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Development of Ultra-fast Assays for RNA-based Disease Targets

Primary Supervisor: Professor James Tucker, School of Chemistry

Secondary supervisors: Professor Tim Dafforn

PhD project title: Development of Ultra-fast Assays for RNA-based Disease Targets

University of Registration: University of Birmingham

Project outline:

The recent COVID-19 pandemic has resulted in intense interest in the development of novel technologies[1] for fast and sensitive nucleic acid (DNA & RNA) detection. The current gold-standard COVID-19 assay uses PCR, where strands of DNA are copied (amplified) many times to generate a read-out signal. However, as the virus genome of SARS-CoV-2 is RNA, first conversion from RNA into DNA is required using reverse transcription (RT), which slows down the assay time. Recently we reported an entirely new method for generating DNA from RNA which bypasses the lengthy RT step, making it reverse-transcriptase free (RTF). We then coupled this process with a much faster amplification process than PCR, called EXPAR. The new combined assay, called RTF-EXPAR (see scheme below), has led to detection times of SARS-CoV-2 RNA in less than 10 minutes.[2]

The current technology for the RTF-EXPAR assay uses fluorescence for the read-out signal. We now wish to apply this technology to other read-out methods (e.g. electrochemistry[3] or other optical methods[4] 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 RNA-based assay where fast detection is required, including the detection of other pathogenic agents and nucleic acid-based cancer biomarkers.

In this project, the student (from a chemistry, molecular biology or biochemistry background) will first learn the RTF-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 alternative RNA targets to SARS-CoV-2. They will have an interest in learning and applying molecular biology techniques as well as performing a range of chemical 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.

References:

  1. For a review of COVID-19 assays, see: Carter et al., Assay Techniques and Test Development for COVID-19 Diagnosis. ACS Cent. Sci. 2020, 6, 591–605, https://doi.org/10.1021/acscentsci.0c00501
  2. 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 electrochemical sensing method for DNA, see: Duprey et al., Macrocyclic Metal Complex–DNA Conjugates for Electrochemical Sensing of Single Nucleobase Changes in DNA. J. Am. Chem. Soc., 2016, 138, 746-749, https://doi.org/10.1021/jacs.5b11319
  4. 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. Ali et al., RSC Chem. Biol., 2020, 1, 449-454, https://doi.org/10.1039/D0CB00135J

BBSRC Strategic Research Priority: Renewable Resources and Clean Growth: Industrial Biotechnology & Understanding the Rules of Life: Structural Biology & Integrated Understanding of Health: Diet and Health

Techniques that will be undertaken during the project:

  • Bioconjugation chemistry
  • DNA synthesis, characterisation and bioconjugation techniques
  • Enzyme catalysis
  • Spectroscopy (e.g. NMR, UV, CD, LD)
  • Mass spectrometry, HPLC, electrochemistry, electrophoresis

Contact: Professor James Tucker, University of Birmingham