Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

Dynamics of G-Quadruplex Formation under Molecular Crowding.

G-quadruplex (G4) structures assemble from guanine-rich DNA sequences and are believed to regulate several key cellular processes. G4 formation and conformational interconversions are well-established to occur dynamically in vitro. However, a clear understanding of G4 formation dynamics in cells as well as under conditions mimicking the cellular environment is missing. To fill this gap, we have investigated the G4 dynamics in molecularly crowded solutions, thus replicating the effect of the excluded volume present in cells. The results show that the volume exclusion exerted by large crowding agents accelerates the rate of G4 formation by at least an order of magnitude, leading to significant G4 stabilization. Extrapolation from our experimental data predicts crowding-induced G4 stabilization by more than 3 kcal/mol, under crowding levels found in the cellular environment. Such effects are likely to be important for G4-driven regulatory functions.

Inhibited complete folding of consecutive human telomeric G-quadruplexes.

Noncanonical DNA structures, termed G-quadruplexes, are present in human genomic DNA and are important elements in many DNA metabolic processes. Multiple sites in the human genome have G-rich DNA stretches able to support formation of several consecutive G-quadruplexes. One of those sites is the telomeric overhang region that has multiple repeats of TTAGGG and is tightly associated with both cancer and aging. We investigated the folding of consecutive G-quadruplexes in both potassium- and sodium-containing solutions using single-molecule FRET spectroscopy, circular dichroism, thermal melting and molecular dynamics simulations. Our observations show coexistence of partially and fully folded DNA, the latter consisting of consecutive G-quadruplexes. Following the folding process over hours in sodium-containing buffers revealed fast G-quadruplex folding but slow establishment of thermodynamic equilibrium. We find that full consecutive G-quadruplex formation is inhibited by the many DNA structures randomly nucleating on the DNA, some of which are off-path conformations that need to unfold to allow full folding. Our study allows describing consecutive G-quadruplex formation in both nonequilibrium and equilibrium conditions by a unified picture, where, due to the many possible DNA conformations, full folding with consecutive G-quadruplexes as beads on a string is not necessarily achieved.

Ion-mediated control of structural integrity and reconfigurability of DNA nanostructures.

Nucleic acid-based biomolecular self-assembly enables the creation of versatile functional architectures. Electrostatic screening of the negative charges of nucleic acids is essential for their folding and stability; thus, ions play a critical role in nucleic acid self-assembly in both biology and nanotechnology. However, the ion-DNA interplay and the resulting ion-specific structural integrity and responsiveness of DNA constructs are underexploited. Here, we harness a wide range of mono- and divalent ions to control the structural features of DNA origami constructs. Using atomic force microscopy and Förster resonance energy transfer (FRET) spectroscopy down to the single-molecule level, we report on the global and local structural performance and responsiveness of DNA origami constructs following self-assembly, upon post-assembly ion exchange and post-assembly ion-mediated reconfiguration. We determined the conditions for highly efficient DNA origami folding in the presence of several mono- (Li+, Na+, K+, Cs+) and divalent (Ca2+, Sr2+, Ba2+) ions, expanding the range where DNA origami structures can be exploited for custom-specific applications. We then manipulated fully folded constructs by exposing them to unfavorable ionic conditions that led to the emergence of substantial disintegrity but not to unfolding. Moreover, we found that poorly assembled nanostructures at low ion concentrations undergo substantial self-repair upon ion addition in the absence of free staple strands. This reconfigurability occurs in an ion type- and concentration-specific manner. Our findings provide a fundamental understanding of the ion-mediated structural responsiveness of DNA origami at the nanoscale enabling applications under a wide range of ionic conditions.

A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

Tuning of bandgaps and emission properties of light-emitting diode materials through homogeneous alloying in molecular crystals.

