Refine
Has Fulltext
- yes (30)
Is part of the Bibliography
- yes (30)
Document Type
- Journal article (19)
- Preprint (10)
- Book article / Book chapter (1)
Language
- English (30) (remove)
Keywords
- RNA (8)
- in vitro selection (6)
- RNA modification (3)
- deoxyribozymes (3)
- Chili RNA Aptamer (2)
- Deoxyribozymes (2)
- Epitranscriptomics (2)
- RNA Enzymes (2)
- X-ray crystallography (2)
- epitranscriptomics (2)
- ribozymes (2)
- site-specific RNA cleavage (2)
- Aldehyde Bioconjugation (1)
- Alkyltransferase Ribozyme SAMURI (1)
- Analysis of RNA Modifications (1)
- Antiviral nucleoside analogues (1)
- Barbituric Acid Merocyanines (1)
- Biochemistry (1)
- Bioorthogonal Tag (1)
- Chemical modification (1)
- Chromophore Assembly (1)
- Co-Crystal Structures of Chili RNA (1)
- Cryoelectron Microscopy (1)
- Cryoelectron microscopy (1)
- Crystal structure of MTR1 (1)
- DNA (1)
- DNA catalysis (1)
- DNA catalyst (1)
- DNA-based nanostructures (1)
- DNA-processing enzymes (1)
- Deoxyribozyme (1)
- Enzymes (1)
- Fluorescence and Crosslinking (1)
- Fluoreszenzresonanz-Energietransfer (1)
- Fluorogenic RNA Aptamers (1)
- Functional nucleic acids (1)
- High-Throughput Sequencing Method, DZ-seq (1)
- Higher-order Transient Absorption Spectroscopy (1)
- Isomorphe Nukleobasen-Analoga (1)
- METTL8 (1)
- Merocyanine (1)
- Methyltransferase Ribozyme (1)
- Methyltransferase Ribozyme MTR1 (1)
- Mitochondrial Matrix Protein (1)
- Modified Nucleotides in tRNAs (1)
- Molecular mechanism (1)
- Molnupiravir (1)
- Molnupiravir-Induced RNA Mutagenesis Mechanism (1)
- N6-methyladenosine (1)
- N6-methyladenosine (m6A) (1)
- Nucleic Acids (1)
- Nucleobase Analogue (1)
- Nucleobase Surrogate Incorporation (1)
- Optical Spectroscopy (1)
- Organelles (1)
- Photoresponsive DNA Crosslinker (1)
- RNA Aptamer (1)
- RNA Labelling (1)
- RNA Methyltransferase (1)
- RNA Modification (1)
- RNA aptamers (1)
- RNA cleavage (1)
- RNA labeling (1)
- RNA ligation (1)
- RNA-Aptamere (1)
- RNA-Cleaving Deoxyribozymes (1)
- RNA-Dependent RNA Polymerase (1)
- RNA-catalyzed RNA methylation (1)
- RNA-dependent RNA polymerase (1)
- Remdesivir (1)
- Ribozyme (1)
- Ribozyme-catalyzed RNA labeling (1)
- SARS-CoV-2 polymerase (1)
- SARS-CoV2 Replication Impairment (1)
- Site-Specific RNA Cleavage (1)
- Site-specific RNA labelling (1)
- Stokes-Verschiebung (1)
- Stokes-shifted fluorescence emission (1)
- Structural Biology (1)
- Struktursonden (1)
- Supramolecular Element (1)
- Synthetic Functional RNAs (1)
- Tolane-Modified Fluorescent Nucleosides (1)
- X-ray Crystallography (1)
- XNA (1)
- YTH reader proteins (1)
- alkene-alkyne [2+2] photocycloaddition (1)
- arene-fluoroarene (1)
- artificial base pair (1)
- atomic mutagenesis (1)
- bioorthogonal SAM analogue ProSeDMA (1)
- catalytic DNA (1)
- chemical modification (1)
- covalent and site-specific RNA labeling (1)
- demethylase enzymes FTO and ALKBH5 (1)
- dipole-dipole interaction (1)
- duplex structure (1)
- dye assembly (1)
- fluorescence (1)
- fluorescence resonance energy transfer (1)
- fluorescent protein (1)
- fluorescent resonance energy transfer (1)
- fluorogen-activating RNA aptamer (FLAP) (1)
- in vitro Selection (1)
- in vitro selection from a structured RNA library (1)
- intermolecular applications of ribozymes (1)
- isomorphic nucleobase analog (1)
- key structure - fluorescence activation relationships (SFARs) (1)
- large Stokes shift (1)
- large stokes shift (1)
- ligand binding (1)
- light-induced interstrand DNA crosslinking (1)
- merocyanine (1)
- modified RNA nucleotides (1)
- modified nucleosides (1)
- nucleic acids (1)
- nucleoside modification recognition (1)
- optical spectroscopy (1)
- organic chemistry (1)
- peptide backbone (1)
- position-specific installation of m1A in RNA (1)
- rBAM2-labeled RNA strands (1)
- sSupramolecular interaction (1)
- site-specific RNA labeling (1)
- stokes shift (1)
- structural biology (1)
- structure probes (1)
- structure probing (1)
- tenofovir (1)
