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

Descrizione del progetto

La tecnologia dei nanopori consentirà il rilevamento portatile e rapido delle proteine a bassa abbondanza

La tecnologia dei nanopori si basa sui pori proteici naturali che possono essere assemblati in superfici lipidiche ricostituite. Con la tensione applicata, le macromolecole caricate attraversano i pori e alterano il flusso ionico. Le modifiche nella corrente ionica sono caratteristiche della molecola di passaggio e sono state applicate con grande successo nel sequenziamento del DNA a singola molecola. Il progetto nanoEx, finanziato dall’UE, sta valorizzando la tecnologia per il rilevamento di proteine con la modificazione razionale dei nanopori attraverso l’evoluzione e l’inserimento pilotati di amminoacidi non canonici, con successiva caratterizzazione biofisica e strutturale di tali nanopori. Il biorilevamento migliorato dei nanopori per il sequenziamento delle proteine avrà, nel settore, un impatto significativo sul rilevamento delle proteine in tempo reale, per la diagnostica medica e ambientale nonché per le applicazioni di monitoraggio.

Obiettivo

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.