Project description
Nanopore technology will enable portable and rapid sensing of low-abundance proteins
Nanopore technology is based on the naturally occurring protein pores that can be assembled in reconstituted lipid surfaces. Under applied voltage charged macromolecules move through the pore and disrupt the ionic flow. Changes in the ionic current are characteristic of the passing molecule and have been applied for single-molecule DNA sequencing with great success. The EU-funded nanoEx project is enhancing the technology for protein detection by rational modification of the nanopores via directed evolution and insertion of noncanonical amino acids with subsequent biophysical and structural characterisation of such nanopores. Improved nanopore biosensing for protein sequencing will have significant impact on real-time protein detection in the field for medical and environmental diagnostic and monitoring applications.
Objective
Accurate detection of low-abundance proteins in biological samples obtained from patients or invasive species relies on appropriate handling and fixation techniques. Due to their inherent instability and propensity to denature upon freeze-thaw treatments, important protein markers tend to escape detection. Analogous challenges in RNA sequencing have been mitigated by the use of nanopore technology. European academic research and industry actions led to the creation of the widely used MinION devices that employ nanopore technology to enable DNA and RNA sequencing to be executed on site. Through this application I seek to improve nanopores as biosensors for the detection of proteins that will facilitate immediate protein analysis from clinical or ecological samples. I will use my expertise in genetic code expansion (GCE) techniques to engineer two well-characterized cytolysins with distinct architectures: an α-helical actinoporin and β-barrel containing lysenin. To improve these pores for protein identification and sequencing, the proposed research will advance through four stages: Incorporation of noncanonical amino acid (ncAA) to covalently stabilize smaller pores (1) and modulate the pore’s sensing region (2), directed evolution of residues lining the channel walls (3) and structural characterization of the identified variants (4). This is a multidisciplinary project that combines GCE techniques with advanced biophysical and structural characterization of the evolved nanopores. The host’s expertise in nanopore analysis together with my experience with GCE and directed evolution methods provide an important two-way transfer of knowledge. Action includes a training-through-research essential to advance my future academic career and enhance my employability in the biomedical industry. I anticipate the results of nanoEx project will have a major impact on the progress of nanopore biosensing, increasing the competitiveness of the European industry in protein sequencing.
Fields of science
Programme(s)
Funding Scheme
MSCA-IF-EF-RI - RI – Reintegration panelCoordinator
1000 Ljubljana
Slovenia