@article{OkudaLenzSeitzetal.2023,
  author    = {Okuda, Takumi and Lenz, Ann-Kathrin and Seitz, Florian and Vogel, J{\"o}rg and H{\"o}bartner, Claudia},
  title     = {A SAM analogue-utilizing ribozyme for site-specific RNA alkylation in living cells},
  series = {Nature Chemistry},
  journal   = {Nature Chemistry},
  doi       = {10.1038/s41557-023-01320-z},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-328762},
  year      = {2023},
  abstract  = {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.},
  language  = {en}
}
@article{NeitzBessiKachleretal.2022,
  author    = {Neitz, Hermann and Bessi, Irene and Kachler, Valentin and Michel, Manuela and H{\"o}bartner, Claudia},
  title     = {Tailored tolane-perfluorotolane assembly as supramolecular base pair replacement in DNA},
  series = {Angewandte Chemie International Edition},
  volume    = {62},
  journal   = {Angewandte Chemie International Edition},
  number    = {1},
  doi       = {10.1002/anie.202214456},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-312575},
  year      = {2022},
  abstract  = {Arene-fluoroarene interactions offer outstanding possibilities for engineering of supramolecular systems, including nucleic acids. Here, we implement the tolane-perfluorotolane interaction as base pair replacement in DNA. Tolane (THH) and perfluorotolane (TFF) moieties were connected to acyclic backbone units, comprising glycol nucleic acid (GNA) or butyl nucleic acid (BuNA) building blocks, that were incorporated via phosphoramidite chemistry at opposite positions in a DNA duplex. Thermodynamic analyses by UV thermal melting revealed a compelling stabilization by THH/TFF heteropairs only when connected to the BuNA backbone, but not with the shorter GNA linker. Detailed NMR studies confirmed the preference of the BuNA backbone for enhanced polar π-stacking. This work defines how orthogonal supramolecular interactions can be tailored by small constitutional changes in the DNA backbone, and it inspires future studies of arene-fluoroarene-programmed assembly of DNA.},
  language  = {en}
}
@article{LiaqatSednevStilleretal.2021,
  author    = {Liaqat, Anam and Sednev, Maksim V. and Stiller, Carina and H{\"o}bartner, Claudia},
  title     = {RNA-cleaving deoxyribozymes differentiate methylated cytidine isomers in RNA},
  series = {Angewandte Chemie International Edition},
  volume    = {60},
  journal   = {Angewandte Chemie International Edition},
  doi       = {10.1002/anie.202106517},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-256519},
  pages     = {19058-19062},
  year      = {2021},
  abstract  = {Deoxyribozymes are emerging as modification-specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA-cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3-methylcytidine (m\(^3\)C), N\(^4\)-methylcytidine (m\(^4\)C), and 5-methylcytidine (m\(^5\)C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10- to 30-fold accelerated cleavage of their target m\(^3\)C-, m\(^4\)C- or m\(^5\)C-modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mXC-detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m\(^3\)C and m\(^5\)C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.},
  language  = {en}
}
@article{KleiberLemusDiazStilleretal.2022,
  author    = {Kleiber, Nicole and Lemus-Diaz, Nicolas and Stiller, Carina and Heinrichs, Marleen and Mong-Quyen Mai, Mandy and Hackert, Philipp and Richter-Dennerlein, Ricarda and H{\"o}bartner, Claudia and Bohnsack, Katherine E. and Bohnsack, Markus T.},
  title     = {The RNA methyltransferase METTL8 installs m\(^3\)C\(_{32}\) in mitochondrial tRNAs\(^{Thr/Ser(UCN)}\) to optimise tRNA structure and mitochondrial translation},
  series = {Nature Communication},
  volume    = {13},
  journal   = {Nature Communication},
  doi       = {10.1038/s41467-021-27905-1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254592},
  year      = {2022},
  abstract  = {Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m\(^3\)C\(_{32}\) in the human mitochondrial (mt-)tRNA\(^{Thr}\) and mt-tRNA\(^{Ser(UCN)}\). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mttRNA recognition elements revealed U\(_{34}\)G\(_{35}\) and t\(^6\)A\(_{37}\)/(ms\(^2\))i\(^6\)A\(_{37}\), present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C\(_{32}\). Several lines of evidence demonstrate the influence of U\(_{34}\), G\(_{35}\), and the m\(^3\)C\(_{32}\) and t\(^6\)A\(_{37}\)/(ms\(^2\))i\(^6\)A\(_{37}\) modifications in mt-tRNA\(^{Thr/Ser(UCN)}\) on the structure of these mt-tRNAs. Although mt-tRNA\(^{Thr/Ser(UCN)}\) lacking METTL8-mediated m\(^3\)C\(_{32}\) are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m\(^3\)C\(_{32}\) within mt-tRNAs.},
  language  = {en}
}
@article{KabingerStillerSchmitzovaetal.2021,
  author    = {Kabinger, Florian and Stiller, Carina and Schmitzov{\´a}, Jana and Dienemann, Christian and Kokic, Goran and Hillen, Hauke S. and H{\"o}bartner, Claudia and Cramer, Patrick},
  title     = {Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis},
  series = {Nature Structural \& Molecular Biology},
  volume    = {28},
  journal   = {Nature Structural \& Molecular Biology},
  doi       = {10.1038/s41594-021-00651-0},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254603},
  pages     = {740-746},
  year      = {2021},
  abstract  = {Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-d-\(N^4\)-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.},
  language  = {en}
}
@article{DietzschBialasBandorfetal.2022,
