TY - JOUR A1 - Steinmetzger, Christian A1 - Bäuerlein, Carmen A1 - Höbartner, Claudia T1 - Supramolecular fluorescence resonance energy transfer in nucleobase-modified fluorogenic RNA aptamers JF - Angewandte Chemie, International Edition N2 - RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence-based strategies reveal information on structure and dynamics of RNA aptamers. Here we report the incorporation of the universal emissive nucleobase analog 4-cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to bound ligands DMHBI+ or DMHBO+. The photophysical properties of the new nucleobase-ligand-FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET-based readout of ligand binding. This strategy is generally suitable for binding site mapping and may also be applied for responsive aptamer devices. KW - RNA aptamers KW - fluorescence resonance energy transfer KW - large stokes shift KW - isomorphic nucleobase analog KW - structure probing KW - structure probes KW - stokes shift KW - Fluoreszenzresonanz-Energietransfer KW - Isomorphe Nukleobasen-Analoga KW - RNA-Aptamere KW - Stokes-Verschiebung KW - Struktursonden Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203084 N1 - Parallel erschienen in Angewandte Chemie 2020,132, 6826–6830. DOI: 10.1002/ange.201916707 (Deutsche Ausgabe). VL - 59 ER - TY - JOUR A1 - Steinmetzger, Christian A1 - Bessi, Irene A1 - Lenz, Ann-Kathrin A1 - Höbartner, Claudia T1 - Structure-fluorescence activation relationships of a large Stokes shift fluorogenic RNA aptamer JF - Nucleic Acids Research N2 - The Chili RNA aptamer is a 52 nt long fluorogen-activating RNA aptamer (FLAP) that confers fluorescence to structurally diverse derivatives of fluorescent protein chromophores. A key feature of Chili is the formation of highly stable complexes with different ligands, which exhibit bright, highly Stokes-shifted fluorescence emission. In this work, we have analyzed the interactions between the Chili RNA and a family of conditionally fluorescent ligands using a variety of spectroscopic, calorimetric and biochemical techniques to reveal key structure - fluorescence activation relationships (SFARs). The ligands under investigation form two categories with emission maxima of ~540 nm or ~590 nm, respectively, and bind with affinities in the nanomolar to low-micromolar range. Isothermal titration calorimetry was used to elucidate the enthalpic and entropic contributions to binding affinity for a cationic ligand that is unique to the Chili aptamer. In addition to fluorescence activation, ligand binding was also observed by NMR spectroscopy, revealing characteristic signals for the formation of a G-quadruplex only upon ligand binding. These data shed light on the molecular features required and responsible for the large Stokes shift and the strong fluorescence enhancement of red and green emitting RNA-chromophore complexes. KW - Chili RNA Aptamer KW - fluorogen-activating RNA aptamer (FLAP) KW - Stokes-shifted fluorescence emission KW - key structure - fluorescence activation relationships (SFARs) KW - ligand binding Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192340 ER - TY - INPR A1 - Sednev, Maksim V. A1 - Mykhailiuk, Volodymyr A1 - Choudhury, Priyanka A1 - Halang, Julia A1 - Sloan, Katherine E. A1 - Bohnsack, Markus T. A1 - Höbartner, Claudia T1 - N\(^6\)-methyladenosine-sensitive RNA-cleaving deoxyribozymes T2 - Angewandte Chemie, International Edition N2 - Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Here we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N\(^6\)-methyladenosine, which is the most wide-spread and extensively investigated natural RNA modification. Using in vitro selection from random DNA, we developed deoxyribozymes that are sensitive to the presence of N\(^6\)-methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m\(^6\)A as a regulatory element that influences RNA folding and protein binding. KW - N6-methyladenosine KW - RNA modification KW - deoxyribozymes KW - in vitro selection KW - DNA catalyst Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171753 N1 - This is the pre-peer reviewed version of the following article: M.V. Sednev, V. Mykhailiuk, P. Choudhury, J. Halang, K. E. Sloan, M. T. Bohnsack, C. Höbartner, Angew. Chem. Int. Ed. 2018, 57, 15117-15121, which has been published in final form at https://doi.org/10.1002/anie.201808745. