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Accurate and Ultra-Sensitive DNA Mutation Detection by Nanopore Barcode Recording

   Faculty of Environment

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  Dr M Castronovo, Dr J L Thorne, Dr N Thomson, Dr P Actis, Dr R Tooze  No more applications being accepted  Competition Funded PhD Project (Students Worldwide)

About the Project

Mutations of DNA within the genome are defining features of cancers, yet their accurate, quantitative, multiplexed detection with amplification-based methods is only possible above a threshold of tumour burden where sufficient material is available. Nucleic acid amplification-based methods introduce errors, which are more pronounced the lower the concentration of starting material, thus limiting the time primary or secondary disease can be detected after tumour growth has begun. Therefore, the quest has intensified for rapid, sensitive and affordable high-throughput techniques for the accurate and sensitive detection of single-base genetic mutations in suboptimal samples.

Emerging nanopore technologies such as solid-state nanopores (SSNP) have the potential to meet this challenge. SSNP are non-biological devices that can measure ionic current fluctuations from picoampere to nanoampere, occurring in milliseconds due to the transit of molecular-scale objects through a pore of 20-80 nanometre (nm) in diameter. SSNP can detect single, large biomolecules with high throughput and sensitivity. They hold great potential for medical diagnostic applications owing to their high durability, low-cost and pocketsize equipment. However, due to relatively large nanopore size compared to the diameter of the DNA double-helix (approx. 2 nm) and the distance between two consecutive bases in its sequence (approx. 3.4 nm), SSNP technologies are unable to directly analyse DNA sequences with single-base resolution (Xue et al. Nature Reviews Materials 2020).

Our integrated DNA nanotechnology and SSNP approach:

pioneering work led by the Dr Castronovo lab on the allosteric properties of self-assembled DNA origami nanostructures (Stopar et al. NAR 2018, Suma et al. NAR 2020) offers a solution to link the sequence of a DNA molecule trapped in a DNA nanostructure to an enzymatic transformation of the same nanostructure, a new concept termed allosteric DNA recording. Inspired by these findings, the key idea underpinning this project is to use nanotechnology methods to cage each copy of gene target in the sample inside a mutation-sensing, DNA-based nanostructure. We will expand our knowledge of enzymatic reactions with DNA origami nanostructures to design and optimise an enzymatic process that accurately inspects the whole gene sequence trapped by the cage, including the inherent presence/absence of the mutation of interest. Rather than amplifying the genetic material of liquid biopsies that may contain the single-base DNA mutation(s) of interest, this process will associate each copy of a target mutation in solution with a unique DNA nanostructure barcode that can be detected by nanopore transit with single-copy resolution, based on pioneering work led by Dr Actis’ lab (Raveendran et al. Nature Communications 2020). This will lead to a step-change in clinical capacity to detect cancers in minimal samples including blood.

The technology will be developed and optimised to improve the diagnosis of two prototypical examples of where early diagnosis is critical to addressing the poor survival rates faced by patients: diffuse large B-cell lymphoma (DLBCL) (Prof. R.Tooze) and secondary breast cancer (BC) (Dr J. Thorne), using plasma cell-free DNA (cfDNA). Initially, we will detect synthetic DNA sequences in solution, then in cell cultures and, finally, in matched tumour/blood samples from cancer patients. The sensitivity and specificity of the technology will be assessed by independent sample analysis by means of qPCR and next-generation sequencing.

You will apply current methods in DNA nanotechnology (Dr. M.Castronovo), solid-state nanopore (Dr. P.Actis), atomic force microscopy (Dr N. Thomson), molecular biology methods for DNA sample preparation (Dr. J.Thorne and Prof. R.Tooze). You will become part of an international group of scientists, working within cutting-edge interdisciplinary research at the frontier between biomedical and physical sciences.

Funding Notes

This 3.5 years EPSRC DTP award will provide full tuition fees, a stipend at the UK research council rate (UK Sterling 15,840 for 2022/23), and a research training and support grant.

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