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Accurate extraction of multiplet parameters, such as J-couplings and chemical shifts, play a vital role in small molecule analysis using nuclear magnetic resonance (NMR) spectroscopy. These parameters provide essential quantitative information about molecular structures, interatomic interactions, and chemical environments, enabling precise characterization of small organic compounds. This poster presents an innovative omputational approach that utilizes state-of-the-art deep learning techniques, specifically detection transformers, to automate and optimize the extraction of multiplet parameters from 1D NMR spectra of small molecules. By incorporating these advanced computational methods, experimenters can achieve improved efficiency, accuracy, and speed in analyzing and characterizing small organic compounds using NMR spectroscopy.

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.

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.

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.

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.

We introduce a deep learning-based deconvolution approach for 1H NMR spectra, developed by leveraging concepts from the field of physics informed-learning, intelligent labeling, and tailored high dynamic range (HDR) spectral preprocessing. Since automation and faster workflows are major concerns in NMR spectroscopy, the algorithm handles uncorrected spectra without strict assumptions on phase and baseline correction as well as line shape. Due to the lack of high quality and consistently labeled experimental spectra in quantities needed to train modern deep learning models, we relied on synthetic spectra creation. Moreover, instead of training with synthetic spectra consisting of single lines, we created synthetic multiples that further supported a realistic deconvolution. We achieved super-human performance on corrected and uncorrected synthetic spectra. Finally, and most importantly, the results on synthetic data translate well to experimental spectra despite the covariate shift. Thus, this tool is a promising candidate for automated expert-level deconvolution of experimental HDR 1H NMR spectra.

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.

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.

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).

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.

This chapter focuses on the interaction of metal ions mainly with RNA. It is an update of the previous Chapter 3.21 in the 2nd Edition of Comprehensive Inorganic Chemistry II (2013) but focusing solely on RNA. Metal ions are key to folding, structure, and function of any nucleic acid. These interactions are generally of a weak and highly dynamic nature as they concern mostly K+ and Mg2+ in living organisms. Aside from the large excess of loosely bound ions for charge compensation, a network of inner-sphere and outer-sphere interactions holds more specifically bound ions in place. Hence, metal ion binding to larger RNAs is rather complicated and has many facets. After a few general considerations on the basic properties of metal ions and the potential coordination sites on the RNA, the thermodynamics of metal ion binding to RNA and known metal ion binding motifs in RNA are described. This is followed by today's knowledge on the role of metal ions in folding, dynamics, sensing, and/or catalysis of riboswitches and ribozymes, respectively, is summarized.

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.

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.

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.

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.

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.

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.

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.

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

Although lead(II) is naturally not associated with nucleic acids, this metal ions has been applied with DNA and RNA in various contexts. Pb2+ is an excellent hydrolytic metal ion for nucleic acids, which is why it is mainly used as probing agent for secondary structure and to determine metal ion binding sites both in vitro and in vivo. A further application of lead(II) is in structural studies, i.e., NMR, but also in X-ray crystallography, mostly using this heavy metal to solve the phase problem in the latter method. The structures of tRNAPhe, RNase P, HIV-1 DIS, and the leadzyme are discussed here in detail. A major part of this review is devoted to the cleavage properties of lead(II) with RNA because of its excellence in catalyzing phosphodiester cleavage. Metal ion binding sites in large naturally occurring ribozymes are regularly determined by Pb2+ cleavage, and also in the in vitro selected socalled leadzyme, this metal ion is the decisive key to backbone cleavage at a specific site. Lead(II) was used in the first in vitro selection that yielded a catalytic DNA, i.e., the DNAzyme named GR5. Next to the GR5, the so-called 8-17E is the second most prominent DNAzyme today. Derivatives of these two lead(II)-dependent DNAzymes, as well as the G-quadruplex forming PS2.M have been applied to detect lead(II) in the lower nanomolar range not only in the test tube but also in body fluids. Due to the toxicity of lead(II) for living beings, this is a highly active research field. Finally, further applications of lead(II)-dependent DNAzymes, e.g., in the construction of nanocomputers, are also discussed.

