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Charges in lipid head groups generate electrical surface potentials at cell membranes, and changes in their composition are involved in various signaling pathways, such as T-cell activation or apoptosis. Here, we present a DNA origami-based sensor for membrane surface charges with a quantitative fluorescence read-out of single molecules. A DNA origami plate is equipped with modifications for specific membrane targeting, surface immobilization, and an anionic sensing unit consisting of single-stranded DNA and the dye ATTO542. This unit is anchored to a lipid membrane by the dye ATTO647N, and conformational changes of the sensing unit in response to surface charges are read out by fluorescence resonance energy transfer between the two dyes. We test the performance of our sensor with single-molecule fluorescence microscopy by exposing it to differently charged large unilamellar vesicles. We achieve a change in energy transfer of ∼10% points between uncharged and highly charged membranes and demonstrate a quantitative relation between the surface charge and the energy transfer. Further, with autocorrelation analyses of confocal data, we unravel the working principle of our sensor that is switching dynamically between a membrane-bound state and an unbound state on the timescale of 1-10 ms. Our study introduces a complementary sensing system for membrane surface charges to previously published genetically encoded sensors. Additionally, the single-molecule read-out enables investigations of lipid membranes on the nanoscale with a high spatial resolution circumventing ensemble averaging.

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.

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. Using DNA origami, we incorporate and optimize different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we use a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA connecting the DNA origami and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit is read out by changes of Fluorescence Resonance Energy Transfer (FRET) of single sensors attached to liposomes. They show a FRET change of ∼5% for ΔΨ=100 mV and allow adapting the potential range of highest sensitivity. Further, the working mechanism is rationalized by molecular dynamics simulations. Our approach holds potential for the application as non-genetically encoded sensors at membranes.

In recent years, DNA nanotechnology has matured to enable robust production of complex nanostructures and hybrid materials. We have combined DNA nanotechnology with sensitive optical detection to create functional single-molecule devices that enable new applications in single-molecule biosensing and superresolution microscopy. Starting with superresolution nanorulers and brightness reference samples we determined the resolving power of superresolution microscopes and evaluated the sensitivity of smartphone cameras. To improve the sensitivity we created DNA origami optical antennas for metal enhanced fluorescence. The unique ability of our DNA origami nanoantennas to place molecular assays specifically in the plasmonic hotspot is used for detecting Zika-virus nucleic acids. The fluorescence enhancement is further exploited to achieve ultra-sensitive detection on low-cost devices such as a modified smartphone leading to the democratization of single-molecule detection.

We explore the potential of DNA nanotechnology for developing novel optical voltage sensing nanodevices that convert a local change of electric potential into optical signals. As a proof-of-concept of the sensing mechanism, we assembled voltage responsive DNA origami structures labeled with a single pair of FRET dyes. The DNA structures were reversibly immobilized on a nanocapillary tip and underwent controlled structural changes upon application of an electric field. The applied field was monitored through a change in FRET efficiency. By exchanging the position of a single dye, we could tune the voltage sensitivity of our DNA origami structure, demonstrating the flexibility and versatility of our approach. The experimental studies were complemented by coarse-grained simulations that characterized voltage-dependent elastic deformation of the DNA nanostructures and the associated change in the distance between the FRET pair. Our work opens a novel pathway for determining the mechanical properties of DNA origami structures and highlights potential applications of dynamic DNA nanostructures as voltage sensors.

Because of the limited signal-to-background ratio, molecular diagnostics requires molecular amplification of the target molecules or molecular signal amplification after target recognition. For direct molecular detection, we demonstrate a purely physical fluorescence enhancement process which can elevate the fluorescence signal of single fluorescent dyes by several orders of magnitude. To this end, DNA origami-based optical antennas with a height of around 125 nm are used, which utilize metallic nanoparticles to create a hotspot where fluorescence signals are enhanced by plasmonic effects. By equipping the hotspot with a molecular beacon-like structure, we combine the plasmonic signal enhancement with a specific signal generation, leading to an enhanced and therefore easy to detect signal only in the presence of the specific target nucleic acid. Exemplified with Zika virus detection, we show the applicability of this approach by detecting Zika-specific artificial DNA and RNA both in buffer and in heat-inactivated human blood serum. We show the sensitivity against small nucleotide variations of this approach, allowing the discrimination of closely related pathogens. Furthermore, we show how the modularity offered by DNA nanotechnology enables multiplexing by incorporating orthogonal fluorescent labels for the simultaneous detection of different sequences. The achieved signal enhancement will allow technically simplified signal detection, paving the way for single molecule-based point-of-care diagnosis.

The integrity of the nuclear envelope barrier relies on membrane remodeling by the ESCRTs, which seal nuclear envelope holes and contribute to the quality control of nuclear pore complexes (NPCs); whether these processes are mechanistically related remains poorly defined. Here, we show that the ESCRT-II/III chimera, Chm7, is recruited to a nuclear envelope subdomain that expands upon inhibition of NPC assembly and is required for the formation of the storage of improperly assembled NPCs (SINC) compartment. Recruitment to sites of NPC assembly is mediated by its ESCRT-II domain and the LAP2-emerin-MAN1 (LEM) family of integral inner nuclear membrane proteins, Heh1 and Heh2. We establish direct binding between Heh2 and the "open" forms of both Chm7 and the ESCRT-III, Snf7, and between Chm7 and Snf7. Interestingly, Chm7 is required for the viability of yeast strains where double membrane seals have been observed over defective NPCs; deletion of CHM7 in these strains leads to a loss of nuclear compartmentalization suggesting that the sealing of defective NPCs and nuclear envelope ruptures could proceed through similar mechanisms.

Mechanisms that ensure the integrity of the nuclear envelope rely on membrane remodeling proteins like the ESCRTs and the AAA ATPase Vps4, which help seal the nuclear envelope at the end of mitosis and prevent the formation of defective nuclear pore complexes (NPCs). Here, we show that the integral inner nuclear membrane proteins Heh1 and Heh2 directly bind the ESCRT-III, Snf7, and the ESCRT-II/III chimera, Chm7, in their ‘open’ forms. Moreover, Heh1 is required for Chm7-recruitment to the nuclear envelope. As Chm7 accumulates on the nuclear envelope upon blocks to NPC assembly, but not to nuclear transport, interactions between ESCRTs and the Heh proteins might form a biochemically distinct nuclear envelope subdomain that delimits regions of assembling NPCs. Interestingly, deletion of CHM7 suppresses the formation of the storage of improperly assembled NPC compartment prevalent in vps4Δ strains. Thus, our data support that the Heh1-dependent recruitment of Chm7 is a key component of a quality control pathway whose local regulation by Vps4 and the transmembrane nup, Pom152, prevents loss of nuclear compartmentalization by defective NPCs.