Richard Börner

Efficient simultaneous fluorescence orientation, spectrum, and lifetime detection for single molecule dynamics.

We report on the simultaneous detection of the fluorescence lifetime, spectrum, and three-dimensional dipole orientation determination of single perylene diimide molecules deposited on a silica surface as a model system for studying fluorophore internal and orientational dynamics. We employ a multi-parameter detection scheme to demonstrate how jumps in the orientation of the molecule can be disentangled from spectral jumps, both leading to changes of the detected total fluorescence intensity. The fluorescence lifetime determined simultaneously from the same photons is also sensitive to the orientation of the dipole with respect to the interface between media with different refractive indices. The correlated changes of the lifetime and orientation we observe are in good agreement with theory.

An atomistic view on carbocyanine photophysics in the realm of RNA

Carbocyanine dyes have a long-standing tradition in fluorescence imaging and spectroscopy, due to their photostability and large spectral separation between individual dye species. Herein, we explore the versatility of cyanine dyes to probe the dynamics of nucleic acids and we report on the interrelation of fluorophores, RNA, and metal ions, namely K+ and Mg2+. Photophysical parameters including the fluorescence lifetime, quantum yield and dynamic anisotropy are monitored as a function of the nucleic acid composition, conformation, and metal ion abundance. Occasional excursions to a non-fluorescent cis-state hint at the remarkable sensitivity of carbocyanines to their local environment. Comparison of time-correlated single photon experiments with all-atom molecular dynamics simulations demonstrate that the propensity of photoisomerization is dictated by sterical constraints imposed on the fluorophore. Structural features in the vicinity of the dye play a crucial role in RNA recognition and have far-reaching implications on the mobility of the fluorescent probe. An atomic level description of the mutual interactions will ultimately benefit the quantitative interpretation of single-molecule FRET measurements on large RNA systems.

Distinct differences in metal ion specificity of RNA and DNA G-quadruplexes.

RNA G-quadruplexes, as their well-studied DNA analogs, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li+, Na+, K+, Rb+, Cs+, NH4+, Mg2+, Ca2+, Sr2+, and Ba2+. Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes, and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na+, K+, Rb+, NH4+, as well as Sr2+ and Ba2+ in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K+ and Sr2+ lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na+ ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As the method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, such that the use of fluorescent lipid dyes (DiO) allows to establish a a wide range of dipole orientations experimentally. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach.

Site-specific dual-color labeling of long RNAs for single-molecule spectroscopy

Abstract Labeling of long RNA molecules in a site-specific yet generally applicable manner is integral to many spectroscopic applications. Here we present a novel covalent labeling approach that is site-specific and scalable to long intricately folded RNAs. In this approach, a custom-designed DNA strand that hybridizes to the RNA guides a reactive group to target a preselected adenine residue. The functionalized nucleotide along with the concomitantly oxidized 3′-terminus can subsequently be conjugated to two different fluorophores via bio-orthogonal chemistry. We validate this modular labeling platform using a regulatory RNA of 275 nucleotides, the btuB riboswitch of Escherichia coli, demonstrate its general applicability by modifying a base within a duplex, and show its site-selectivity in targeting a pair of adjacent adenines. Native folding and function of the RNA is confirmed on the single-molecule level by using FRET as a sensor to visualize and characterize the conformational equilibrium of the riboswitch upon binding of its cofactor adenosylcobalamin. The presented labeling strategy overcomes size and site constraints that have hampered routine production of labeled RNA that are beyond 200 nt in length.

Detailed analysis of complex single molecule FRET data with the software MASH

The processing and analysis of surface-immobilized single molecule FRET (Förster resonance energy transfer) data follows systematic steps (e.g. single molecule localization, clearance of different sources of noise, selection of the conformational and kinetic model, etc.) that require a solid knowledge in optics, photophysics, signal processing and statistics. The present proceeding aims at standardizing and facilitating procedures for single molecule detection by guiding the reader through an optimization protocol for a particular experimental data set. Relevant features were determined from single molecule movies (SMM) imaging Cy3- and Cy5-labeled Sc.ai5γ group II intron molecules synthetically recreated, to test the performances of four different detection algorithms. Up to 120 different parameterizations per method were routinely evaluated to finally establish an optimum detection procedure. The present protocol is adaptable to any movie displaying surface-immobilized molecules, and can be easily reproduced with our home-written software MASH (multifunctional analysis software for heterogeneous data) and script routines (both available in the download section of www.chem.uzh.ch/rna).

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.

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).

A macromolecular crowding study of RNA folding and activity: polymer pore size matters!(Conference Presentation)

Catalytic RNAs, like the group IIB intron ribozyme of S. cerevesiae, require a high magnesium(II) concentration to show folding and function in vitro [1]. In contrast, in vivo conditions are characterized by a highly crowded cellular environment and much lower ion concentration. Molecular crowding agents are a widespread tool to mimic cellular crowding [2]. However, particular physical/chemical properties explaining the crowders influence are mostly not understood. In this study, we gain new insights on how polymer properties like viscosity, pore size etc. influence the activity and folding of a large RNA. We combined bulk activity assays and single-molecule Förster Resonance Energy Transfer experiments, screening the PEG volume fraction (%) and molecular weight (MW). Our results revealed that upon the influence of crowding agents, a compaction of the underlying structure depends on the PEG % and the presence of different PEG MW and % unveiled an optimal pore size in terms of catalytic activity. In summary, an increasing density of the crowding environment shifts the RNA towards the most compact state, but the ribozyme is only active if the crowders network matches its size [4]. We interpret the most compact state as necessary, but not sufficient, to keep the ribozyme active. Financial support from the European Research Council (MIRNA N° 259092, to RKOS), the Swiss National Fund (SNF), and the Forschungskredit Grant of the University of Zürich (FK-14-096 and 15-092 to RB) are gratefully acknowledged. [1] Swisher J.F., Su L.J., Brenowitz M., Anderson V.E., Pyle A.M., J. Mol. Bio., 315, 297-310 (2002). [2] Kilburn D., Roh J.H., Guo L., Briber R.M., Woodson S.A., JACS, 132, 8690-6 (2010). [3] Steiner M., Karunatilaka K.S., Sigel R.K.O., Rueda D., Proc. Natl. Acad. Sci. U.S.A.,105, 13853-8 (2008). [4] aBörner R, Fiorini E, Sigel R.K.O., Chimia, 69, 207-212 (2015).; bFiorini E., Paudel B., Börner R., Rueda D., Sigel R.K.O., submitted. [5] König S.L.B., Hadzic M., Fiorini E., Börner R., Kowerko D., Blanckenhorn W.U., Sigel R.K.O., PLoS ONE, 8, e84157 (2013).