Claudia Höbartner

Multimodal optical sensing and analyte specificity using single-walled carbon nanotubes

Nanoscale sensing elements offer promise for single-molecule analyte detection in physically or biologically constrained environments. Single-walled carbon nanotubes have several advantages when used as optical sensors, such as photostable near-infrared emission for prolonged detection through biological media and single-molecule sensitivity. Molecular adsorption can be transduced into an optical signal by perturbing the electronic structure of the nanotubes. Here, we show that a pair of single-walled nanotubes provides at least four modes that can be modulated to uniquely fingerprint agents by the degree to which they alter either the emission band intensity or wavelength. We validate this identification method in vitro by demonstrating the detection of six genotoxic analytes, including chemotherapeutic drugs and reactive oxygen species, which are spectroscopically differentiated into four distinct classes, and also demonstrate single-molecule sensitivity in detecting hydrogen peroxide. Finally, we detect and identify these analytes in real time within live 3T3 cells, demonstrating multiplexed optical detection from a nanoscale biosensor and the first label-free tool to optically discriminate between genotoxins.

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

The majority of structural efforts addressing RNAs catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

DNA-Catalyzed Formation of Nucleopeptide Linkages

Covalent linkages between nucleic acids and amino acid side chains are important in many biological contexts. For example, phosphodiester bonds between the 5’-end of DNA or RNA and the side chain of tyrosine are formed during topoisomerase activity and in genomes of picornaviruses, such as poliovirus, and an RNA 5’-terminus is linked to a serine side chain of the tumor-suppressor protein p53. In these and other cases, the ability to form nucleopeptide linkages will enable biochemical experiments that require conjugation of nucleic acids and proteins. Although chemical methods for nucleopeptide synthesis have been devised, the routes are often lengthy, and synthesis of larger nucleoproteins is quite challenging. A more biologically inspired approach to formation of nucleopeptide linkages seeks enzymes that can directly and specifically create the intended bonds, without requiring orthogonal protecting groups and multistep fragment-assembly pathways. Towards this goal, we considered whether deoxyribozymes (DNA enzymes) might be applied to form nucleopeptide linkages. We also envisioned that identifying deoxyribozymes which form nucleopeptide linkages would expand the scope of reactions known to be catalyzed by DNA. The first artificial deoxyribozyme (DNA enzyme) was identified by in vitro selection in 1994. Although many deoxyribozymes have subsequently been discovered, most are restricted to catalyzing reactions of oligonucleotide functional groups. RNA cleavage is the most common DNA-catalyzed activity; our laboratory has focused on RNA ligation. Deoxyribozymes that phosphorylate or cap DNA oligonucleotides using ATP have been identified, as have DNA enzymes that ligate, cleave, or deglycosylate DNA, cleave a phosphoramidate bond within DNA, photochemically cleave a DNA thymine dimer, or metalate a porphyrin. A fundamental yet unanswered question is the extent to which DNA can catalyze reactions of functional groups that are not normally part of oligonucleotides. For example, no known deoxyribozyme catalyzes the reaction of an amino acid side chain in any context. With the long-term objective of deoxyribozyme-catalyzed nucleopeptide synthesis, here we addressed the immediate goal of understanding the ability of DNA to catalyze covalent modification of amino acid side chains. We performed in vitro selection experiments that assess DNA-catalyzed formation of nucleopeptide linkages involving the side chains of tyrosine, serine, and lysine (Tyr, Ser, and Lys). We previously reported 7S11 and related deoxyribozymes that create 2’,5’-branched RNA in a three-helix-junction (3HJ) architecture by catalyzing the reaction of a branch-site RNA 2’-OH group with an RNA 5’-triphosphate. Here we sought to reveal the intrinsic catalytic ability of DNAwith amino acid side chains while avoiding the need for a discrete peptide binding site, either by design or by selection. Therefore, we separately placed each potentially reactive amino acid Tyr, Ser, and Lys at the intersection of the 3HJ formed from candidate deoxyribozyme sequences and two nucleic acid strands (Figure 1). One of these strands is a