Alloy formation is ubiquitous in inorganic materials science, and it strongly depends on the similarity between the alloyed atoms. Since molecules have widely different shapes, sizes and bonding properties, it is highly challenging to make alloyed molecular crystals. Here we report the generation of homogenous molecular alloys of organic light emitting diode materials that leads to tuning in their bandgaps and fluorescence emission. Tris(8-hydroxyquinolinato)aluminium (Alq3) and its Ga, In and Cr analogues (Gaq3, Inq3, and Crq3) form homogeneous mixed crystal phases thereby resulting in binary, ternary and even quaternary molecular alloys. The M x M'(1-x)q3 alloy crystals are investigated using X-ray diffraction, energy dispersive X-ray spectroscopy and Raman spectroscopy on single crystal samples, and photoluminescence properties are measured on the exact same single crystal specimens. The different series of alloys exhibit distinct trends in their optical bandgaps compared with their parent crystals. In the Al x Ga(1-x)q3 alloys the emission wavelengths lie in between those of the parent crystals, while the Al x In(1-x)q3 and Ga x In(1-x)q3 alloys have red shifts. Intriguingly, efficient fluorescence quenching is observed for the M x Cr(1-x)q3 alloys (M = Al, Ga) revealing the effect of paramagnetic molecular doping, and corroborating the molecular scale phase homogeneity.

A Single Molecule Polyphenylene-Vinylene Photonic Wire.

Nanoscale transport of light through single molecule systems is of fundamental importance for light harvesting, nanophotonic circuits, and for understanding photosynthesis. Studies on organization of molecular entities for directional transfer of excitation energy have focused on energy transfer cascades via multiple small molecule dyes. Here, we investigate a single molecule conjugated polymer as a photonic wire. The phenylene-vinylene-based polymer is functionalized with multiple DNA strands and immobilized on DNA origami by hybridization to a track of single-stranded staples extending from the origami structure. Donor and acceptor fluorophores are placed at specific positions along the polymer which enables energy transfer from donor to polymer, through the polymer, and from polymer to acceptor. The structure is characterized by atomic force microscopy, and the energy transfer is studied by ensemble fluorescence spectroscopy and single molecule TIRF microscopy. It is found that the polymer photonic wire is capable of transferring light over distances of 24 nm. This demonstrates the potential residing in the use of conjugated polymers for nanophotonics.

Bandgap Tuning in Molecular Alloy Crystals Formed by Weak Chalcogen Interactions.

We demonstrate systematic tuning in the optical bandgaps of molecular crystals achieved by the generation of molecular alloys/solid solutions of a series of diphenyl dichalcogenides-characterized by weak chalcogen bonding interactions involving S, Se, and Te atoms. Despite the variety in chalcogen bonding interactions found in this series of dichalcogenide crystals, they show isostructural interaction topologies, enabling the formation of solid solutions. The alloy crystals exhibit Vegard's law-like trends of variation in their unit cell dimensions and a nonlinear trend for the variation in optical bandgaps with respect to their compositions. Energy-dispersive X-ray and spatially resolved Raman spectroscopic studies indicate significant homogeneity in the domain structure of the solid solutions. Quantum periodic calculations of the projected density of states provide insights into the bandgap tuning in terms of the mixing of states in the alloy crystal phases.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Ligand Binding to Dynamically Populated G-Quadruplex DNA.

Several small-molecule ligands specifically bind and stabilize G-quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand-induced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.

Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay.

Two types of clinically important nucleic acid biomarkers, microRNA (miRNA) and circulating tumor DNA (ctDNA) were detected and quantified from human serum using an amplification-free fluorescence hybridization assay. Specifically, miRNAs hsa-miR-223-3p and hsa-miR-486-5p with relevance for rheumatoid arthritis and cancer related mutations BRAF and KRAS of ctDNA were directly measured. The required oligonucleotide probes for the assay were rationally designed and synthesized through a novel "clickable" approach which is time and cost-effective. With no need for isolating nucleic acid components from serum, the fluoresence-based assay took only 1 hour. Detection and absolute quantification of targets was successfully achieved despite their notoriously low abundance, with a precision down to individual nucleotides. Obtained miRNA and ctDNA amounts showed overall a good correlation with current techniques. With appropriate probes, our novel assay and signal boosting approach could become a useful tool for point-of-care measuring other low abundance nucleic acid biomarkers.

Single molecule analysis of structural fluctuations in DNA nanostructures.