Although cisplatin and its analogues have been widely utilized as anticancer metallodrugs in clinics, their serious side effects and damage to normal tissues cannot be avoided because cisplatin kills cancer cells by attacking genomic DNA. Thus the design of metallodrugs possessing different actions of anti-cancer mechanism is promising. G-quadruplex nucleic acid, which is formed by self-assembly of guanine-rich nucleic acid sequences, has recently been considered as an attractive target for anticancer drug design. The basic unit of a G-quadruplex is a G-quartet, a planar motif generated from four guanine residues pairing together through Hoogsteen like hydrogen bonds. DNA G-quadruplex (G4) structures exist in the chromosomal telomeric sequences and the promoter regions of numerous genes, including oncogenetic promoters. Formation of G4 structures within the 3′-overhang of telomeric DNA can inhibit the telomerase activity, which is silent in normal cells but up-regulated in most cancer cells, thus significantly shortening telomeres and preventing cancer cell proliferation and immortalization. Intramolecular G4 structures formed within the oncogene promoter regions can effectively inhibit oncogenen transcription and expression. Thus rational design of small molecular ligands to selectively interact, stabilize or cleave G4 structures is a promising strategy for developing potent anti-cancer drugs with selective toxicity towards cancer cells over normal ones. This review will highlight the recent development of G4-interacting metal complexes, termed G4-ligands, discussing their binding modes with G-quadruplex DNA and their potential to serve as anticancer drugs in the medical field. The collaboration between Prof. Zong-Wan Mao from Sun Yat-Sen University, P. R. China and Prof. Roland K. O. Sigel from the University of Zurich, Switzerland officially began in January, 2014. The international collaborative research project titled “Chemical Biology Research of New Metallodr

Correctly folded into the respective native 3D structure, RNA and DNA are responsible for uncountable key functions in any viable organism. In order to exert their function, metal ion cofactors are closely involved in folding, structure formation and, e.g. in ribozymes, also the catalytic mechanism. The database MINAS, Metal Ions in Nucleic AcidS (http://www.minas.uzh.ch), compiles the detailed information on innersphere, outersphere and larger coordination environment of >70 000 metal ions of 36 elements found in >2000 structures of nucleic acids contained today in the PDB and NDB. MINAS is updated monthly with new structures and offers a multitude of search functions, e.g. the kind of metal ion, metal-ligand distance, innersphere and outersphere ligands defined by element or functional group, residue, experimental method, as well as PDB entry-related information. The results of each search can be saved individually for later use with so-called miniPDB files containing the respective metal ion together with the coordination environment within a 15 Å radius. MINAS thus offers a unique way to explore the coordination geometries and ligands of metal ions together with the respective binding pockets in nucleic acids

Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement.

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.

Most of today's knowledge of the CPEB3 ribozyme, one of the few small self-cleaving ribozymes known to occur in humans, is based on comparative studies with the hepatitis delta virus (HDV) ribozyme, which is highly similar in cleavage mechanism and probably also in structure. Here we present detailed NMR studies of the CPEB3 ribozyme in order to verify the formation of the predicted nested double pseudoknot in solution. In particular, the influence of Mg2+, the ribozyme's crucial cofactor, on the CPEB3 structure is investigated. NMR titrations, Tb3+-induced cleavage, as well as stoichiometry determination by hydroxyquinoline sulfonic acid fluorescence and equilibrium dialysis, are used to evaluate the number, location, and binding mode of Mg2+ ions. Up to eight Mg2+ ions interact site-specifically with the ribozyme, four of which are bound with high affinity. The global fold of the CPEB3 ribozyme, encompassing 80%–90% of the predicted base pairs, is formed in the presence of monovalent ions alone. Low millimolar concentrations of Mg2+ promote a more compact fold and lead to the formation of additional structures in the core of the ribozyme, which contains the inner small pseudoknot and the active site. Several Mg2+ binding sites, which are important for the functional fold, appear to be located in corresponding locations in the HDV and CPEB3 ribozyme, demonstrating the particular relevance of Mg2+ for the nested double pseudoknot structure.

Due to the polyanionic nature of RNA, the principles of charge neutralization and electrostatic condensation require that cations help to overcome the repulsive forces in order for RNA to adopt a three-dimensional structure. A precise structural knowledge of RNA-metal ion interactions is crucial to understand the mechanism of metal ions in the catalytic or regulatory activity of RNA. We solved the crystal structure of an octameric RNA duplex in the presence of the di- and trivalent metal ions Ca2+, Mn2+, Co2+, Cu2+, Sr2+, and Tb3+. The detailed investigation reveals a unique innersphere interaction to uracil and extends the knowledge of the influence of metal ions for conformational changes in RNA structure. Furthermore, we could demonstrate that an accurate localization of the metal ions in the X-ray structures require the consideration of several crystallographic and geometrical parameters as well as the anomalous difference map.

