Towards nanopore proteomics: enhancing cytolysin performance through genetically encoded noncanonical amino acids

Description du projet

La technologie des nanopores permettra la détection rapide et portable de protéines de faible abondance

La technologie des nanopores repose sur les pores protéiques d’origine naturelle qui peuvent être assemblés à la surface de lipides reconstitués. Lorsqu’une tension est appliquée, les macromolécules chargées traversent le pore et perturbent le flux ionique. Les modifications du courant ionique sont caractéristiques de la molécule qui le traverse, et ont été appliquées avec grand succès au séquençage de molécules uniques d’ADN. Le projet nanoEx, financé par l’UE, améliore cette technologie pour la détection de protéines en modifiant de manière rationnelle les nanopores par le biais de l’évolution dirigée et de l’insertion d’acides aminés non canoniques, suivies de la caractérisation structurelle et biophysique de ces nanopores. L’amélioration de la biodétection de nanopores pour le séquençage de protéines aura des répercussions considérables sur la détection de protéines en temps réel dans le domaine du diagnostic environnemental et médical, ainsi que pour des applications de suivi.

Objectif

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.