Bilateral NSF/BIO-BBSRC: Engineering Tunable Portal Hybrid Nanopores for High-Resolution Sequence Mapping

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Genome technology is an important part of modern life and is used routinely in medicine, forensic and crop science. However, despite rapid advances in DNA sequencing technology, large regions of genomes, including the human genome, are poorly characterised. In this proposal we aim to develop a new hybrid nanopore technology for DNA analysis and explore the possibility of using this method for improved analysis of these poorly defined or 'dark' genomic regions.These 'dark' regions are poorly defined largely due to the presence of multiple repeats. These 'repetitive elements' are difficult to analyse with currently available technology. This is because current methods rely on DNA polymerase enzyme activity which is prone to 'stuttering' and 'slipping' on AT rich repeats and 'stalling' on GC rich repeats.Nanopore technology is a polymerase-independent method for mapping at near-basepair resolution of very long DNA fragments (>100 Kb). Indeed it is hoped that this technology will be advanced towards the de novo sequencing of whole genomes. However, despite great promise, these technologies are still in development and currently are between 60-90% accurate. While protein nanopores, such as the alpha-haemolysin protein, are easily reproducible and tunable, they are generally derivatives of membrane pores. Thus they are supported in relatively fragile lipid-like membranes, requiring detergent for handling and high (micromolar) DNA concentration for signal detection. Conversely, ultra-thin layer solid-state (SS) nanopores are more robust and require only nanomolar quantities of DNA. However, they are difficult to fabricate routinely with the tiny diameter (<4nm) required for high-resolution DNA mapping.Hybrid nanopores can combine the advantages of both systems: namely (i) the reproducible production of tunable nanopores with diameters of 1-3 nm with (ii) robust properties that results in low (nanomolar) DNA concentrations required for signal detection. However, current protein nanopores require detergent and substantial chemical modification for integration into SS nanopores. We propose to investigate the suitability of a natural DNA nanopore, the portal protein from a thermophilic virus, for hybrid nanopore production. This protein is the nanopore through which DNA passes during packaging of the viral genome and so naturally processes the characteristics designed for capture and directional transition of dsDNA. Additionally this bionanopore is thermostable, highly soluble, tractable for bioengineering purposes and easy to produce in large highly pure quantities. Furthermore, the available high-resolution X-ray structure of the portal protein allows the design of portal variants with modified DNA transition properties.In this proposal, we specifically aim to explore and optimise the integration of the portal protein into SS nanopores and define the DNA transition dynamics. Bioengineering methods will be used to optimise the portal protein for 3 microseconds/basepair transition speeds. The hybrid nanopore will be calibrated for accurate analysis of DNA sequences containing multiple repeats.