Bistable Secondary Structures of Small RNAs and Their Structural Probing by Comparative Imino Proton NMR Spectroscopy

We investigate 25-34 nucleotide RNA sequences, that have been rationally designed to adopt two different secondary structures that are in thermodynamic equilibrium. Experimental evidence for the co-existence of the two conformers results from the NH...N 1H NMR spectra. When compared to the NH...N 1H NMR spectra of appropriate reference sequences the equilibrium position is easily quantifiable even without the assignment of the individual NH resonances. The reference sequences represent several Watson-Crick base-paired double helical segments, each encountered in either of the two conformers of the bistable target sequence. In addition, we rationalize the influence of nucleotide mutations on the equilibrium position of one of the bistable RNA sequences. The approach further allows a detailed thermodynamic analysis and the evaluation of secondary structure predictions for multistable RNAs obtained by computational methods.

Probing secondary structures of spin-labeled RNA by pulsed EPR spectroscopy.

The ability of RNA to interconvert between multiple conformational states is essential for the diversity of biological functions that have been discovered in the recent past. For example, the correct operation of regulatory RNA elements, such as riboswitches, is based on the precise interplay of alternative RNA conformations. Studying the molecular mechanisms of RNA function entails probing RNA-folding intermediates on the energy landscape. EPR spectroscopy, in particular, has been increasingly applied to obtain structural information on nucleic acids, including local conformational changes in RNA and the identification of metal-ion binding sites. Pulsed EPR techniques (PELDOR/ DEER) have been used to determine distances between paramagnetic centers in specifically modified RNA. PELDOR should therefore be suitable for the detection of alternative RNA conformations that involve distinct changes in base-pairing patterns. The accessibility of spin-labeled RNA still poses the major challenge for the widespread applicability of powerful EPR techniques. Nitroxide radicals are the most commonly used type of paramagnetic labels for nucleic acids. Several methods have been reported for attaching nitroxide groups at internal positions at the ribose, the phosphate backbone, or at nucleobases, often by means of multiatom linkers that provide several unwanted degrees of rotational freedom. Rigid nitroxide spin labels conjugated to the nucleobase or to nucleobase analogues have been reported for DNA. Our RNA spin-labeling approach addresses the direct attachment of nitroxide labels onto RNA nucleobases, such that conformational changes can be directly detected by PELDOR (i.e., by the change in distance between two labeled nucleotides). The nucleobase spin labels used in this study are also designed to preserve the Watson–Crick base-pairing capability of labeled nucleotides and not to interfere with alternative base-pairing patterns in different RNA conformations. Here, we describe the installation of nitroxide spin labels on exocyclic amino groups of the RNA nucleobases guanine, adenine, and cytosine (Figure 1) with unprecedented effi-

