Single molecule characterisation of biological nanopores in action

Descrizione del progetto

Informazioni biofisiche sui nanopori

I nanopori biologici sono i canali formati tramite le proteine transmembrana sulle cellule. Oltre alla loro funzione biologica specifica, tali nanopori sono stati utilizzati in diverse applicazioni per il rilevamento di biomolecole e per il sequenziamento del DNA. Il progetto NOTE, finanziato dall’UE, propone di usare la pinzetta ottica come metodo per studiare come i nanopori biologici interagiscono con i loro substrati durante la traslocazione. La pinzetta ottica impiega una luce laser concentrata per mantenere e muovere le molecole in alta risoluzione, offrendo un importante strumento di studio per i sistemi biologici. NOTE genererà conoscenze fondamentali necessarie per un proficuo utilizzo di nanopori biologici nell’ambito di tecnologie basate su biosensori per la medicina personalizzata.

Obiettivo

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.