Corrin Ring Modifications Reveal the Chemical and Spatial Requirements for the B12-btuB Riboswitch Interaction.

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i.e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.

FRET-guided modeling of nucleic acids.

The functional diversity of RNAs is encoded in their innate conformational heterogeneity. The combination of single-molecule spectroscopy and computational modeling offers new attractive opportunities to map structural transitions within nucleic acid ensembles. Here, we describe a framework to harmonize single-molecule Förster resonance energy transfer (FRET) measurements with molecular dynamics simulations and de novo structure prediction. Using either all-atom or implicit fluorophore modeling, we recreate FRET experiments in silico, visualize the underlying structural dynamics and quantify the reaction coordinates. Using multiple accessible-contact volumes as a post hoc scoring method for fragment assembly in Rosetta, we demonstrate that FRET can be used to filter a de novo RNA structure prediction ensemble by refuting models that are not compatible with in vitro FRET measurement. We benchmark our FRET-assisted modeling approach on double-labeled DNA strands and validate it against an intrinsically dynamic manganese(II)-binding riboswitch. We show that a FRET coordinate describing the assembly of a four-way junction allows our pipeline to recapitulate the global fold of the riboswitch displayed by the crystal structure. We conclude that computational fluorescence spectroscopy facilitates the interpretability of dynamic structural ensembles and improves the mechanistic understanding of nucleic acid interactions.

From Enigma to Revelation: Unravelling Biological Functions of Ubiquitous Small Ribozymes.

RNA, widely recognized as an information-carrier molecule, is capable of catalyzing essential biological processes through ribozymes. Despite their ubiquity, specific functions in a biological context and phenotypes based on the ribozymes' activity are often unknown. Here, we present the discovery of a subgroup of minimal HDV-like ribozymes, which reside 3' to viral tRNAs and appear to cleave the 3'-trailers of viral premature tRNA transcripts. This proposed tRNA-processing function is unprecedented for any ribozymes, thus, we designate this subgroup as theta ribozymes. Most theta ribozymes were identified in Caudoviricetes bacteriophages, the main constituent (>90%) of the mammalian gut virome. Intriguingly, our findings further suggest the involvement of theta ribozymes in the transition of certain bacteriophages between distinct genetic codes, thus possibly contributing to the phage lysis trigger. Our discovery expands the limited repertoire of biological functions attributed to HDV-like ribozymes and provides insights into the fascinating world of RNA catalysis.

Identification of HDV-like theta ribozymes involved in tRNA-based recoding of gut bacteriophages.

Trillions of microorganisms, collectively known as the microbiome, inhabit our bodies with the gut microbiome being of particular interest in biomedical research. Bacteriophages, the dominant virome constituents, can utilize suppressor tRNAs to switch to alternative genetic codes (e.g., the UAG stop-codon is reassigned to glutamine) while infecting hosts with the standard bacterial code. However, what triggers this switch and how the bacteriophage manipulates its host is poorly understood. Here, we report the discovery of a subgroup of minimal hepatitis delta virus (HDV)-like ribozymes - theta ribozymes - potentially involved in the code switch leading to the expression of recoded lysis and structural phage genes. We demonstrate their HDV-like self-scission behavior in vitro and find them in an unreported context often located with their cleavage site adjacent to tRNAs, indicating a role in viral tRNA maturation and/or regulation. Every fifth associated tRNA is a suppressor tRNA, further strengthening our hypothesis. The vast abundance of tRNA-associated theta ribozymes - we provide 1753 unique examples - highlights the importance of small ribozymes as an alternative to large enzymes that usually process tRNA 3'-ends. Our discovery expands the short list of biological functions of small HDV-like ribozymes and introduces a previously unknown player likely involved in the code switch of certain recoded gut bacteriophages.

Group II Introns: Highly Structured yet Dynamic.

