Single-Molecule Experiments on Biological Nanopores Embedded on a Capillary
Author
Jankünaite, Dainora
Term
4. term
Education
Publication year
2019
Abstract
Motorproteiner, der translokerer nukleinsyrer, driver centrale cellulære processer, men i enkeltmolekyleteknikken SPRNT (single-molecule picometer resolution nanopore tweezers) er den netto kraft, der virker på DNA, ikke kendt, hvilket vanskeliggør tolkning af kinetik. Dette speciale havde til formål at muliggøre kraftmåling ved at kombinere optiske pincetter med en kapillærbaseret flowcelle, hvor en biologisk nanopore indlejres i et lipid dobbeltlag. Vi designede og fremstillede en flowcelle, udviklede en DNA‑konstruktion til optiske pincetforsøg og optimerede protokoller for lipidmembrandannelse. Ved kapillæråbningen dannede vi lipidmembraner og rekonstituerede MspA‑porinen, hvorefter vi målte og sammenlignede strøm‑spændings‑karakteristikker for poren i vores flowcelle med dem kendt fra SPRNT‑opstillingen. Vi demonstrerede desuden, at optiske pincetter er kompatible med bioporen og lipidmembranen i denne konfiguration. Samlet lægger arbejdet et eksperimentelt grundlag for fremtidig kalibrering af den kraft, der virker på DNA i SPRNT, som funktion af spænding, temperatur og saltkoncentration.
Motor proteins that translocate nucleic acids underpin key cellular processes, but in the single‑molecule SPRNT (single‑molecule picometer resolution nanopore tweezers) technique the net force acting on DNA is unknown, complicating kinetic interpretation. This thesis set out to enable force measurement by combining optical tweezers with a capillary‑based flow cell in which a biological nanopore is embedded in a lipid bilayer. We designed and fabricated the flow cell, developed a DNA construct for optical tweezers experiments, and optimized lipid bilayer formation. At the capillary opening we formed lipid membranes and reconstituted the MspA porin, then measured and compared the pore’s current–voltage characteristics in our flow cell with those from the SPRNT setup. We further demonstrated that optical tweezers are compatible with the biological pore and lipid bilayer in this configuration. Together, these results establish an experimental foundation for future calibration of the force experienced by DNA in SPRNT as a function of voltage, temperature, and salt concentration.
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