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Nucleic acid delivery to plants. Towards improved protection and development of crops

Primary Supervisor: Dr Paco Fernandez-Trillo (PFT), School of Chemistry

Secondary supervisor: Estrella Luna-Diez (ELD) and Sara Jabbari (SJ)

PhD project title: Nucleic acid delivery to plants. Towards improved protection and development of crops

University of Registration: University of Birmingham

Project outline:

Plant genetic engineering is an important tool used in current efforts in crop improvement, pharmaceutical product biosynthesis and sustainable agriculture. Exogenous biomolecule delivery into plants is difficult because the plant cell wall poses a dominant transport barrier, thereby limiting the efficiency of plant genetic engineering. Traditional DNA delivery methods for plants suffer from host-species limitations, low transformation efficiencies, tissue damage, or unavoidable and uncontrolled DNA integration into the host genome. While nanomaterials have been studied for gene delivery into animal cells their potential for plant systems remains under-studied. PFT’s group has recently developed an innovative screening technology, that allow us to identify transfection agents in a timely and efficient way. This methodology allows us to screen for transfection efficiency and toxicity without having to purify any of the candidate transfection agents, simplifying this way the discovery process.

In this multidisciplinary project, we will identify novel transfection agents capable of delivering plasmid DNA to plant cells and tissues that are classically thought of as difficult or impossible to transfect.

Research Objectives: Our high-throughput strategy is based on the modification of a polymer scaffold (P1) to give us access to a library of modulated transfection agents (PM) with a broad range of chemical functionalities in highly efficient way (Scheme). Chemical functionality will be selected from a range of moieties known to mediate conjugation to nucleic acids (i.e. cationic residues) and transport across membranes (i.e. hydrophobic moieties). Detailed objectives are:

O1. Synthesis of Polymer Scaffolds (P1): In this part of the project we will explore the use of other polymer backbones (e.g. degradable polyesters, helical polyacetylenes) and functionalities (e.g. alkoxyamine) that can improve biocompatibility and stability of the formed hydrazones. All new compounds will be characterised by NMR, HRMS, GPC and IR.

O2. Synthesis of Modulated Transfection agents (PM): We will prepare candidate polymers Pm simply by incubation of P1 with the desired aldehydes, and the activity tested by diluting this mixture in the relevant assay. Loading efficiencies will be evaluated by NMR and GPC, and the ability of PM to facilitate the transport of nucleic acids across membranes monitored first with a fluorogenic assay, to identify any candidate polymers with potential to damage membranes.

O3. High-throughput evaluation of Transfection: The initial screening will be done by incubation of the peptide-plasmid complexes with somatic embryos of model plants. An easy- to-monitor cargo will be used, such as plasmids encoding for reporter genes (EGFP, luciferase, etc.). The efficiency of the transfection will be followed by confocal microscopy (EGFP) or enzymatic activity (luciferase). The compounds identified in this first round of screening will be thoroughly characterized. The biological (toxicity, integration of DNA, mRNA expression levels, etc.) and biophysical properties (hydrodynamic radius, zeta-potential, stability against nucleases and heparin displacement, etc.). We will employ an iterative process so that toxicity and transfection efficiency will inform the choice of polymer scaffolds (P1), aldehydes and their compositions. Mathematical modelling will be employed to correlate how these parameters affect transfection and toxicity and optimise PM design. Lead compounds will be then taken and transfection in whole tissues (roots and leaves) will be explored.

BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Plant and Crop Science

Techniques that will be undertaken during the project:

Due to the multidisciplinary approach, the PhD student will develop a wide range of experimental skill described in the following training objectives:

  • Training 1. Cell culture and cell phenotyping including culture of J774 macrophages and primary myeloid cells.
  • Training 2. Fluorescence and Confocal Microscopy including live cell imaging, multidimensional imaging and quantitative fluorescence work
  • Training 3. Computational Modelling: The skills developed as part of the Foundation Skills Modules (i.e.: Large Dataset Handling, Quantitative Biology) will be supported by training in multinomial logistic and multivariate regression.
  • Training 4. (Polymer) synthesis and Characterisation: Controlled polymerisation (i.e. free radical), combinatorial chemistry, (bio)conjugation using efficient chemistries (i.e. hydrazine-aldehyde condensation), polymer characterisation (NMR, GPC, IR). This expertise will be key for the completion of Objective 1.

Contact: Dr Paco Fernandez Trillo, University of Birmingham