Controlled Assembly of Fluorophores inside a Nanoliposome

Abstract

Cellular compartmentalization plays an essential role in organizing the complex and multiple biochemical reactions in the cell. An artificial compartment would provide powerful strategies to develop new biochemical tools for material production and diagnosis, but it is still a great challenge to synthesize the compartments that encapsulate materials of interest while controlling their accurate locations, numbers, and stoichiometry. In this study, we evaluated chemical characteristics of a liposome-encapsulated compartment, which has great potential to locate various materials of interest with precise control of their locations and numbers in the compartment. A nanoliposome was constructed inside a ring-shaped DNA origami skeleton according to the method of Yang et al., and further equipped with a double-stranded DNA platform to assemble molecules of interest in the nanoliposome. Upon formation of the nanoliposome, a pH-sensitive fluorophore on the bridged platform showed little or no response to the pH change of the outer buffer, ensuring that the molecules assembled on the platform are effectively shielded from the outer environment. The ring-shaped DNA skeleton equipped with a double-stranded DNA platform allows spatial assembly of several functional molecules inside the nanoliposome to isolate them from the outer environment.

An Artificial Liposome Compartment with Size Exclusion Molecular Transport

“…The estimated pK a value was shifted to the basic side compared to free CF (pK a = 6.5) due to the destabilization of the dianionic form of CF by the negative charge of the DNA phosphate backbone of WS, which is consistent with that of CF on a DNA nanostructure as reported previously. [32,39] The result indicated that the fluorescence emission of CF on naked-WS retained its sensitivity to pH changes and was effectively decreased at pH 6.0. Fluorescence microscopy analyses of naked-WS were carried out by changing the pH of the outer buffer (Figure 4a and S9).…”

“…190 μL of the recovered solution was mixed with 110 μL of 54 % iodixanol in 1× hydration buffer and placed at the bottom of a centrifuge tube (11×34 mm, Beckman Coulter Inc.). Six additional layers of 0 to 18 % iodixanol solution [29,32] in buffer were added to the centrifuge tube (Figure S6). The tube was spun in an MLA-130 rotor (Beckman Coulter Inc.) at 150,000 × g at 4 °C for 5 h, and the fractions (55 μL per fraction) were collected.…”

“…[37] The lipid bilayer was reconstituted by a dialysis step. The dialyzed solution was loaded onto an iodixanol gradient, optimized based on our previous work [32] to maximize recovery yield, and spun in an ultracentrifuge to separate liposomecoated WS (lipo-WS) (Figure S6a).…”

“…[28] DNA nanostructures have served as high-precision templates for membrane engineering and have provided a promising route for the construction of confined membrane structures. [29][30][31][32] In this study, an artificial liposome compartment with a uniform diameter of ~80 nm was constructed using an internal DNA origami skeleton prepared by modifying the design reported by Perrault et al [33] to realize the following features: its interior is isolated from the external environment and it provides a scaffold for organizing the molecules of interest. The internal DNA origami skeleton was modified with a defined number of pH-sensitive and/or pH-insensitive fluorophores to monitor pH changes around the skeleton.…”

“…At the same time, rationally designed DNA nanostructures were modified with lipid molecules at controlled positions to promote the formation of liposomes of defined size [28] . DNA nanostructures have served as high‐precision templates for membrane engineering and have provided a promising route for the construction of confined membrane structures [29–32] …”