Temperature-Independent Porous Nanocontainers for Single-Molecule Fluorescence Studies

Abstract

In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to perform single molecule fluorescence measurements over a biologically relevant temperature range. Lipid vesicles can serve as an ideal nanocontainer for single molecule fluorescence measurements of bio-macromolecules. 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 in equilibrium with the initial buffer condition. In order to permeabilize the barrier, Staphylococcal aureus toxin α-hemolysin (aHL) channels have been incorporated into the membrane. These aHL channels have been characterized using single molecule fluorescence resonance energy transfer (smFRET) signals from vesicle encapsulated guanine-rich DNA that folds in G-quadruplex motif as well as from 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 °C). Ions can efficiently pass through preformed aHL channels in order to initiate DNA folding without any detectable delay. With addition of the cholesterol to the membrane, we also report 35-fold improvement in 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 Rep helicase repetitive shuttling dynamic along a single stranded DNA at various temperature values. 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.

Regulation of the Membrane Insertion and Conductance Activity of the Metamorphic Chloride Intracellular Channel Protein CLIC1 by Cholesterol

“…Addition of equal amounts of the protein α-hemolysin to membranes containing varying amounts of cholesterol (0–50 mol%) clearly demonstrated an increase in membrane conductance due to α-hemolysin. Although α-hemolysin is not classed as a member of the cholesterol-dependent-cytolysins, recent studies in the literature indicate cholesterol does impact upon its activity and membrane interactions [31], with depletion of cholesterol from membranes resulting in arrest in the assembly of the α-hemolysin [32]. In the current study however, the highest levels of membrane conductance due to α-hemolysin were observed in the sample containing no cholesterol in the membrane (Figure 1).…”

Single-molecule investigation of G-quadruplex using a nanopore sensor

“…The G-quadruplex has also been studied using other single-molecule methods, including fluorescence resonance energy transfer (FRET) spectroscopy [82,84,85,92], and atomic force microscopy (AFM) [93-95]. Unlike these single molecule approaches, the nanopore electrical detection is a label-free method and does not need the attachment of probing agents such as fluorophores or immobilization on solid surfaces for observation.…”

High‐throughput single‐molecule studies of protein–DNA interactions

“…Most methods that seek to image individual molecules at higher fluorophore concentrations either increase the local protein concentration, or further confine the laser illumination volume. For example, the reaction volume can be reduced by encapsulating the biochemical reaction of interest in a porous lipid vesicle [41][42][43][44][45], within a PDMS nanochannel [46], or within a confined volume induced by a convex lens and a coverslip [47]. Alternatively, the laser excitation can be confined to an attoliter volume within a zero-mode waveguide [48][49][50], or near plasmonic nano-structures that locally enhance the light excitation [51,52].…”