DNA origami is an excellent tool for building complex artificial nanostructures. Functionalization of these structures provides the possibility of precise organization of matter at the nanoscale. In practice, efforts in this endeavour can be impeded by electrostatic repulsion or other dynamics at the molecular scale, resulting in uncompliant local structures. Using single molecule FRET microscopy combined with coarse-grained Brownian dynamics simulations, we investigated here the local structure around the lid of a DNA origami box, which can be opened by specific DNA keys. We found that FRET signals for the closed box depend on buffer ion concentrations and small changes to the DNA structure design. Simulations provided a view of the global and local structure and showed that the distance between the box wall and lid undergoes fluctuations. These results provide methods to vizualise and improve the local structure of three-dimensional DNA origami assemblies and offer guidance for exercising control over placement of chemical groups and ligands.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Multifluorophore DNA Origami Beacon as a Biosensing Platform.

Biosensors play increasingly important roles in many fields, from clinical diagnosis to environmental monitoring, and there is a growing need for cheap and simple analytical devices. DNA nanotechnology provides methods for the creation of sophisticated biosensors, however many of the developed DNA-based sensors are limited by cumbersome and time-consuming readouts involving advanced experimental techniques. Here we describe design, construction, and characterization of an optical DNA origami nanobiosensor device exploiting arrays of precisely positioned organic fluorophores. Two arrays of donor and acceptor fluorophores make up a multifluorophore Förster resonance energy-transfer pair that results in a high-output signal for microscopic detection of single devices. Arrangement of fluorophores into arrays increases the signal-to-noise ratio, allowing detection of signal output from singular biosensors using a conventional fluorescence microscopy setup. Single device analysis enables detection of target DNA sequences in concentrations down to 100 pM in <45 min. We expect that the presented nanobiosensor can function as a general platform for incorporating sensor modules for a variety of targets and that the strong signal amplification properties may allow detection in portable microscope systems to be used for biosensor applications in the field.

Controlled aggregation of DNA functionalized poly(phenylene-vinylene).

The morphology of conjugated polymers strongly influences their optical and electronic properties and affects their performance in polymer devices. Using optical spectroscopy and atomic force microscopy, we investigate the fluorescence properties and the aggregation state of DNA-functionalized poly(phenylene-vinylene). We show that polymer aggregation can be controlled in solution through ion and DNA interactions; aggregation is induced in the presence of divalent cations and can be reversed by adding sequence specific DNA. These interactions provide ways to tune polymer aggregation on the timescale of minutes and allows tuning of the polymer's optical properties.

Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions.

The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes.

G-quadruplexes (G4s) are DNA secondary structures that are capable of forming and function in vivo The propensity of G4s to exhibit extreme polymorphism and complex dynamics is likely to influence their cellular function, yet a clear microscopic picture of their folding process is lacking. Here we employed single-molecule FRET microscopy to obtain a direct view of the folding and underlying conformational dynamics of G4s formed by the human telomeric sequence in potassium containing solutions. Our experiments allowed detecting several folded states that are populated in the course of G4 folding and determining their folding energetics and timescales. Combining the single-molecule data with molecular dynamics simulations enabled obtaining a structural description of the experimentally observed folded states. Our work thus provides a comprehensive thermodynamic and kinetic description of the folding of G4s that proceeds through a complex multi-route pathway, involving several marginally stable conformational states.

Optimal Background Estimators in Single-Molecule FRET Microscopy.

Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Förster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique.

Engineering a Prototypic P-type ATPase Listeria monocytogenes Ca(2+)-ATPase 1 for Single-Molecule FRET Studies.

Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrolysis in the cytoplasmic domains to ion transport across the membrane. The Ca(2+)-ATPase from Listeria monocytogenes, LMCA1, was found to be a suitable model of P-type ATPases and was engineered to facilitate single-molecule FRET studies of transport-related structural changes. Mutational analyses of the endogenous cysteine residues in LMCA1 were performed to reduce background labeling without compromising activity. Pairs of cysteines were introduced into the optimized low-reactivity background, and labeled with maleimide derivatives of Cy3 and Cy5 resulting in site-specifically double-labeled protein with moderate activity. Ensemble and confocal single-molecule FRET studies revealed changes in FRET distribution related to structural changes during the transport cycle, consistent with those observed by X-ray crystallography for the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). Notably, the cytosolic headpiece of LMCA1 was found to be distinctly more compact in the E1 state than in the E2 state. Thus, the established experimental system should allow future real-time FRET studies of the structural dynamics of LMCA1 as a representative P-type ATPase.