Temperature-Independent Porous Nanocontainers for Single-Molecule Fluorescence Studies

In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to per form single molecule fluorescence measurements over a biologically relevant temperature range Lipid vesicles can serve as an ideal nanocontainer for single molecule fluorescence measurements of biomacromolecules One serious limitation of the vesicle encapsulation method has been that the lipid membrane is practically impermeable to most ions and small molecules, limiting its application to observing reactions m equilibrium with the initial buffer condition To permeabilize the barrier, Staphylococcus aureus toxin alpha-hemolysin (aHL) channels have been incorporated into the membrane These aHL channels have been characterized using single molecule fluorescence resonance energy transfer signals from vesicle encapsulated guanine-rich DNA that folds in a G-quadruplex motif as well as from the Rep helicase -DNA system We show that these aHL channels are permeable to monovalent ions and small molecules, such as ATP, over the biologically relevant temperature range (17-37 degrees C) Ions can efficiently pass through preformed aHL channels to initiate DNA folding without any detectable delay With addition of the cholesterol to the membrane, we also report a 35 fold improvement in the aHL channel formation efficiency, making this approach more practical for wider applications Finally, the temperature-dependent single molecule enzymatic study inside these nanocontainers is demonstrated by measuring the Rep helicase repetitive shuttling dynamics along a single stranded DNA at various temperatures The permeability of the biofriendly nanocontainer over a wide range of temperature would be effectively applied to other surface-based high throughput measurements and sensors beyond the single molecule fluorescence measurements