Phil Holzmeister

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.

Distance Dependence of Single-Fluorophore Quenching by Gold Nanoparticles Studied on DNA Origami

We study the distance-dependent quenching of fluorescence due to a metallic nanoparticle in proximity of a fluorophore. In our single-molecule measurements, we achieve excellent control over structure and stoichiometry by using self-assembled DNA structures (DNA origami) as a breadboard where both the fluorophore and the 10 nm metallic nanoparticle are positioned with nanometer precision. The single-molecule spectroscopy method employed here reports on the co-localization of particle and dye, while fluorescence lifetime imaging is used to directly obtain the correlation of intensity and fluorescence lifetime for varying particle to dye distances. Our data can be well explained by exact calculations that include dipole-dipole orientation and distances. Fitting with a more practical model for nanosurface energy transfer yields 10.4 nm as the characteristic distance of 50% energy transfer. The use of DNA nanotechnology together with minimal sample usage by attaching the particles to the DNA origami directly on the microscope coverslip paves the way for more complex experiments exploiting dye-nanoparticle interactions.

Fluorescence and super-resolution standards based on DNA origami

To the Editor: In recent years, the ability to validate microscopy techniques as well as distinguish instrument-specific from sample-specific error sources has not kept up with the pace of progress in fluorescence, and especially super-resolution, imaging. Commonly, new methods are demonstrated by imaging cellular filaments whose labeling and preparation cannot be easily reproduced. A desired validation standard requires, on one hand, structural control to position distinct marks at defined distances. On the other hand, the validation standard has to provide stoichiometric control, which implies the ability to place a defined number of molecules of equal brightness per mark. We present molecular rulers based on self-assembled DNA origami structures as a general and highly versatile platform for fluorescence and super-resolution standards. Examples include fluorescence brightness standards as well as standards covering a distance range from 6 to 386 nm that are adapted to the needs of the specific microscopy technique, including stimulated emission depletion (STED), localization-based super-resolution and diffractionlimited microscopy. Scaffolded DNA origamis are produced by hybridizing ~200 staple oligonucleotides to a long single-stranded DNA scaffold to yield a predefined shape1. DNA origamis can be labeled at specific sites with fluorescent dyes by using dye-modified staple strands (Supplementary Methods). For DNA origami brightness standards, we imaged rectangular DNA origamis (Fig. 1a) with 1–36 ATTO647N molecules (minimal interdye distance is 6 nm) immobilized on coverslips by fluorescence lifetime imaging.

Molecular force spectroscopy with a DNA origami–based nanoscopic force clamp

Many tiny force sensors Several techniques can measure forces on biomolecules, but the need to connect the molecule to the macroscopic world often limits the rate at which data can be taken. Nickels et al. created large arrays of nanoscale force sensors by using DNA origami structures. Single-stranded DNA molecules of different lengths attached to the molecule of interest acted as entropic springs, with shorter strands exerting more force. The authors used their setup to study the bending of DNA induced by the TATA-binding protein. Science, this issue p. 305 A self-assembled molecular force clamp built from DNA enables highly parallelized force spectroscopy measurements. Forces in biological systems are typically investigated at the single-molecule level with atomic force microscopy or optical and magnetic tweezers, but these techniques suffer from limited data throughput and their requirement for a physical connection to the macroscopic world. We introduce a self-assembled nanoscopic force clamp built from DNA that operates autonomously and allows massive parallelization. Single-stranded DNA sections of an origami structure acted as entropic springs and exerted controlled tension in the low piconewton range on a molecular system, whose conformational transitions were monitored by single-molecule Förster resonance energy transfer. We used the conformer switching of a Holliday junction as a benchmark and studied the TATA-binding protein–induced bending of a DNA duplex under tension. The observed suppression of bending above 10 piconewtons provides further evidence of mechanosensitivity in gene regulation.

DNA origami as biocompatible surface to match single-molecule and ensemble experiments

Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements.

Single-molecule FRET supports the two-state model of Argonaute action

Argonaute can be found in all three domains of life and is the functional core of the eukaryotic RNA-silencing machinery. In order to shed light on the conformational changes that drive Argonaute action, we performed single-molecule FRET measurements employing a so far uncharacterized member of the Argonaute family, namely Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo). We show that MjAgo is a catalytically active Argonaute variant hydrolyzing exclusively DNA target strands out of a DNA/DNA hybrid. We studied the interplay between Argonaute and nucleic acids using fluorescent dyes covalently attached at different positions of the DNA guide as steric reporters. This allowed us to determine structurally confined parts of the protein scaffold and flexible regions of the DNA guide. Single-molecule FRET measurements demonstrate that the 3′end of the DNA guide is released from the PAZ domain upon target strand loading. This conformational change does not necessitate target strand cleavage but a fully complementary target strand. Thus, our data support the two state model for Argonaute action.

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.

Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways

During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP–DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions. Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in the absence and presence of TFB in real-time. We observed that the lifetime of the TBP–DNA interaction differs significantly between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear, stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes the fully bent TBP–promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP–DNA interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the way to the formation of the pre-initiation complex.

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.

Counting fluorescent dye molecules on DNA origami by means of photon statistics.

Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single-molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1-20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well-defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non-invasively within 150 ms. The method might become a quantifying add-on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT-type microscopy.