Chemical RNA Modifications for Studies of RNA Structure and Dynamics

The growing number of RNAs known to be involved in complex biological processes has increased the appreciation for the structural and functional diversity of RNA. In recent years, the scientific community has witnessed the discovery of various RNAs playing integral roles in catalysis, metabolite binding, regulation of transcription and translation events, protein recognition, and epigenetic gene regulation. A fundamental basis for many of these unanticipated biological functions is the ability of RNA to adopt complex three-dimensional structures and to interconvert rapidly between multiple functional states. However, at a molecular level, the current understanding of conformational rearrangement mechanisms and RNA folding pathways is often limited. Recent intensive biochemical and biophysical research efforts have been dedicated to the collection of much-needed information on RNA structures of different hierarchical levels and at different resolutions. An extensive set of experimental and theoretical tools is available to improve our understanding of dynamic RNA folding events and to help uncover the fundamentals of the exquisite specificity of RNA recognition processes. Many biochemical and biophysical techniques for the investigation of RNA structures and mechanisms not only benefit from, but often depend on, the availability of site-specifically modified RNA samples. Fluorescence spectroscopy is a prominent example of an effective biophysical method for monitoring global structure and conformational dynamics of RNA. Fluorescence-based assays always depend on the incorporation of fluorescent nucleoside analogues or the attachment of extrinsic chromophores to RNA residues. The applications of fluorescently labeled RNA range from measurement of fluorescence emission quenching, to fluorescence anisotropy and fluorescence lifetime, shifting of fluorescence spectra, and fluorescence resonance energy transfer. In addition to steady-state fluorescence measurements, new developments in time-resolved (e.g. , femtosecond time-resolved fluorescence spectroscopy) and advanced single-molecule techniques have shown high potential to improve our understanding of the fundamentally important dynamic aspects of RNA structures. Magnetic resonance techniques have become indispensable for the analysis of structure and dynamics of RNA. NMR spectroscopy has proven to be a powerful tool for determining the structure of small RNA motifs. Recent developments to directly detect hydrogen-bonding interactions, also in combination with time-resolved NMR spectroscopic techniques, have been used to distinguish possible secondary structures and to study mechanisms of ligand-binding events. Probing RNA dynamics in solution is based on NMR relaxation data, and information about long-range structural interactions and orientations of helical domains can be obtained by NMR residual dipolar coupling techniques. The successful application of these advanced techniques essentially relies on RNA modification strategies for the incorporation of stable isotopes, such as C, N, H, and F to alleviate the effects of signal degeneracy. Electron paramagnetic resonance (EPR) spectroscopy provides information on local and global dynamic properties and can be used to probe metal ion binding sites in RNA. Detailed information about the binding site geometry can be obtained by advanced EPR methods (e.g. , ENDOR, electron nuclear double resonance). Pulsed electron double resonance (PELDOR) yields long-range structural restraints by measuring the distance between two spin labels. The application of EPR spectroscopy is directly linked to the ability to incorporate paramagnetic centers into RNA at specific positions. Impressive high-resolution structures of small and large RNAs have recently been obtained by X-ray crystallography. De novo determination of 3D RNA structures requires sophisticated phasing techniques, which are based on derivatization with heavy atoms, by soaking crystals in salt solutions, or by chemical derivatization of nucleosides with halogen or selenium atoms. The increasing number of reported applications of modified RNAs for biophysical experiments shows that site-specific incorporation of artificial RNA modifications constitute important enabling technologies for the detailed investigation of functional RNAs. In this review, we discuss recent examples of chemical RNA modifications that were used to monitor conformational changes by NMR spectroscopy, with a focus on fluorine-modified RNA. In addition, we describe modifications to deliberately manipulate RNA secondary structure populations, and RNA analogues to observe conformational changes by fluorescence spectroscopy. Furthermore, we summarize recent developments in the areas of spin-labeled RNA for EPR spectroscopy and selenium-modified RNA for X-ray crystallography.

NSUN3 and ABH1 modify the wobble position of mt‐tRNAMet to expand codon recognition in mitochondrial translation

Mitochondrial gene expression uses a non‐universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt‐)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt‐tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt‐tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt‐tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt‐tRNAMet function. Together, our data reveal how modifications in mt‐tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine.

Orientation selection in distance measurements between nitroxide spin labels at 94 GHz EPR with variable dual frequency irradiation

Pulsed electron-electron double resonance (PELDOR, also known as DEER) has become a method of choice to measure distances in biomolecules. In this work we show how the performance of the method can be improved at high EPR frequencies (94 GHz) using variable dual frequency irradiation in a dual mode cavity in order to obtain enhanced resolution toward orientation selection. Dipolar evolution traces of a representative RNA duplex and an α-helical peptide were analysed in terms of possible bi-radical structures by considering the inherent ambiguity of symmetry-related solutions.

Crystal structure of a DNA catalyst.

Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms.

Synthesis and characterization of RNA containing a rigid and nonperturbing cytidine-derived spin label.

The nitroxide-containing nucleoside Çm is reported as the first rigid spin label for paramagnetic modification of RNA by solid-phase synthesis. The spin label is well accommodated in several RNA secondary structures as judged by its minor effect on the thermodynamic stability of hairpin and duplex RNA. Electron paramagnetic resonance (EPR) spectroscopic characterization of mono-, bi-, and trimolecular RNA structures shows that Çm will be applicable for advanced EPR studies to elucidate structural and dynamic aspects of folded RNA.