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. ER - TY - INPR A1 - Sednev, Maksim V. A1 - Liaqat, Anam A1 - Höbartner, Claudia T1 - High-Throughput Activity Profiling of RNA-Cleaving DNA Catalysts by Deoxyribozyme Sequencing (DZ-seq) T2 - Journal of the American Chemical Society N2 - RNA-cleaving deoxyribozymes have found broad application as useful tools for RNA biochemistry. However, tedious in vitro selection procedures combined with laborious characterization of individual candidate catalysts hinder the discovery of novel catalytic motifs. Here, we present a new high-throughput sequencing method, DZ-seq, which directly measures activity and localizes cleavage sites of thousands of deoxyribozymes. DZ-seq exploits A-tailing followed by reverse transcription with an oligo-dT primer to capture the cleavage status and sequences of both deoxyribozyme and RNA substrate. We validated DZ-seq by conventional analytical methods and demonstrated its utility by discovery of novel deoxyribozymes that allow for cleaving challenging RNA targets or the analysis of RNA modification states. KW - RNA-Cleaving Deoxyribozymes KW - High-Throughput Sequencing Method, DZ-seq KW - Analysis of RNA Modifications Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-258520 ER - TY - INPR A1 - Scheitl, Carolin P.M. A1 - Ghaem Maghami, Mohammad A1 - Lenz, Ann-Kathrin A1 - Höbartner, Claudia T1 - Site-specific RNA methylation by a methyltransferase ribozyme T2 - Nature N2 - Nearly all classes of coding and non-coding RNA undergo post-transcriptional modification including RNA methylation. Methylated nucleotides belong to the evolutionarily most conserved features of tRNA and rRNA.1,2 Many contemporary methyltransferases use the universal cofactor S-adenosylmethionine (SAM) as methyl group donor. This and other nucleotide-derived cofactors are considered as evolutionary leftovers from an RNA World, in which ribozymes may have catalysed essential metabolic reactions beyond self-replication.3 Chemically diverse ribozymes seem to have been lost in Nature, but may be reconstructed in the laboratory by in vitro selection. Here, we report a methyltransferase ribozyme that catalyses the site-specific installation of 1-methyladenosine (m1A) in a substrate RNA, utilizing O6-methylguanine (m6G) as a small-molecule cofactor. The ribozyme shows a broad RNA sequence scope, as exemplified by site-specific adenosine methylation in tRNAs. This finding provides fundamental insights into RNA’s catalytic abilities, serves a synthetic tool to install m1A in RNA, and may pave the way to in vitro evolution of other methyltransferase and demethylase ribozymes. KW - Methyltransferase Ribozyme KW - RNA Enzymes KW - position-specific installation of m1A in RNA Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-218687 ER - TY - INPR A1 - Scheitl, Carolin P. M. A1 - Mieczkowski, Mateusz A1 - Schindelin, Hermann A1 - Höbartner, Claudia T1 - Structure and mechanism of the methyltransferase ribozyme MTR1 T2 - Nature Chemical Biology N2 - RNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (m\(^1\)A) in RNA, using O\(^6\)-methylguanine (m\(^6\)G) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution. KW - Methyltransferase Ribozyme MTR1 KW - Crystal structure of MTR1 KW - RNA-catalyzed RNA methylation KW - X-ray crystallography KW - RNA Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-272170 ET - submitted version ER - TY - JOUR A1 - Scheitl, Carolin P. M. A1 - Lange, Sandra A1 - Höbartner, Claudia T1 - New deoxyribozymes for the native ligation of RNA JF - Molecules N2 - Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalysing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5’-adenylated RNA (5’-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3’-5’-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5’-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations. KW - RNA ligation KW - DNA catalysis KW - in vitro selection KW - Deoxyribozyme Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-210405 VL - 25 IS - 16 ER - TY - JOUR A1 - Ronald, Micura A1 - Höbartner, Claudia T1 - Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes JF - Chemical Society Reviews N2 - This review aims at juxtaposing common versus distinct structural and functional strategies that are applied by aptamers, riboswitches, and ribozymes/DNAzymes. Focusing on recently discovered systems, we begin our analysis with small-molecule binding aptamers, with emphasis on in vitro-selected fluorogenic RNA aptamers and their different modes of ligand binding and fluorescence activation. Fundamental insights are much needed to advance RNA