RNA splicing, the removal of introns and ligation of exons, is a crucial process during mRNA maturation. Group II introns are large ribozymes that self-catalyze their splicing, as well as their transposition. They are living fossils of spliceosomal introns and eukaryotic retroelements. The yeast mitochondrial Sc.ai5γ is the first identified and best-studied self-splicing group II intron. A combination of biochemical, biophysical, and computational tools enables studying its catalytic properties, structure, and dynamics, while also serving to develop new therapeutic and biotechnological tools. We survey the history of group II intron studies paralleling the trends in RNA methodology with Sc.ai5γ in the spotlight.

Organometallic Pillarplexes That Bind DNA 4-Way Holliday Junctions and Forks.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination, and repair) and are dynamic structures that adopt either open or closed conformations, the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core, an ideal structure to interact with open DNA junction cavities. Combining experimental studies and MD simulations, we show that an Au pillarplex can bind DNA 4-way (Holliday) junctions in their open form, a binding mode not accessed by synthetic agents before. Pillarplexes can bind 3-way junctions too, but their large size leads them to open up and expand that junction, disrupting the base pairing, which manifests in an increased hydrodynamic size and lower junction thermal stability. At high loading, they rearrange both 4-way and 3-way junctions into Y-shaped forks to increase the available junction-like binding sites. Isostructural Ag pillarplexes show similar DNA junction binding behavior but lower solution stability. This pillarplex binding contrasts with (but complements) that of metallo-supramolecular cylinders, which prefer 3-way junctions and can rearrange 4-way junctions into 3-way junction structures. The pillarplexes' ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures. In human cells, the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher-order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction binders into organometallic chemistry.

The structural features of the ligand-free moaA riboswitch and its ion-dependent folding.

Riboswitches are structural elements of mRNA involved in the regulation of gene expression by responding to specific cellular metabolites. To fulfil their regulatory function, riboswitches prefold into an active state, the so-called binding competent form, that guarantees metabolite binding and allows a consecutive refolding of the RNA. Here, we describe the folding pathway to the binding competent form as well as the ligand free structure of the moaA riboswitch of E. coli. This RNA proposedly responds to the molybdenum cofactor (Moco), a highly oxygen-sensitive metabolite, essential in the carbon and sulfur cycles of eukaryotes. K+- and Mg2+-dependent footprinting assays and spectroscopic investigations show a high degree of structure formation of this RNA already at very low ion-concentrations. Mg2+ facilitates additionally a general compaction of the riboswitch towards its proposed active structure. We show that this fold agrees with the earlier suggested secondary structure which included also a long-range tetraloop/tetraloop-receptor like interaction. Metal ion cleavage assays revealed specific Mg2+-binding pockets within the moaA riboswitch. These Mg2+ binding pockets are good indicators for the potential Moco binding site, since in riboswitches, Mg2+ was shown to be necessary to bind phosphate-carrying metabolites. The importance of the phosphate and of other functional groups of Moco is highlighted by binding assays with tetrahydrobiopterin, the reduced and oxygen-sensitive core moiety of Moco. We demonstrate that the general molecular shape of pterin by its own is insufficient for the recognition by the riboswitch.

A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories.

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We test them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

Magnesium(II)-ATP Complexes in 1-Ethyl-3-Methylimidazolium Acetate Solutions Characterized by 31 Mg ß-Radiation-Detected NMR Spectroscopy.

The complexation of MgII with adenosine 5'-triphosphate (ATP) is omnipresent in biochemical energy conversion, but is difficult to interrogate directly. Here we use the spin- 1/2 ß-emitter 31 Mg to study MgII -ATP complexation in 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) solutions using ß-radiation-detected nuclear magnetic resonance (ß-NMR). We demonstrate that (nuclear) spin-polarized 31 Mg, following ion-implantation from an accelerator beamline into EMIM-Ac, binds to ATP within its radioactive lifetime before depolarizing. The evolution of the spectra with solute concentration indicates that the implanted 31 Mg initially bind to the solvent acetate anions, whereafter they undergo dynamic exchange and form either a mono- (31 Mg-ATP) or di-nuclear (31 MgMg-ATP) complex. The chemical shift of 31 Mg-ATP is observed up-field of 31 MgMg-ATP, in accord with quantum chemical calculations. These observations constitute a crucial advance towards using ß-NMR to probe chemistry and biochemistry in solution.

