Environmental, bystander and resident exposure from orchard applications using an agricultural unmanned aerial spraying system.

Unmanned aerial spraying systems (UASS), i.e., unmanned aerial vehicles designed for pesticide applications, are widely used in East Asia and increasingly prevalent in other regions of the world, including North America and Europe. However, according to a recent report of the Organization for Economic Co-operation and Development, spray drift and exposure caused by these systems are not yet fully understood. In particular, there are at present no peer-reviewed reports on direct exposure of residents and bystanders to spray drift following UASS applications. This lack of data results in regulatory concerns with respect to the environment and human safety. The objective of this study was to quantify environmental, resident and bystander exposure following the application of a plant protection product to an orchard using a commercial UASS under field conditions. Using a fluorescent tracer, horizontal and vertical downwind drift data were collected and direct exposure of residents and bystanders located downwind the sprayed area to spray drift was quantified using display mannequins equipped with personal air sampling pumps. Spray drift and exposure inversely correlated with sampling height and downwind distance. Furthermore, drift and exposure were strongly influenced by wind speed and direction, albeit hardly affected by the growth stage of the trees. In addition, substantially less tracer was extracted from the filters of the air sampling pumps than from the coveralls worn by mannequins, suggesting that direct resident/bystander exposure to spray drift may predominantly occur via the dermal route. This report provides essential data on UASS spray drift potential that are relevant for environmental and health risk assessments related to these systems. The results are compared to predicted values of current regulatory models and previously reported field data on drift and exposure caused by different spraying equipment.

Reliable State Identification and State Transition Detection in Fluorescence Intensity-Based Single-Molecule Förster Resonance Energy-Transfer Data.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique to probe biomolecular structure and dynamics. A popular implementation of smFRET consists of recording fluorescence intensity time traces of surface-immobilized, chromophore-tagged molecules. This approach generates large and complex data sets, the analysis of which is to date not standardized. Here, we address a key challenge in smFRET data analysis: the generation of thermodynamic and kinetic models that describe with statistical rigor the behavior of FRET trajectories recorded from surface-tethered biomolecules in terms of the number of FRET states, the corresponding mean FRET values, and the kinetic rates at which they interconvert. For this purpose, we first perform Monte Carlo simulations to generate smFRET trajectories, in which a relevant space of experimental parameters is explored. Then, we provide an account on current strategies to achieve such model selection, as well as a quantitative assessment of their performances. Specifically, we evaluate the performance of each algorithm (change-point analysis, STaSI, HaMMy, vbFRET, and ebFRET) with respect to accuracy, reproducibility, and computing time, which yields a range of algorithm-specific referential benchmarks for various data qualities. Data simulation and analysis were performed with our MATLAB-based multifunctional analysis software for handling smFRET data (MASH-FRET).

Simulations of camera-based single-molecule fluorescence experiments.

Single-molecule microscopy has become a widely used technique in (bio)physics and (bio)chemistry. A popular implementation is single-molecule Förster Resonance Energy Transfer (smFRET), for which total internal reflection fluorescence microscopy is frequently combined with camera-based detection of surface-immobilized molecules. Camera-based smFRET experiments generate large and complex datasets and several methods for video processing and analysis have been reported. As these algorithms often address similar aspects in video analysis, there is a growing need for standardized comparison. Here, we present a Matlab-based software (MASH-FRET) that allows for the simulation of camera-based smFRET videos, yielding standardized data sets suitable for benchmarking video processing algorithms. The software permits to vary parameters that are relevant in cameras-based smFRET, such as video quality, and the properties of the system under study. Experimental noise is modeled taking into account photon statistics and camera noise. Finally, we survey how video test sets should be designed to evaluate currently available data analysis strategies in camera-based sm fluorescence experiments. We complement our study by pre-optimizing and evaluating spot detection algorithms using our simulated video test sets.

Curvature of designed armadillo repeat proteins allows modular peptide binding.

Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n=27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.

Sequence-Specific Post-Synthetic Oligonucleotide Labeling for Single-Molecule Fluorescence Applications.

