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

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.

Nanopore based sequencing is proving useful in sequencing long genomic regions (>100 Kb) and may provide a method to accurately map regions containing repetitive DNA sequences. However, this technology is currently in development with several challenges to overcome. Hybrid nanopores may combine the advantages of both protein and solid-state (SS) nanopores to form a reproducible, robust nanopore that requires nanomolar concentrations of DNA for detection. However, to date the only successful hybrid nanopore reported was created using the membrane-associated alpha-haemolysin protein in a complex process that required detergents.

We propose to exploit the stable and soluble portal protein from the thermophilic bacteriophage G20C, as an alternative protein component in the production of hybrid nanopores. This protein is a natural DNA nanopore, serving as the gate through which DNA passes during the viral packaging process. Indeed preliminary experiments using voltage differentials and sensitive electronic detection have proven very promising and we aim to capitalise on this work by defining the method for efficient and stable insertion of portal in solid-state nanopores. Time resolved ion current signals will be used to characterise the DNA transition dynamics. We will use bioengineering and chemical biology approaches to tune the tunnel loops of the portal protein to acheive DNA transition rates of 3 microseconds/basepair and to create a stable portal SS-nanopre interface. Engineered proteins will be characterised using biophysical techniques, prior to characterisation of the DNA transition dynamics. Portal variants with the most suitable properties will be further characterised by X-ray crystallography. Finally, hybrid nanopores will be used to map the position and size of protein-bound DNA regions in selected viral genomes and to define the characteristic ion current signals for transition of homopolymeric ssDNA.

Beneficiaries and interested parties:
Inspired by the powerful DNA motors present in viruses, we aim to exploit a synthetic molecular nanomachine for creation of a novel tool for DNA analysis. Specifically, we aim to create a hybrid nanopore that combines the advantages of both protein and solid-state nanopores as a DNA mapping tool. Such a tool would be used to define the repetitive elements in DNA that are not well characterised with currently available methods. To this end, we propose to combine a recently characterised natural DNA pore found in thermostable bacterial viruses with a synthetic nanopore drilled into an ultrathin metal membrane fabricated with the latest photolithographic technology. Such a hybrid nanopore is expected to have superior stability, sensitivity and resolution for DNA mapping uses compared with either protein or synthetic nanopores alone. It is hoped that this would lead to the development of rapid and accurate methods for mapping multiple repeat regions within genomes. In the future this technology could potentially lead to devices for sequencing whole genomes.

(1) The immediate beneficiaries include those researchers in academia (national and international) and in the private commercial sector (pharmaceutical companies), who are developing new hybrid biological and synthetic molecular nanotechnology approaches, in particular novel DNA and protein sequencing tools. These researchers will benefit from the research outcomes that we plan to publish in months 18, 28 and 36. They will also benefit from new structural data on modified viral portal proteins which will be deposited with the Protein Data Bank and made publicly accessible upon publication. Engagement with industry will involve participation in the BBSRC Knowledge Transfer Network for Nanotechnology events and the provision of information for both web-based and print media articles.

(2) Long-term direct and indirect beneficiaries would include: (i) Researchers in pharmaceutical companies targeting rapid, cost-effective genome sequencing technology for personalised medicine approaches and therapeutic gain; (ii) Crop scientists who rely on genome sequencing technologies for improvement of yield, drought and disease tolerance; (iii) Researchers in academia and industry that seek to understand the genetic regulation of potentially useful natural products; (iv) Industrial researchers interested in understanding the composition and function of difficult-to-sequence and non-protein-coding genomic regions how this relates to human disease; (v) Forensic scientists that require superior accuracy for defining repeat regions used to identify individuals; and (vi) The wider population who will benefit from improved health, nutrition and justice that would accompany accurate definition of these previously poorly characterised repetitive regions.

Engagement with the wider community will be facilitated by participation in Forum for the Future, a non-profit sustainability network that connects government, business and community groups. Finally, participation in general public engagement events, such as the Cambridge Science Fair and YorNight events will provide opportunities for communication with the general public about the potential contribution of nanopore sequencing technology to improvements in everyday life.

Wider Social and Economic Benefits:
Development of a sequencing technology capable of accurately mapping whole genomes, including the regions that are currently poorly defined, would have broad economic and social impact. The life sciences (including health, pharmaceutical and forensic industries) and agri-food industries would benefit from the sale of new genetic tests and treatments for disease, and from superior crop species, leading to a healthier and well-nourished society.