Single Molecule Nanocontainers Made Porous Using a Bacterial Toxin

Abstract

Encapsulation of a biological molecule or a molecular complex in a vesicle provides a means of biofriendly immobilization for single molecule studies and further enables new types of analysis if the vesicles are permeable. We previously reported on using DMPC (dimyristoylphosphatidylcholine) vesicles for realizing porous bioreactors. Here, we describe a different strategy for making porous vesicles using a bacterial pore-forming toxin, α-hemolysin. Using RNA folding as a test case, we demonstrate that protein-based pores can allow exchange of magnesium ions through the vesicle wall while keeping the RNA molecule inside. Flow measurements indicate that the encapsulated RNA molecules rapidly respond to the change in the outside buffer condition. The approach was further tested by coencapsulating a helicase protein and its single-stranded DNA track. The DNA translocation activity of E. coli Rep helicase inside vesicles was fueled by ATP provided outside the vesicle, and a dramatically higher number of translocation cycles could be observed due to the minuscule vesicle volume that facilitates rapid rebinding after dissociation. These pores are known to be stable over a wide range of experimental conditions, especially at various temperatures, which is not possible with the previous method using DMPC vesicles. Moreover, engineered mutants of the utilized toxin can potentially be exploited in the future applications.

A practical guide to studying G-quadruplex structures using single-molecule FRET

“…On a related note, it was shown that hGQ can be encapsulated within lipid vesicles, typically dimyristoylphosphatidylcholine (DMPC), that are immobilized on a supported lipid bilayer (such as EggPC), PEG, or BSA surface (Ishitsuka et al ; Okumus et al 2004; Cisse et al 2007; Okumus et al 2009; Okumus and Ha 2010). Comparative studies of hGQ that is encapsulated within a vesicle or immobilized on a BSA surface yielded consistent results in terms of the observed FRET levels (Lee et al 2005).…”

Single-molecule approaches to characterizing kinetics of biomolecular interactions

“…Following a similar reasoning, researchers have been able to capture fluorescently labeled binding partners in small (100-200 nm), surface-tethered lipid nanovesicles and study their interactions through single-molecule FRET measurements (Figure 2b) [51][52][53]54 ]. The volume of a 100-nm vesicle is two orders of magnitude smaller than that of a diffraction-limited volume and thus allows proteins to be visualized at the single-molecule level at much higher effective concentrations than hitherto possible.…”

Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC)

“…Furthermore, lipid vesicles or trapped droplets do not generally allow exchange of buffer within the confined volume, which is problematic for studying enzymes, since the reactants may quickly become consumed. Although nanovesicles can be adapted to allow exchange of small molecules through nanopores with the buffer in which the vesicles are immersed, this is not useful in the case that small molecule reactants, such as ATP, are the fluorescent substrates to be imaged [7]. Another alternative strategy for increasing the concentration limit that avoids three-dimensional confinement, PhotoActivation, Diffusion, and Excitation (PhADE), was recently demonstrated [8].…”