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A new bioconjugation agent for RNA analysis.

Primary Supervisor: Professor Martin Wills, Department of Chemistry

Secondary supervisor: Professor Karuna Sampath (WMS), Other academic staff may become involved as the project develops

PhD project title: A new bioconjugation agent for RNA analysis.

University of Registration: University of Warwick

Project outline:

Background to project. There is a need for efficient chemical reactions which permit the attachment of chemical groups such as dyes, fluorescent markers, drug molecules etc., to large biomolecules such as proteins, RNA or DNA. Transformations of this type, i.e. ‘bioconjugation’ reactions,1 are used for both the identification and treatment of a range of diseases and medical conditions. A fluorescent group attached to a targeting protein, for example, can help identify the location of the molecule during early development or during progression of a specific illness such as a cancer. The attachment of a small-molecule drug to an antibody with specificity for cancer cell antigens provides a tool for the development of antibody-drug conjugate medicines (ADCs).2 However, efficient bioconjugations are generally difficult– multiple sites may react and undesired side-reactions may occur. For this reason, efficient reactions, which do not interfere with normal biological processes (i.e. ‘bioorthogonal’ reactions), have been developed. An important ‘bioorthogonal’ reaction is the cycloaddition of a ‘strained’ alkyne with functionalised azides without the need for a copper catalyst (Figure 1).3

Programme of work. In recent work at Warwick, a new class of fluorescent strained alkyne reagent has been developed and a number of derivatives are now available for assessment as bioconjugation reagents.4 This project shall involve a study of their attachment to a range of RNA derivatives together with analysis of the properties of the resulting adducts (Figure 1). Analysis of these shall involve the use of a range of methods including mass spectrometry, NMR and fluorescence spectroscopy. The labelled fluorescent compounds will be analysed by UV-vis and fluorescence spectroscopy. The rates of addition of the strained alkynes to the biomolecule azides will be measured, in order to gain structure-reactivity information. In addition, a series of compounds will be prepared in which Cu-catalysed addition reactions have been used to functionalise the RNA base.

Overall a rapid picture will be established of the effectiveness of the new bioconjugation agents on example RNA using a range of analytical methods, and of the potential value of the methodology for the development of diagnostic agents with medical applications. The selected RNAs will include those which have been recently identified as being of high therapeutic and diagnostic value in studies within the Department of Chemistry (Chem) and Warwick Medical School (WMS). It would also involve the expertise of the Warwick Centre for Mechanochemical Cell Biology (CMCB) and the Centre for Early Life (CfEL). This interdisciplinary project would be ideal for a Biochemistry or Chemistry graduate student with an interest in working at the boundaries between these areas. The student would gain training in synthetic chemistry and cell and molecular biology, as well as a range of analytical techniques in both disciplines, a range of analytical methods, and would become well equipped to work efficiently as a research across these areas.

References:

  1. Reviews on click chemistry and applications: (a) P. Thirumurugan, D. Matosiuk and K. Jowiak, Rev. 2013, 113, 4905-4979. (b) J.M. Palomo, Org. Biomol. Chem. 2012, 10, 9309-9318.
  2. Click reactions in peptide-based drug design; J. D. Thomas, H. Cui, P. J. North, T. Hofer, C. Rader, and T. R. Burke Jr. Bioconjugate Chem. 2012, 23, 2007–2013.
  3. Cu-free click cycloadditions in chemical biology; (a) J.C. Jewett and C. R. Bertozzi, Soc. Rev. 2010, 39, 1272-1279. (b) X. Zhang and Y. Zhang, Molecules 2013, 18, 7145-7159. (c) Y. Gong and L. Pan, Tetrahedron Lett. 2015, 56, 2123-2132. (d) X. Chen, K. Muthoosamy, A. Pfisterer, B. Neumann and T. Weil, Bioconjugate Chem. 2012, 23, 500-508. (e) J. Dommerholt, F. P. J. T. Rutjes and F. L. van Delft, Top. Curr. Chem (Z) 2016, 374:16.
  4. relevant publications by the applicants: (a) A. Del Grosso, L.-D. Galanopoulos, C. K. C. Chiu, G. J. Clarkson, P. B. O′ Connor and M. Wills, Org. Biomol.Chem. 2017, 15, 4517 – 4521. (b) A. Mistry, R. C. Knighton, S. Forshaw, Z. Dualeh, J. S. Parker and M. Wills, Org. Biomol. Chem. 2018, 16, 8965 - 8975.

BBSRC Strategic Research Priority: Renewable Resources and Clean Growth: Industrial Biotechnology. Understanding the Rules of Life: Stem Cells, Structural Biology & Systems Biology

Techniques that will be undertaken during the project:

Synthetic organic chemistry; Synthesis of substrates for testing, detection, isolation and analysis of organic reaction products, use of analytical methods including Nuclear Magnetic Resonance, Mass Spectrometry and chromatographic methods. Supervisor; Prof Martin Wills.

Cell & Molecular biology with respect to protein and RNA, including use of restriction enzymes, cloning, PCR, protein/RNA interactions and RNA synthesis, in vivo tests of RNAs in cells and zebrafish embryos. Supervisor; Karuna Sampath (WMS/CMCB/CfEL).

In all cases, the respective laboratories are equipped to a high standard with the facilities and instruments to support this project.

Interested students should discuss the project in detail with M. Wills and K. Sampath before making a selection.

Contact: Professor Martin Wills, University of Warwick