Single molecule characterisation of biological nanopores in action

Descripción del proyecto

Conocimientos biofísicos sobre los nanoporos

Los nanoporos biológicos son los canales formados a través de proteínas de transmembrana en las células. Aparte de su función biológica inherente, se han empleado en múltiples aplicaciones para la detección de biomoléculas, así como para la secuenciación de ADN. El objetivo del proyecto NOTE, financiado con fondos europeos, es emplear pinzas ópticas como método para estudiar la interacción de los nanoporos biológicos con sus sustratos durante la translocación. Las pinzas ópticas emplean luz láser enfocada para sostener y mover moléculas en alta resolución, por lo que constituyen una herramienta importante para el estudio de sistemas biológicos. El trabajo del equipo de NOTE proporcionará conocimientos fundamentales para el empleo satisfactorio de nanoporos biológicos en tecnologías de biosensores para la medicina personalizada.

Objetivo

Biological nanopores are nanometre-scale holes in membranes created by transmembrane proteins. In nature, nanopores come from pore-forming toxins or exist as transporters in bacteria. To date, they have been successfully used for an impressive variety of applications ranging from DNA sequencing through to the measurement of function-related motions of enzymes. Planned developments using nanopores include their integration into biosensors for the detection of analytes from blood. However, to reach their potential, miniaturisation and parallelisation of electrophysiology setups and an in-depth understanding of how nanopores exert forces on their substrates during trapping and translocation are required. There are currently no experimental techniques which provide access to this information. Single molecule studies using the optical tweezers (OT) have, over the past decade, become the gold standard in high resolution measurements of forces in biological systems, including observations of sub-nanometre enzyme kinetics, protein folding and protein degradation machinery in action. I propose the development of low-cost, microfluidic-based tools for the study of biological nanopores using OT. The technologies arising from this work will provide an unprecedented insight into the function of biological nanopores and will enable studies of any other transmembrane protein system, many of which are also of great medical importance. The findings will also lay the groundwork for peptide-sequencing and biosensor technologies for personalised medicine. The combination of my skills in single molecule biophysics and Prof. Maglia’s expertise at the forefront of biological nanopore technologies is ideal for the successful completion of the research objectives. Through the planned interdisciplinary work and high-quality training, I will increase both my research- and transferable skill sets enabling me to reach my goal of establishing a leading independent research group within the EU.