Andreas Gietl

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.

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.

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.

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Significance DNA-dependent RNA polymerases (RNAPs) are complex enzymes that synthesize RNA in a factor-dependent fashion. Like mechanical engines, RNAPs consist of rigid and flexible parts; the catalytic function of RNAPs critically relies on conformational changes. Based on single-molecule FRET measurements that directly report on the movements of the RNAP clamp of the archaeal 12-subunit RNAP, we show that the clamp domain exists in alternative states distinguishable by the width of the DNA binding channel. The conformation of the clamp is adjusted through the transcription cycle; more precisely, it varies as a function of (i) RNA subunits Rpo4/7, (ii) the presence of the DNA nontemplate strand, and (iii) transcription initiation and elongation factors TFE and Spt4/5, respectively. Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle.

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.

Geminate Recombination as a Photoprotection Mechanism for Fluorescent Dyes

Despite common presumption due to fast photodestruction pathways through higher excited states, we show that further improvement of photostability is still achievable with diffusion-limited photoprotection formulas. Single-molecule fluorescence spectroscopy reveals that thiolate ions effectively quench triplet states of dyes by photoinduced electron transfer. Interestingly, this reaction rarely yields a radical anion of the dye, but direct return to the ground state is promoted by an almost instantaneous back electron transfer (geminate recombination). This type of mechanism is not detected for commonly used reductants such as ascorbic acid and trolox. The mechanism avoids the formation of radical cations and improves the photostability of single fluorophores. We find that a combination of β-mercaptoethanol and classical reducing and oxidizing systems yields the best results for several dyes including Atto532 and Alexa568.

Modern biophysical approaches probe transcription-factor-induced DNA bending and looping

The genetic information of every living organism is stored in its genomic DNA that is perceived as a chemically stable and robust macromolecule. But at the same time, to fulfil its functions properly, it also needs to be highly dynamic and flexible. This includes partial melting of the double helix or compaction and bending of the DNA often brought about by protein factors that are able to interact with DNA stretches in a specific and non-specific manner. The conformational changes in the DNA need to be understood in order to describe biological systems in detail. As these events play out on the nanometre scale, new biophysical approaches have been employed to monitor conformational changes in this regime at the single-molecule level. Focusing on transcription factor action on promoter DNA, we discuss how current biophysical techniques are able to quantitatively describe this molecular process.