In vitro studies on macromolecules, like proteins and nucleic acids, are mostly carried out in highly diluted systems where the molecules are studied under artificial conditions. These experimental conditions are optimized for both the system under investigation and the technique used. However, these conditions often do not reflect the in vivo situation and are therefore inappropriate for a reliable prediction of the native behavior of the molecules and their interactions under in vivo conditions. The intracellular environment is packed with cosolutes (macromolecules, metabolites, etc.) that create ‘macromolecular crowding’. The addition of natural or synthetic macromolecules to the sample solution enables crowding to be mimicked. In this surrounding most of the studied biomolecules show a more compact structure, an increased activity, and a decrease of salt requirement for structure formation and function. Herein, we refer to a collection of examples for proteins and nucleic acids and their interactions in crowding environments and present in detail the effect of cosolutes on RNA folding and activity using a group II intron ribozyme as an example.

Group II intron ribozymes catalyze the cleavage of (and their reinsertion into) DNA and RNA targets using a Mg$^{2+}$-dependent reaction. The target is cleaved 3′ to the last nucleotide of intron binding site 1 (IBS1), one of three regions that form base pairs with the intron's exon binding sites (EBS1 to -3). We solved the NMR solution structure of the d3′ hairpin of the Sc.ai5γ intron containing EBS1 in its 11-nucleotide loop in complex with the dIBS1 DNA 7-mer and compare it with the analogous RNA·RNA contact. The EBS1·dIBS1 helix is slightly flexible and non-symmetric. NMR data reveal two major groove binding sites for divalent metal ions at the EBS1·dIBS1 helix, and surface plasmon resonance experiments show that low concentrations of Mg$^{2+}$ considerably enhance the affinity of dIBS1 for EBS1. Our results indicate that identification of both RNA and DNA IBS1 targets, presentation of the scissile bond, and stabilization of the structure by metal ions are governed by the overall structure of EBS1·dIBS1 and the surrounding loop nucleotides but are irrespective of different EBS1·(d)IBS1 geometries and interstrand affinities.

The unique feature of riboswitches is to control selected gene expression by specific recognition of a cognate ligand. AdoCbl (adenosyl cobalamin, coenzyme B12) riboswitches regulate the expression of enzymes and transporters involved in bacterial AdoCbl biosynthesis or uptake on a transcriptional and/or translational level. The analysis of ligand recognition and the induced conformational changes requires a detailed knowledge of the kinetics and thermodynamics of ligand binding and of the secondary structure rearrangements. This chapter describes the investigation of coenzyme B12 binding to the btuB riboswitch from Escherichia coli by in-line probing assays and surface plasmon resonance spectroscopy. The experimental conditions, requirements, and performance of both methods are presented together with the evaluation of the experimental data to determine the associated conformational changes of the RNA and the kinetic and thermodynamic parameters of ligand binding. Owing to the light sensitivity of the cobalt(I)′single bondcarbon bond, these methods were specifically modified to ensure the chemical integrity of AdoCbl.

In this chapter we describe the use of two methods, in-line probing as well as terbium(III) cleavage. Both methods can be applied to RNAs of any size, structure, and function. Aside from revealing directly metal ion-binding sites these techniques also provide structural information for longer RNA sequences that are out of range to be analyzed with other techniques such as NMR. The cleavage pattern derived from in-line probing experiments reflects local and overall conformational changes in RNA upon the addition of metal ions, metal complexes, or other ligands. On the other side, terbium(III) cleavage experiments are applied to locate specific metal ion-binding sites in RNA molecules.

