions occur. MinION technology allows highly parallel data on single molecule measurements at nucleotide resolution to be collected quickly and easily, facilitating rapid progress on this project.
To facilitate our understanding of the process at a molecular scale, the student will utilise a range of biophysical techniques including state-of-the-art NMR approaches, EM and cryo-EM reconstructions to study the structure and function of these large protein assemblies in solution. These techniques will be used to map at atomic resolution the path taken by nucleic acids through the MCM complex and the conformational changes that the protein must undergo in order for DNA unwinding to be realized.
Our project builds on an initial collaborative study with ONT that supports the concept that MCM motor function can be measured on MinION devices. Support for the project provided by ONT, including training in sample preparation, nanopore assays and data analysis at ONT's site in Oxford will generate data that will demonstrate the efficacy of the ONT flow cell system as a single molecule research tool.
People |
ORCID iD |
| James Chong (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M015831/1 | 01/10/2015 | 30/09/2019 | |||
| 2119240 | Studentship | BB/M015831/1 | 01/10/2017 | 30/09/2021 |