  author    = {Dietzsch, Julia and Bialas, David and Bandorf, Johannes and W{\"u}rthner, Frank and H{\"o}bartner, Claudia},
  title     = {Tuning Exciton Coupling of Merocyanine Nucleoside Dimers by RNA, DNA and GNA Double Helix Conformations},
  series = {Angewandte Chemie International Edition},
  journal   = {Angewandte Chemie International Edition},
  doi       = {10.1002/anie.202116783},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254565},
  pages     = {e202116783},
  year      = {2022},
  abstract  = {Exciton coupling between two or more chromophores in a specific environment is a key mechanism associated with color tuning and modulation of absorption energies. This concept is well exemplified by natural photosynthetic proteins, and can also be achieved in synthetic nucleic acid nanostructures. Here we report the coupling of barbituric acid merocyanine (BAM) nucleoside analogues and show that exciton coupling can be tuned by the double helix conformation. BAM is a nucleobase mimic that was incorporated in the phosphodiester backbone of RNA, DNA and GNA oligonucleotides. Duplexes with different backbone constitutions and geometries afforded different mutual dye arrangements, leading to distinct optical signatures due to competing modes of chromophore organization via electrostatic, dipolar, - stacking and hydrogen-bonding interactions. The realized supramolecular motifs include hydrogenbonded BAM-adenine base pairs and antiparallel as well as rotationally stacked BAM dimer aggregates with distinct absorption, CD and fluorescence properties.},
  language  = {en}
}
@article{LiaqatSednevStilleretal.2021,
  author    = {Liaqat, Anam and Sednev, Maksim V. and Stiller, Carina and H{\"o}bartner, Claudia},
  title     = {RNA-Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA},
  series = {Angewandte Chemie International Edition},
  volume    = {60},
  journal   = {Angewandte Chemie International Edition},
  number    = {35},
  doi       = {10.1002/anie.202106517},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254544},
  pages     = {19058-19062},
  year      = {2021},
  abstract  = {Deoxyribozymes are emerging as modification-specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA-cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3-methylcytidine (m\(^3\)C), N\(^4\)-methylcytidine (m\(^4\)C), and 5-methylcytidine (m\(^5\)C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10- to 30-fold accelerated cleavage of their target m\(^3\)C-, m\(^4\)C- or m\(^5\)C-modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The m\(^X\)C-detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m\(^3\)C and m\(^5\)C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.},
  language  = {en}
}
@article{MieczkowskiSteinmetzgerBessietal.2021,
  author    = {Mieczkowski, Mateusz and Steinmetzger, Christian and Bessi, Irene and Lenz, Ann-Kathrin and Schmiedel, Alexander and Holzapfel, Marco and Lambert, Christoph and Pena, Vladimir and H{\"o}bartner, Claudia},
  title     = {Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine},
  series = {Nature Communications},
  volume    = {12},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-021-23932-0},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254527},
  pages     = {3549},
  year      = {2021},
  abstract  = {Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.},
  language  = {en}
}
@article{KokicHillenTegunovetal.2021,
  author    = {Kokic, Goran and Hillen, Hauke S. and Tegunov, Dimitry and Dienermann, Christian and Seitz, Florian and Schmitzova, Jana and Farnung, Lucas and Siewert, Aaron and H{\"o}bartner, Claudia and Cramer, Patrick},
  title     = {Mechanism of SARS-CoV-2 polymerase stalling by remdesivir},
  series = {Nature Communications},
  volume    = {12},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-020-20542-0},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-220979},
  year      = {2021},
  abstract  = {Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryoelectron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.},
  language  = {en}
}
@article{LiaqatStillerMicheletal.2020,
  author    = {Liaqat, Anam and Stiller, Carina and Michel, Manuela and Sednev, Maksim V. and H{\"o}bartner, Claudia},
  title     = {N\(^6\)-Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes},
  series = {Angewandte Chemie International Edition},
  journal   = {Angewandte Chemie International Edition},
  edition   = {Early View},
  doi       = {10.1002/ange.202006218},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212121},
  year      = {2020},
  abstract  = {RNA-cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site-specific endonuclease deoxyribozymes that selectively cleave posttranscriptionally modified RNA are extremely rare and their specificity over unmodified RNA is low. In this study, we report that the native tRNA modification N\(^6\)-isopentenyladenosine (i\(^6\)A) strongly enhances the specificity and has the power to reconfigure the active site of an RNA-cleaving deoxyribozyme. Using in vitro selection, we identified a DNA enzyme that cleaves i\(^6\)A-modified RNA at least 2500-fold faster than unmodified RNA. Another deoxyribozyme shows unique and unprecedented behaviour by shifting its cleavage site in the presence of the i\(^6\)A RNA modification. Together with deoxyribozymes that are strongly inhibited by i\(^6\)A, these results highlight intricate ways of modulating the catalytic activity of DNA by posttranscriptional RNA modifications.},
  language  = {en}
}