Coordination Chemistry of Nucleotides and Antivirally Active Acyclic Nucleoside Phosphonates, including Mechanistic Considerations.

Considering that practically all reactions that involve nucleotides also involve metal ions, it is evident that the coordination chemistry of nucleotides and their derivatives is an essential corner stone of biological inorganic chemistry. Nucleotides are either directly or indirectly involved in all processes occurring in Nature. It is therefore no surprise that the constituents of nucleotides have been chemically altered-that is, at the nucleobase residue, the sugar moiety, and also at the phosphate group, often with the aim of discovering medically useful compounds. Among such derivatives are acyclic nucleoside phosphonates (ANPs), where the sugar moiety has been replaced by an aliphatic chain (often also containing an ether oxygen atom) and the phosphate group has been replaced by a phosphonate carrying a carbon-phosphorus bond to make the compounds less hydrolysis-sensitive. Several of these ANPs show antiviral activity, and some of them are nowadays used as drugs. The antiviral activity results from the incorporation of the ANPs into the growing nucleic acid chain-i.e., polymerases accept the ANPs as substrates, leading to chain termination because of the missing 3'-hydroxyl group. We have tried in this review to describe the coordination chemistry (mainly) of the adenine nucleotides AMP and ATP and whenever possible to compare it with that of the dianion of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA2- = adenine(N9)-CH2-CH2-O-CH2-PO32) [or its diphosphate (PMEApp4-)] as a representative of the ANPs. Why is PMEApp4- a better substrate for polymerases than ATP4-? There are three reasons: (i) PMEA2- with its anti-like conformation (like AMP2-) fits well into the active site of the enzyme. (ii) The phosphonate group has an enhanced metal ion affinity because of its increased basicity. (iii) The ether oxygen forms a 5-membered chelate with the neighboring phosphonate and favors thus coordination at the Pα group. Research on ANPs containing a purine residue revealed that the kind and position of the substituent at C2 or C6 has a significant influence on the biological activity. For example, the shift of the (C6)NH2 group in PMEA to the C2 position leads to 9-[2-(phosphonomethoxy)ethyl]-2-aminopurine (PME2AP), an isomer with only a moderate antiviral activity. Removal of (C6)NH2 favors N7 coordination, e.g., of Cu2+, whereas the ether O atom binding of Cu2+ in PMEA facilitates N3 coordination via adjacent 5- and 7-membered chelates, giving rise to a Cu(PMEA)cl/O/N3 isomer. If the metal ions (M2+) are M(α,ß)-M(γ)-coordinated at a triphosphate chain, transphosphorylation occurs (kinases, etc.), whereas metal ion binding in a M(α)-M(ß,γ)-type fashion is relevant for polymerases. It may be noted that with diphosphorylated PMEA, (PMEApp4-), the M(α)-M(ß,γ) binding is favored because of the formation of the 5-membered chelate involving the ether O atom (see above). The self-association tendency of purines leads to the formation of dimeric [M2(ATP)]2(OH)- stacks, which occur in low concentration and where one half of the molecule undergoes the dephosphorylation reaction and the other half stabilizes the structure-i.e., acts as the "enzyme" by bridging the two ATPs. In accord herewith, one may enhance the reaction rate by adding AMP2- to the [Cu2(ATP)]2(OH)- solution, as this leads to the formation of mixed stacked Cu3(ATP)(AMP)(OH)- species, in which AMP2- takes over the structuring role, while the other "half" of the molecule undergoes dephosphorylation. It may be added that Cu3(ATP)(PMEA) or better Cu3(ATP)(PMEA)(OH)- is even a more reactive species than Cu3(ATP)(AMP)(OH)-. - The matrix-assisted self-association and its significance for cell organelles with high ATP concentrations is summarized and discussed, as is, e.g., the effect of tryptophanate (Trp-), which leads to the formation of intramolecular stacks in M(ATP)(Trp)3- complexes (formation degree about 75%). Furthermore, it is well-known that in the active-site cavities of enzymes the dielectric constant, compared with bulk water, is reduced; therefore, we have summarized and discussed the effect of a change in solvent polarity on the stability and structure of binary and ternary complexes: Opposite effects on charged O sites and neutral N sites are observed, and this leads to interesting insights.

