Development of Rapid Assays for Nucleic Acid-based Disease Targets

Secondary Supervisor(s): Professor Tim Dafforn

University of Registration: University of Birmingham

BBSRC Research Themes: Sustainable Agriculture and Food (Microbial Food Safety, Plant and Crop Science)

Project Outline

The recent COVID-19 pandemic has resulted in intense interest in the development of novel technologies for fast and sensitive nucleic acid (DNA & RNA) detection.[1] The current gold-standard for nucleic acid detection uses lab-based PCR, where strands of DNA are copied (amplified) many times to generate a read-out signal. On the other hand, home-based viral antigen tests using lateral flow devices may be faster and more convenient than PCR, but are generally much less sensitive. What is required for pandemics of the future and for other scenarios where rapid testing is imperative, are point-of-care assays that are both rapid and highly sensitive.

The development of a viable universal nucleic acid assay that could operate at a single temperature, while still amplifying its target, would provide a rapid advance in diagnostics technology. Such a test would be much faster than PCR as it would not have to rely on lengthy heating and cooling cycles, but be just as sensitive due to its target amplification step. Isothermal assays offer such a solution, with some based on Loop-mediated isothermal amplification (LAMP) being recently reported.[1] Our research is based on an even faster nucleic acid amplification process based on the exponential amplification reaction (EXPAR), for which we already reported an assay for SARS-CoV-2.[2]

Current technologies for EXPAR use fluorescence for the read-out signal. We now wish to harness and apply this technology to other read-out methods[3] that could lead to more advanced for multiplexed assays, where different disease targets can be detected simultaneously. The technology could in principle be applied to any assay where fast detection is required, including the detection of plant-based pathogenic agents or nucleic acid biomarkers for cancer.

In this project, the student (from a biology, biochemistry or chemistry background) will first learn the EXPAR protocol and then develop and adapt it further for multiplexed sensing. He/she will also vary the sequences and reagents used in the assay to adapt it for various disease targets. They will have an interest in learning and applying molecular biology techniques as well as performing a range of biochemical and analytical techniques, including DNA synthesis, gel electrophoresis, mass spectrometry, electrochemistry and UV/vis spectroscopy. He/she will enjoy working in a multi-disciplinary team and interacting with people in different laboratories at Birmingham, including those in the School of Chemistry, School of Biosciences and the Institute of Cancer and Genomic Sciences.

[1] L. Cheng et al, A review of current effective COVID‐19 testing methods and quality control, Archives of Microbiology (2023) 205:239, https://doi.org/10.1007/s00203-023-03579-9.

[2] J. G. Carter et al., Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription–free exponential amplification reaction, RTF-EXPAR. PNAS, 2021, 118, e2100347118, https://doi.org/10.1073/pnas.2100347118.

[3] For an example from our group of an optical sensing method for DNA, see: Combining bacteriophage engineering and linear dichroism spectroscopy to produce a DNA hybridisation assay. A. Ali et al., RSC Chem. Biol., 2020, 1, 449-454, https://doi.org/10.1039/D0CB00135J.

Molecular Machines from Adapted Transcription Factor-DNA Complexes

Secondary Supervisor(s): Dr Anna Peacock

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Structural Biology)

Project Outline

Scientists are investing significant effort into developing small molecular machines capable of translational or rotational motion at the molecular level in response to an external stimulus, with potential future applications in molecular electronics and data storage.[1] In chemistry, these systems are often based on supramolecular architectures such as catenanes (two mechanically interlocked macrocycles) or rotaxanes (a threaded macrocycle with stoppers) which contain individual components held together by a topological bond. Chemical docking stations which utilise recognition motifs are required in order to control molecular motion. However, most docking stations are not that sophisticated nor selective and can be difficult to make. In contrast, biomolecular recognition in nature is extremely sophisticated, whilst being highly selective. This project therefore sets out to utilise DNA-peptide recognition, using protein and DNA segments as the building blocks, to generate novel molecular machines from biological components.

Proteins such as transcription factors (e.g. the bZIP family) can bind to DNA with a high degree of sequence selectivity.[2] Often direct contact is made between the DNA target site and a relatively short protein segment, commonly an alpha-helix, despite the original protein being much larger. A number of examples now exist which demonstrate that this small protein fragment alone can still display similar sequence-selective DNA binding, and therefore these fragments and DNA represent attractive building blocks for assembling molecular machine-type architectures.[3] In this project the student will therefore study shortened versions of the bZIP family of transcription factor proteins, prepared using automated peptide synthesis. He/she will then assess how their adaptation and cyclisation affects their binding to DNA sequences. Synthetic DNA containing two target sites with different affinities (e.g. double stranded DNA, dsDNA vs single stranded DNA, ssDNA) will also be prepared to assess how selective the adapted transcription factors are in their binding to DNA. This is turn will lead to the design of machines displaying molecular motion in which peptide motion along the DNA axis between docking stations can be controlled using enzymatic synthesis of dsDNA.

References

[1] https://www.nobelprize.org/uploads/2018/06/popular-chemistryprize2016-1.pdf.

[2] M. A. Schumacher et al., The Structure of a CREB bZIPzSomatostatin CRE Complex Reveals the Basis for Selective Dimerization and Divalent Cation-enhanced DNA Binding, J. Biol. Chem., 2000, 275, 35242–35247, https://doi.org/10.1074/jbc.M007293200.

[3] G. A. Bullen, et al., Exploiting anthracene photodimerization within peptides: light induced sequence-selective DNA binding. Chem. Comm., 2015, 51, 8130-8133, https://doi.org/10.1039/C5CC01618E.