imaging probes for detection of exo- and endogenous RNA and for RNA process tracking. Secondly, we discuss the latest gene expression–regulating mRNA riboswitches that respond to the alarmone ppGpp, to PRPP, to NAD+, to adenosine and cytidine diphosphates, and to precursors of thiamine biosynthesis (HMP-PP), and we outline new subclasses of SAM and tetrahydrofolate-binding RNA regulators. Many riboswitches bind protein enzyme cofactors that, in principle, can catalyse a chemical reaction. For RNA, however, only one system (glmS ribozyme) has been identified in Nature thus far that utilizes a small molecule – glucosamine-6-phosphate – to participate directly in reaction catalysis (phosphodiester cleavage). We wonder why that is the case and what is to be done to reveal such likely existing cellular activities that could be more diverse than currently imagined. Thirdly, this brings us to the four latest small nucleolytic ribozymes termed twister, twister-sister, pistol, and hatchet as well as to in vitro selected DNA and RNA enzymes that promote new chemistry, mainly by exploiting their ability for RNA labelling and nucleoside modification recognition. Enormous progress in understanding the strategies of nucleic acids catalysts has been made by providing thorough structural fundaments (e.g. first structure of a DNAzyme, structures of ribozyme transition state mimics) in combination with functional assays and atomic mutagenesis. KW - Functional nucleic acids KW - RNA Enzymes KW - RNA labeling KW - nucleoside modification recognition Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212133 ET - Advance Article ER - TY - JOUR A1 - Okuda, Takumi A1 - Lenz, Ann-Kathrin A1 - Seitz, Florian A1 - Vogel, Jörg A1 - Höbartner, Claudia T1 - A SAM analogue-utilizing ribozyme for site-specific RNA alkylation in living cells JF - Nature Chemistry N2 - Post-transcriptional RNA modification methods are in high demand for site-specific RNA labelling and analysis of RNA functions. In vitro-selected ribozymes are attractive tools for RNA research and have the potential to overcome some of the limitations of chemoenzymatic approaches with repurposed methyltransferases. Here we report an alkyltransferase ribozyme that uses a synthetic, stabilized S-adenosylmethionine (SAM) analogue and catalyses the transfer of a propargyl group to a specific adenosine in the target RNA. Almost quantitative conversion was achieved within 1 h under a wide range of reaction conditions in vitro, including physiological magnesium ion concentrations. A genetically encoded version of the SAM analogue-utilizing ribozyme (SAMURI) was expressed in HEK293T cells, and intracellular propargylation of the target adenosine was confirmed by specific fluorescent labelling. SAMURI is a general tool for the site-specific installation of the smallest tag for azide-alkyne click chemistry, which can be further functionalized with fluorophores, affinity tags or other functional probes. KW - Alkyltransferase Ribozyme SAMURI KW - Site-specific RNA labelling KW - bioorthogonal SAM analogue ProSeDMA KW - Chemical modification KW - RNA Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-328762 ER - TY - INPR A1 - Neitz, Hermann A1 - Bessi, Irene A1 - Kuper, Jochen A1 - Kisker, Caroline A1 - Höbartner, Claudia T1 - Programmable DNA interstrand crosslinking by alkene-alkyne [2+2] photocycloaddition T2 - Journal of the American Chemical Society N2 - Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2’-deoxyuridine (\(^{Phe}\)dU). The crosslinking ability of \(^{Phe}\)dU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2+2] cycloaddition within the DNA duplex, resulting in efficient formation of a \(^{Phe}\)dU-dimer after short irradiation times of a few seconds. A \(^{Phe}\)dU dimer-containing DNA was shown to efficiently bind a helicase complex, but the covalent crosslink completely prevented DNA unwinding, suggesting possible applications in biochemistry or structural biology. KW - light-induced interstrand DNA crosslinking KW - alkene-alkyne [2+2] photocycloaddition KW - DNA-based nanostructures KW - DNA-processing enzymes Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311822 N1 - This document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication in Journal of the American Chemical Society, copyright © 2023 The Authors. Published by American Chemical Society. after peer review. To access the final edited and published work see https://doi.org/10.1021/jacs.3c01611. ET - submitted version ER -