Three ribozymes are known to occur in humans, the CPEB3 ribozyme, the CoTC ribozyme, and the hammerhead ribozyme. Here, we present the NMR solution structure of a well-conserved motif within the CPEB3 ribozyme, the P4 domain. In addition, we discuss the binding sites and impact of Mg2+ and [Co(NH3)6]3+, a spectroscopic probe for [Mg(H2O)6]2+, on the structure. The well-defined P4 region is a hairpin closed with a UGGU tetraloop that shows a distinct electrostatic surface potential and a characteristic, strongly curved backbone trajectory. The P4 hairpin contains two specific Mg2+ binding sites: one outer-sphere binding site close to the proposed CPEB3 ribozyme active site with potential relevance for maintaining a compact fold of the ribozyme core, and one inner-sphere binding site, probably stabilizing the tetraloop structure. The structure of the tetraloop resembles an RNase III recognition structure, as previously described for an AGUU tetraloop. The detailed knowledge of the P4 domain and its metal ion binding preferences thus brings us closer to understanding the importance of Mg2+ binding for the CPEB3 ribozyme’s fold and function in the cell.

In the last decades, a large number of organometallic complexes have shown promising anti-proliferative activity towards different cancer cell lines. However, these compounds generally had low cellular uptake and low selectivity towards cancer cells over healthy cells. The use of external triggers (e.g. light, ultra-sound, temperature, etc.) to modify the cytotoxic effect of a prodrug and the coupling of a targeting vector (e.g. peptides, antibodies, etc.) to a drug were found to be very successful techniques to tackle these drawbacks. Here, we envisioned combining these two methods, namely an external trigger (i.e. light activation) and a targeting vector, in an organometallic compound. More specifically, a Re(I) tricarbonyl N,N-bis(quinolinoyl) complex (Re-NH2) was derivatised with a photo-labile protecting group (PLPG) to cage Re-NH2 by formation of Re-PLPG. For organelle/cellular specificity, Re-PLPG was then further coupled to a nuclear localization sequence (NLS) or a bombesin peptide derivative to give Re-PLPG-NLS or Re-PLPG-Bombesin, respectively. Photolysis experiments in PBS buffer (pH 7.4) demonstrated that Re-NH2 was completely photo-released from Re-PLPG-NLS and Re-PLPG-Bombesin using a very low irradiation dose (1.2 J cm−2). To the best of our knowledge, these are the first two examples of the selective photo-release of an intact organometallic compound from a bioconjugate. Of high interest, both derivatives showed toxicity comparable to that of cisplatin towards cervical cancer cells (HeLa) upon light irradiation, although the phototoxic index (PTI) varied greatly with the targeting peptide. The cell death mechanism of Re-PLPG-NLS was explored using different techniques, including fluorescence microscopy, ICP-MS, gel electrophoresis, flow cytometry and transmission electron microscopy (TEM). It could be demonstrated that HeLa cells treated with Re-PLPG-NLS in the dark and upon irradiation showed severe cell stress (nucleolar segregation, pyknosis and

Ribozymes are enzymes made of RNA although they can be also associated with auxiliary proteins. After their discovery in the early 1980s, ribozymes have been found in the genomes of many species from all kingdoms of life. In addition, numerous artificial ribozymes were developed by in vitro evolution in the past years. All natural ribozymes fall into two major groups that are the small and large ribozymes. Ribozymes catalyze reactions such as RNA splicing, RNA cleavage, and protein synthesis. Catalysis is mainly achieved by forming complex tertiary structures that provide an active site with ideal geometrical restraints to perform nucleophilic substitution reactions for phosphoryltransfers. The catalytic mechanism relies on acid–base catalysis assisted by metal ions and the ribonucleosides.

Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromatic-nitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono- to the di- and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono- or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand contains

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.

Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromatic-nitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono- to the di- and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono- or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand contains

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.

A series of hybrid quantum mechanical/molecular mechanical (QM/MM) calculations was performed on models of a DNA duplex with artificial silver(I)-mediated imidazole base pairs. The optimized structures were compared to the original experimental NMR structure (Nat. Chem. 2 (2010) 229-234). The metal⋯metal distances are significantly shorter (~0.5Å) in the QM/MM model than in the original NMR structure. As a result, argentophilic interactions are feasible between the silver(I) ions of neighboring metal-mediated base pairs. Using the computationally determined metal⋯metal distances, a re-refined NMR solution structure of the DNA duplex was obtained. In this new NMR structure, all experimental constraints remain fulfilled. The new NMR structure shows less deviation from the regular B-type conformation than the original one. This investigation shows that the application of QM/MM models to generate additional constraints to be used during NMR structural refinements represents an elegant approach to obtaining high-resolution NMR structures.