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Quantitative interpretation of single-molecule FRET experiments requires a model of the dye dynamics to link experimental energy transfer efficiencies to distances between atom positions. We have developed FRETraj, a Python module to predict FRET distributions based on accessible-contact volumes (ACV) and simulated photon statistics. FRETraj helps to identify optimal fluorophore positions on a biomolecule of interest by rapidly evaluating donor-acceptor distances. FRETraj is scalable and fully integrated into PyMOL and the Jupyter ecosystem. Here, we describe the conformational dynamics of a DNA hairpin by computing multiple ACVs along a molecular dynamics trajectory and compare the predicted FRET distribution with single-molecule experiments. FRET-assisted modeling will accelerate the analysis of structural ensembles in particular dynamic, non-coding RNAs and transient protein-nucleic acid complexes.

The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. By single-molecule FRET, we explore the binding kinetics of the most important, structurally conserved contact, the exon and intron binding site 1 (EBS1/IBS1). A comparison of RNA-RNA and RNA-DNA hybrid contacts identifies transient metal ion binding as a major source of kinetic heterogeneity which typically appears in the form of degenerate FRET states. Molecular dynamics simulations suggest a structural link between heterogeneity and the sugar conformation at the exon-intron binding interface. While Mg2+ ions lock the exon in place and give rise to long dwell times in the exon bound FRET state, sugar puckering alleviates this structural rigidity and likely promotes exon release. The interplay of sugar puckering and metal ion coordination may be an important mechanism to balance binding affinities of RNA and DNA interactions in general.

Imaging fluorescently labeled biomolecules on a single-molecule level is a well-established technique to follow intra- and intermolecular processes in time, usually hidden in the ensemble average. The classical approach comprises surface immobilization of the molecule of interest, which increases the risk of restricting the natural behavior due to surface interactions. Encapsulation of such biomolecules into surface-tethered phospholipid vesicles enables to follow one molecule at a time, freely diffusing and without disturbing surface interactions. Further, the encapsulation allows to keep reaction partners (reactants and products) in close proximity and enables higher temperatures otherwise leading to desorption of the direct immobilized biomolecules. Here, we describe a detailed protocol for the encapsulation of a catalytically active RNA starting from surface passivation over RNA encapsulation to data evaluation of single-molecule FRET experiments in TIRF microscopy. We present an optimized procedure that preserves RNA functionality and applies to investigations of, e.g., large ribozymes and RNAs, where direct immobilization is structurally not possible.

Labeling of large RNAs with reporting entities, e.g., fluorophores, has significant impact on RNA studies in vitro and in vivo. Here, we describe a minimally invasive RNA labeling method featuring nucleotide and position selectivity, which solves the long-standing challenge of how to achieve accurate site-specific labeling of large RNAs with a least possible influence on folding and/or function. We use a custom-designed reactive DNA strand to hybridize to the RNA and transfer the alkyne group onto the targeted adenine or cytosine. Simultaneously, the 3′-terminus of RNA is converted to a dialdehyde moiety under the experimental condition applied. The incorporated functionalities at the internal and the 3′-terminal sites can then be conjugated with reporting entities via bioorthogonal chemistry. This method is particularly valuable for, but not limited to, single-molecule fluorescence applications. We demonstrate the method on an RNA construct of 275 nucleotides, the btuB riboswitch of Escherichia coli.

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-HgII-T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium. The major species contains two (N3)T-HgII-(N3)C base pairs in a predominantly B-form helix. The minor species contains (N3)T-HgII-(N4)C base pairs and greater A-form characteristics. Ten-fold different 1J coupling constants (15N,199Hg) are observed for (N3)C-HgII (114 Hz) versus (N4)C-HgII (1052 Hz) connectivities, reflecting differences in cytosine ionization and metal-bonding strengths. Dynamic interconversion between the two types of C-HgII-T base pairs are coupled to a global conformational exchange between the helices. These observations inspired the design of a repetitive DNA sequence capable of undergoing a global B-to-A-form helical transition upon adding HgII, demonstrating that C-HgII-T has unique switching potential in DNA-based materials and devices.

Exploring the spatiotemporal dynamics of biomolecules on a single-molecule level requires innovative ways to make them spectroscopically visible. Fluorescence resonance energy transfer (FRET) uses a pair of organic dyes as reporters to measure distances along a predefined biomolecular reaction coordinate. For this nanoscopic ruler to work, the fluorescent labels need to be coupled onto the molecule of interest in a bioorthogonal and site-selective manner. Tagging large non-coding RNAs with single-nucleotide precision is an open challenge. Here we summarize current strategies in labeling riboswitches and ribozymes for fluorescence spectroscopy and FRET in particular. A special focus lies on our recently developed, DNA-guided approach that inserts two fluorophores through a stepwise process of templated functionality transfer and click chemistry.

Life depends on metals. While carbon, in terms of abundance and versatility, is considered THE element of life, the vast variety and diversity of the chemistry taking place in living organisms could not be achieved without metal ions. More than twenty metals are found in the human body, most of them being essential, some beneficial, and for others it is still unknown what role they might fulfil in a living cell. Here we give a short introduction into the bioinorganic world of the periodic table, providing just a few examples of key metals for life and aiming to give a flavour to gain further insights into this exciting field of inorganic chemistry at the intersection to the life sciences.

Tc-DNA is a conformationally constrained oligonucleotide analogue which shows significant increase in thermal stability when hybridized with RNA, DNA or tc-DNA. Remarkably, recent studies revealed that tc-DNA antisense oligonucleotides (AO) hold great promise for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy. To date, no high-resolution structural data is available for fully modified tc-DNA duplexes and little is known about the origins of their enhanced thermal stability. Here, we report the structures of a fully modified tc-DNA oligonucleotide paired with either complementary RNA, DNA or tc-DNA. All three investigated duplexes maintain a right-handed helical structure with Watson-Crick base pairing and overall geometry intermediate between A- and B-type, but closer to A-type structures. All sugars of the tc-DNA and RNA residues adopt a North conformation whereas the DNA deoxyribose are found in a South-East-North conformation equilibrium. The conformation of the tc-DNA strand in the three determined structures is nearly identical and despite the different nature and local geometry of the complementary strand, the overall structures of the examined duplexes are very similar suggesting that the tc-DNA strand dominates the duplex structure.

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.

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.