Birka Lalkens

Fluorescence Enhancement at Docking Sites of DNA-Directed Self-Assembled Nanoantennas

Building a Fluorescent Hotspot When two gold nanoparticles come close together, their overlapping plasmonic fields can create a region that acts as a nanoantenna that can enhance the fluorescent emission of a molecule. Acuna et al. (p. 506) used a surface-anchored DNA origami structure to assemble one or two gold nanoparticles next to a dye trapped within the structure. A > 100-fold enhancement in fluorescent emission was observed when the dye molecules were located in a 23-nm gap between two 100-nm gold nanoparticles. A DNA origami structure enhances the emission of a dye molecule by directing the proximate binding of gold nanoparticles. We introduce self-assembled nanoantennas to enhance the fluorescence intensity in a plasmonic hotspot of zeptoliter volume. The nanoantennas are prepared by attaching one or two gold nanoparticles (NPs) to DNA origami structures, which also incorporated docking sites for a single fluorescent dye next to one NP or in the gap between two NPs. We measured the dependence of the fluorescence enhancement on NP size and number and compare it to numerical simulations. A maximum of 117-fold fluorescence enhancement was obtained for a dye molecule positioned in the 23-nanometer gap between 100-nanometer gold NPs. Direct visualization of the binding and unbinding of short DNA strands, as well as the conformational dynamics of a DNA Holliday junction in the hotspot of the nanoantenna, show the compatibility with single-molecule assays.

DNA Origami Nanopillars as Standards for Three-Dimensional Superresolution Microscopy

Nanopillars are promising nanostructures composed of various materials that bring new functionalities for applications ranging from photovoltaics to analytics. We developed DNA nanopillars with a height of 220 nm and a diameter of ~14 nm using the DNA origami technique. Modifying the base of the nanopillars with biotins allowed selective, upright, and rigid immobilization on solid substrates. With the help of site-selective dye labels, we visualized the structure and determined the orientation of the nanopillars by three-dimensional fluorescence superresolution microscopy. Because of their rigidity and nanometer-precise addressability, DNA origami nanopillars qualify as scaffold for the assembly of plasmonic devices as well as for three-dimensional superresolution standards.

A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

Single-molecule positioning in zeromode waveguides by DNA origami nanoadapters.

Nanotechnology is challenged by the need to connect top-down produced nanostructures with the bottom-up world of chemistry. A nanobiotechnological prime example is the positioning of single polymerase molecules in small holes in metal films, so-called zeromode waveguides (ZMWs), which is required for single-molecule real-time DNA sequencing. In this work, we present nanoadapters made of DNA (DNA origami) that match the size of the holes so that exactly one nanoadapter fits in each hole. By site-selective functionalization of the DNA origami nanoadapters, we placed single dye molecules in the ZMWs, thus optimizing the hole usage and improving the photophysical properties of dyes compared to stochastically immobilized molecules.

Simple and aberration-free 4color-STED--multiplexing by transient binding.

Most fluorescence microscopy experiments today require a multicolor-capable setup, e.g. to study the interaction between different proteins. Multicolor capabilities are also well desirable for superresolution images. However, especially for STED (Stimulated Emission Depletion) microscopy, which requires two laser lines for a single color, multicolor imaging is technically challenging. Here we present a straightforward, easy-to-implement method to extend a single-color fluorescence (STED) microscope to a multichannel microscope without the need of modifying the optical setup. Therefore, we use a labeling technique based on complementary DNA sequences: a single-stranded short DNA sequence is attached to each structure to be imaged, different colors for labeling different features are represented by different sequences. Within the imaging process, the corresponding complementary sequence labeled with an organic fluorophore is added and transiently binds to the corresponding structure. After imaging, the labeled sequence is washed away and replaced by a second fluorescently labeled DNA strand complementary to the sequence bound to another feature. This way, multiplexing is achieved using only one arbitrary fluorophore, therefore aberrations are avoided.

DNA-templated nanoantennas for single-molecule detection at elevated concentrations

Abstract. The dynamic concentration range is one of the major limitations of single-molecule fluorescence techniques. We show how bottom-up nanoantennas enhance the fluorescence intensity in a reduced hotspot, ready for biological applications. We use self-assembled DNA origami structures as a breadboard where gold nanoparticle (NP) dimers are positioned with nanometer precision. A maximum of almost 100-fold intensity enhancement is obtained using 100-nm gold NPs within a gap of 23 nm between the particles. The results obtained are in good agreement with numerical simulations. Due to the intensity enhancement introduced by the nanoantenna, we are able to perform single-molecule measurements at concentrations as high as 500 nM, which represents an increment of 2 orders of magnitude compared to conventional measurements. The combination of metallic NPs with DNA origami structures with docking points for biological assays paves the way for the development of bottom-up inexpensive enhancement chambers for single-molecule measurements at high concentrations where processes like DNA sequencing occur.

Functionalizing large nanoparticles for small gaps in dimer nanoantennas

The process of functionalizing gold nanoparticles with DNA commonly competes with nanoparticle aggregation, especially for larger particles of more than 80 nm diameter. Longer DNA strands reduce the tendency for aggregation but commonly lead to larger gaps when applied in certain geometrical arrangements such as gap nanoantennas. Here, we demonstrate that reversing the polarization of one of the strands for hybridization (yielding a zipper-like geometry) is sterically possible with uncompromised yields. Using the single dye molecules fluorescence lifetime as an indicator of the proximity of the nanoparticle in combination with electrodynamic simulations, we determine the distance between the nanoparticle and the dye placed in a DNA origami pillar. Importantly, compared to the common shear geometry smaller distances between the connected structures are obtained which are independent of the length of the DNA connector. Using the zipper geometry, we then arranged nanoparticles of 100 and 150 nm diameter on DNA origami and formed gap nanoantennas. We find that the previously reported trend of increased fluorescence enhancement of ATTO647N with increasing particle size for 20–100 nm nanoparticles is stopped. Gap nanoantennas built with 150 nm nanoparticles exhibit smaller enhancement than those with 100 nm nanoparticles. These results are discussed with the aid of electrodynamic simulations.

Synergistic Combination of Unquenching and Plasmonic Fluorescence Enhancement in Fluorogenic Nucleic Acid Hybridization Probes

Fluorogenic nucleic acid hybridization probes are widely used for detecting and quantifying nucleic acids. The achieved sensitivity strongly depends on the contrast between a quenched closed form and an unquenched opened form with liberated fluorescence. So far, this contrast was improved by improving the quenching efficiency of the closed form. In this study, we modularly combine these probes with optical antennas used for plasmonic fluorescence enhancement and study the effect of the nanophotonic structure on the fluorescence of the quenched and the opened form. As quenched fluorescent dyes are usually enhanced more by fluorescence enhancement, a detrimental reduction of the contrast between closed and opened form was anticipated. In contrast, we could achieve a surprising increase of the contrast with full additivity of quenching of the dark form and fluorescence enhancement of the bright form. Using single-molecule experiments, we demonstrate that the additivity of the two mechanisms depends on the perfect quenching in the quenched form, and we delineate the rules for new nucleic acid probes for enhanced contrast and absolute brightness. Fluorogenic hybridization probes optimized not only for quenching but also for the brightness of the open form might find application in nucleic acid assays with PCR avoiding detection schemes.

Optical Nanoantenna for Single Molecule-Based Detection of Zika Virus Nucleic Acids without Molecular Multiplication

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.