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Super-resolution imaging allows for visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA Point Accumulation In Nanoscale Topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), that allows for faster binding. Ago pre-orders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude while maintaining the high spatial resolution. We envision this tool to be useful not only for super-resolution imaging, but also for other techniques that rely on nucleic-acid interactions.

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.

We developed an efficient, versatile, and accessible super-resolution microscopy method to construct a nanoparticle assembly at a spatial resolution below the optical diffraction limit. The method utilizes DNA and a photoactivated DNA cross-linker. Super-resolution optical techniques have been used only as a means to make measurements below the light diffraction limit. Furthermore, no optical technique is currently available to construct nanoparticle assemblies with a precisely designed shape and internal structure at a resolution of a few tens of nanometers (nm). Here we demonstrate that we can fulfill this deficiency by utilizing spontaneous structural dynamics of DNA hairpins combined with single-molecule fluorescence resonance energy transfer (smFRET) microscopy and a photoactivated DNA cross-linker. The stochastic fluorescence blinking due to the spontaneous folding and unfolding motions of DNA hairpins enables us to precisely localize a folded hairpin and solidify it only when it is within a predesigned target area whose size is below the diffraction limit. As the method is based on an optical microscope and an easily clickable DNA cross-linking reagent, it will provide an efficient means to create large nanoparticle assemblies with a shape and internal structure at an optical super-resolution, opening a wide window of opportunities toward investigating their photophysical and optoelectronic properties and developing novel devices.

DNA barcoding provides a way to label a myriad of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is presumably the most easy-to-access method for DNA barcoding, yet large spectral overlaps between fluorescence dyes severely limit the numbers of barcodes that can be detected simultaneously. We here demonstrate the use of single-molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two-color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak DNA hybridization events, we markedly enhanced accuracy in our determination of the single-molecule FRET efficiency exhibited by each binding event between DNA barcode sequences. This allowed us to unambiguously differentiate six DNA barcodes encoding different FRET values without involving any probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of DNA barcoding.

DNA-PAINT is an optical super-resolution microscopy method that can visualize nanoscale protein arrangements and provide spectrally unlimited multiplexing capabilities. However, current multiplexing implementations based on, for example, DNA exchange (such as Exchange-PAINT) achieves multitarget detection by sequential imaging, limiting throughput. Here, we combine DNA-PAINT with single-molecule FRET and use the FRET efficiency as parameter for multiplexed imaging with high specificity. We demonstrate correlated single-molecule FRET and super-resolution on DNA origami structures, which are equipped with binding sequences that are targeted by pairs of dye-labeled oligonucleotides generating the FRET signal. We futher extract FRET values from single binding sites that are spaced just ∼55 nm apart, demonstrating super-resolution FRET imaging. This combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging with low background and opens the door for accurate distance readout in the 1-10 nm range.

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.

Single-molecule Förster resonance energy transfer (smFRET) has become a powerful nanoscopic tool in studies of biomolecular structures and nanoscale objects; however, conventional smFRET measurements are generally blind to distances above 10 nm thus impeding the study of long-distance phenomena. Here, we report the development of farFRET, a technique that extends the range in smFRET measurements beyond the 10 nm line by enhanced energy transfer using multiple acceptors. We demonstrate that farFRET can be readily employed to quantify FRET efficiencies and conformational dynamics using double-stranded DNA molecules, RecA-filament formation on single-stranded DNA and Holliday junction dynamics. farFRET allows quantitative measurements of large biomolecular complexes and nanostructures thus bridging the remaining gap to superresolution microscopy.

Förster resonance energy transfer (FRET) is widely applied in chemistry, biology, and nanosciences to assess distances on sub-10 nm scale. Extending the range and applicability of FRET requires enhancement of the fluorescence energy transfer at a spatial scale comparable to the donor-acceptor distances. Plasmonic nanoantennas are ideal to concentrate optical fields at a nanoscale fully matching the FRET distance range. Here, we present a resonant aluminum nanogap antenna tailored to enhance single molecule FRET. A 20 nm gap confines light into a nanoscale volume, providing a field gradient on the scale of the donor-acceptor distance, a large 10-fold increase in the local density of optical states, and strong intensity enhancement. With our dedicated design, we obtain 20-fold enhancement on the fluorescence emission of donor and acceptor dyes, and most importantly up to 5-fold enhancement of the FRET rate for donor-acceptor separations of 10 nm. We also provide a thorough framework of the fluorescence photophysics occurring in the nanoscale gap volume. The presented enhancement of energy transfer flow at the nanoscale opens a yet unexplored facet of the various advantages of optical nanoantennas and provides a new strategy toward biological applications of single molecule FRET at micromolar concentrations.

The ability of zero-mode waveguides (ZMWs) to guide light energy into subwavelength-diameter cylindrical nanoapertures has been exploited for single-molecule fluorescence studies of biomolecules at micromolar concentrations, the typical dissociation constants for biomolecular interactions. Although epi-fluorescence microscopy is now adopted for ZMW-based imaging as an alternative to the commercialized ZMW imaging platform, its suitability and performance awaits rigorous examination. Here, we present conical lens-based dark-field fluorescence microscopy in combination with a ZMW/microfluidic chip for single-molecule fluorescence imaging. We demonstrate that compared to epi-illumination, the dark-field configuration displayed diminished background and noise and enhanced signal-to-noise ratios. This signal-to-noise ratio for imaging using the dark-field setup remains essentially unperturbed by the presence of background fluorescent molecules at micromolar concentration. Our design allowed single-molecule FRET studies that revealed weak DNA–protein and protein–protein interactions found with T4 replisomal proteins.

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 or...