Single-Molecule Kinetic Studies of Nucleic Acids by Förster Resonance Energy Transfer.

Single-molecule microscopy is often used to observe and characterize the conformational dynamics of nucleic acids (NA). Due to the large variety of NA structures and the challenges specific to single-molecule observation techniques, the data recorded in such experiments must be processed via multiple statistical treatments to finally yield a reliable mechanistic view of the NA dynamics. In this chapter, we propose a comprehensive protocol to analyze single-molecule trajectories in the scope of single-molecule Förster resonance energy transfer (FRET) microscopy. The suggested protocol yields the conformational states common to all molecules in the investigated sample, together with the associated conformational transition kinetics. The given model resolves states that are indistinguishable by their observed FRET signals and is estimated with 95% confidence using error calculations on FRET states and transition rate constants. In the end, a step-by-step user guide is given to reproduce the protocol with the Multifunctional Analysis Software to Handle single-molecule FRET data (MASH-FRET).

Chemical Dual End-Labeling of Large Ribozymes.

Fast and efficient site-specific labeling of long RNAs is one of the main bottlenecks limiting distance measurements by means of Förster resonance energy transfer (FRET) or electron paramagnetic resonance (EPR) spectroscopy. Here, we present an optimized protocol for dual end-labeling with different fluorophores at the same time meeting the restrictions of highly labile and degradation-sensitive RNAs. We describe in detail the dual-labeling of a catalytically active wild-type group II intron as a typical representative of long functional RNAs. The modular procedure chemically activates the 5'-phosphate and the 3'-ribose for bioconjugation with a pair of fluorophores, as shown herein, or with spin labels. The mild reaction conditions preserve the structural and functional integrity of the biomacromolecule and results in covalent, dual-labeled RNA in its pre-catalytic state in yields suitable for both ensemble and single-molecule FRET experiments.

FRETraj: integrating single-molecule spectroscopy with molecular dynamics.

SUMMARY: 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. AVAILABILITY AND IMPLEMENTATION: FRETraj is implemented as a cross-platform Python package available under the GPL-3.0 on Github (https://github.com/RNA-FRETools/fretraj) and is documented at https://RNA-FRETools.github.io/fretraj. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Puf6 primes 60S pre-ribosome nuclear export at low temperature.

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.

Screening for potential interaction partners with surface plasmon resonance imaging coupled to MALDI mass spectrometry.

We coupled SPR imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to identify new potential RNA binders. Here, we improve this powerful method, especially by optimizing the proteolytic digestion (type of reducing agent, its concentration, and incubation time), to work with complex mixtures, specifically a lysate of the rough mitochondrial fraction from yeast. The advantages of this hyphenated method compared to column-based or separate analyses are (i) rapid and direct visual readout from the SPRi array, (ii) possibility of high-throughput analysis of different interactions in parallel, (iii) high sensitivity, and (iv) no sample loss or contamination due to elution or micro-recovery procedures. The model system used is a catalytically active RNA (group IIB intron from Saccharomyces cerevisiae, Sc.ai5γ) and its cofactor Mss116. The protein supports the RNA folding process and thereby the subsequent excision of the intronic RNA from the coding part. Using the novel approach of coupling SPR with MALDI MS, we report the identification of potential RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach. Our results show that proteins other than the well-known cofactor Mss116 interact with Sc.ai5γ (Dbp8, Prp8, Mrp13, and Cullin-3), suggesting that the intron folding and splicing are regulated by more than one cofactor in vivo.

Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy.

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.

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts.

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.

Site-Specific Dual-Color Labeling of Long RNAs.

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.

Concerted dynamics of metallo-base pairs in an A/B-form helical transition.

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.