The sequence-specific fluorescence labeling of nucleic acids is a prerequisite for various methods including single-molecule Förster resonance energy transfer (smFRET) for the detailed study of nucleic acid folding and function. Such nucleic acid derivatives are commonly obtained by solid-phase methods; however, yields decrease rapidly with increasing length and restrict the practicability of this approach for long strands. Here, we report a new labeling strategy for the postsynthetic incorporation of a bioorthogonal group into single stranded regions of both DNA and RNA of unrestricted length. A 12-alkyne-etheno-adenine modification is sequence-selectively formed using DNA-templated synthesis, followed by conjugation of the fluorophore Cy3 via a copper-catalyzed azide-alkyne cycloaddition (CuAAC). Evaluation of the labeled strands in smFRET measurements shows that the strategy developed here has the potential to be used for the study of long functional nucleic acids by (single-molecule) fluorescence or other methods. To prove the universal use of the method, its application was successfully extended to the labeling of a short RNA single strand. As a proof-of-concept, also the labeling of a large RNA molecule in form of a 633 nucleotide long construct derived from the Saccharomyces cerevisiae group II intron Sc.ai5γ was performed, and covalent attachment of the Cy3 fluorophore was shown with gel electrophoresis.

Cation-induced kinetic heterogeneity of the intron-exon recognition in single group II introns.

RNA is commonly believed to undergo a number of sequential folding steps before reaching its functional fold, i.e., the global minimum in the free energy landscape. However, there is accumulating evidence that several functional conformations are often in coexistence, corresponding to multiple (local) minima in the folding landscape. Here we use the 5'-exon-intron recognition duplex of a self-splicing ribozyme as a model system to study the influence of Mg(2+) and Ca(2+) on RNA tertiary structure formation. Bulk and single-molecule spectroscopy reveal that near-physiological M(2+) concentrations strongly promote interstrand association. Moreover, the presence of M(2+) leads to pronounced kinetic heterogeneity, suggesting the coexistence of multiple docked and undocked RNA conformations. Heterogeneity is found to decrease at saturating M(2+) concentrations. Using NMR, we locate specific Mg(2+) binding pockets and quantify their affinity toward Mg(2+). Mg(2+) pulse experiments show that M(2+) exchange occurs on the timescale of seconds. This unprecedented combination of NMR and single-molecule Förster resonance energy transfer demonstrates for the first time to our knowledge that a rugged free energy landscape coincides with incomplete occupation of specific M(2+) binding sites at near-physiological M(2+) concentrations. Unconventional kinetics in nucleic acid folding frequently encountered in single-molecule experiments are therefore likely to originate from a spectrum of conformations that differ in the occupation of M(2+) binding sites.

Kinetic subpopulations detected by single-molecule spectroscopy: fundamental property of functional nucleic acids or experimental artefact?

Single-molecule spectroscopy allows the direct observation of conformational dynamics in individual biomolecules. Here, we describe how single-molecule Förster resonance energy transfer (smFRET) reveals heterogeneous kinetics in the EBS1*/IBS1* interaction, two RNA sequences that play an important role in group II intron mediated self-cleavage. Further examples of dynamic heterogeneity in functional nucleic acids are provided and the possible origins of this phenomenon are discussed.

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences.

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.

Helicase-mediated changes in RNA structure at the single-molecule level.

RNA helicases are a diverse group of RNA-dependent ATPases known to play a large number of biological roles inside the cell, such as RNA unwinding, remodeling, export and degradation. Understanding how helicases mediate changes in RNA structure is therefore of fundamental interest. The advent of single-molecule spectroscopic techniques has unveiled with unprecedented detail the interplay of RNA helicases with their substrates. In this review, we describe the characterization of helicase-RNA interactions by single-molecule approaches. State-of-the-art techniques are presented, followed by a discussion of recent advancements in this exciting field.

BOBA FRET: bootstrap-based analysis of single-molecule FRET data.

Time-binned single-molecule Förster resonance energy transfer (smFRET) experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR) in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations) from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc.) are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1/IBS1) is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET), as well as a graphical user interface, is available via http://www.aci.uzh.ch/rna/.

Seven essential questions on G-quadruplexes.

The helical duplex architecture of DNA was discovered by Francis Crick and James Watson in 1951 and is well known and understood. However, nucleic acids can also adopt alternative structural conformations that are less familiar, although no less biologically relevant, such as the G-quadruplex. G-quadruplexes continue to be the subject of a rapidly expanding area of research, owing to their significant potential as therapeutic targets and their unique biophysical properties. This review begins by focusing on G-quadruplex structure, elucidating the intermolecular and intramolecular interactions underlying its formation and highlighting several substructural variants. A variety of methods used to characterize these structures are also outlined. The current state of G-quadruplex research is then addressed by proffering seven pertinent questions for discussion. This review concludes with an overview of possible directions for future research trajectories in this exciting and relevant field.