DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity.

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches, including optical voltage-sensing dyes and genetically encoded voltage indicators, have evolved. Here, we present a DNA nanotechnology-based system and demonstrated its functionality on liposomes. Using DNA origami, we incorporated and optimized different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we used a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA to connect the nanostructure and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit was read out by fluorescence resonance energy transfer (FRET) changes of single sensors attached to liposomes. A FRET change of ∼5% for ΔΨ = 100 mV was observed. The working mechanism of the sensor was rationalized by molecular dynamics simulations. Our approach holds potential for an application as nongenetically encoded membrane sensors.

Manipulating Enzymes Properties with DNA Nanostructures.

Nucleic acids and proteins are two major classes of biopolymers in living systems. Whereas nucleic acids are characterized by robust molecular recognition properties, essential for the reliable storage and transmission of the genetic information, the variability of structures displayed by proteins and their adaptability to the environment make them ideal functional materials. One of the major goals of DNA nanotechnology-and indeed its initial motivation-is to bridge these two worlds in a rational fashion. Combining the predictable base-pairing rule of DNA with chemical conjugation strategies and modern protein engineering methods has enabled the realization of complex DNA-protein architectures with programmable structural features and intriguing functionalities. In this review, we will focus on a special class of biohybrid structures, characterized by one or many enzyme molecules linked to a DNA scaffold with nanometer-scale precision. After an initial survey of the most important methods for coupling DNA oligomers to proteins, we will report the strategies adopted until now for organizing these conjugates in a predictable spatial arrangement. The major focus of this review will be on the consequences of such manipulations on the binding and kinetic properties of single enzymes and enzyme complexes: an interesting aspect of artificial DNA-enzyme hybrids, often reported in the literature, however, not yet entirely understood and whose full comprehension may open the way to new opportunities in protein science.

Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions.

The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.