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

Descripción del proyecto

Una tecnología de nanoporos permitirá la detección portátil y rápida de proteínas de baja concentración

La tecnología de nanoporos emplea proteínas formadoras de poros naturales que se pueden ensamblar en superficies lipídicas reconstituidas. Al aplicar tensión eléctrica, las macromoléculas cargadas se desplazan a través del poro e interrumpen el flujo iónico. Los cambios en la corriente iónica son característicos de la molécula que la atraviesa y se han empleado para la secuenciación de una molécula única de ADN con gran éxito. El proyecto nanoEx, financiado con fondos europeos, está mejorando la tecnología de detección de proteínas mediante la modificación racional de los nanoporos a través de la evolución dirigida y la inserción de aminoácidos no canónicos con la consiguiente caracterización biofísica y estructural de dichos nanoporos. La mejora de la biodetección por nanoporos para la secuenciación de proteínas tendrá un impacto significativo en la detección de proteínas en tiempo real para aplicaciones de diagnóstico y supervisión médica y medioambiental.

Objetivo

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.