- trans-acting 2'-5' adenylyl transferase ribozymes (1)
Institute
Schriftenreihe
Sonstige beteiligte Institutionen
- International Max Planck Research School Molecular Biology, University of Göttingen, Germany (2)
- Agricultural Center, BASF SE, 67117 Limburgerhof, Germany (1)
- Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany (1)
- Center for Nanosystems Chemistry (CNC), University of Würzburg (1)
- Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells, Göttingen (1)
- Department of Cellular Biochemistry, University Medical Center Göttingen (1)
- Department of Cellular Biochemistry, University Medical Centre Göttingen (1)
- Department of Molecular Biology, University Medical Center Göttingen, Germany (1)
- Department of Molecular Biology, University Medical Centre Göttingen (1)
- Department of Molecular Biology, University Medical Centre Göttingen, Göttingen 37073, Germany (1)
- Georg August University School of Science (1)
- Göttingen Center for Molecular Biosciences, Georg- August University Göttingen, Göttingen 37077, Germany (1)
- Göttingen Center for Molecular Biosciences, University of Göttingen (1)
- Institut für Molekulare Infektionsbiologie (MIB) der Universität Würzburg (1)
- Institute of Cancer Research (ICR) London (1)
- Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck, CMBI, Leopold-Franzens University Innsbruck, Austria (1)
- Max Planck Institute for Biophysical Chemistry (1)
- Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen (1)
- Max Planck Institute for Biophysical Chemistry, Research Group Structure and Function of Molecular Machines, Göttingen (1)
- Max-Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen (1)
- University Medical Center Göttingen, Department of Cellular Biochemistry, Göttingen (1)
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.
We report the synthesis and spectroscopic analysis of RNA containing the barbituric acid merocyanine rBAM2 as a nucleobase surrogate. Incorporation into RNA strands by solid-phase synthesis leads to fluorescence enhancement compared to the free chromophore. In addition, linear absorption studies show the formation of an excitonically coupled H-type dimer in the hybridized duplex. Ultrafast third- and fifth-order transient absorption spectroscopy of this non-fluorescent dimer suggests immediate (sub-200 fs) exciton transfer and annihilation due to the proximity of the rBAM2 units.
The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.
Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4 hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogs that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functional-ization at the C2 position of the imidazolone, this strategy yielded DMHBO\(^+\), which binds to the Chili aptamer with a low-nanomolar K\(_D\). Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO\(^+\) complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.
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.
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.
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.
Deoxyribozymes are artificially evolved DNA molecules with catalytic abilities. RNA-cleaving deoxyribozymes have been recognized as an efficient tool for detection of modifications in target RNAs and provide an alternative to traditional and modern methods for detection of ribose or nucleobase methylation. However, there are only few examples of DNA enzymes that specifically reveal the presence of a certain type of modification, including N6-methyladenosine, and the knowledge about how DNA enzymes recognize modified RNAs is still extremely limited. Therefore, DNA enzymes cannot be easily engineered for the analysis of desired RNA modifications, but are instead identified by in vitro selection from random DNA libraries using synthetic modified RNA substrates. This protocol describes a general in vitro selection stagtegy to evolve new RNA-cleaving DNA enzymes that can efficiently differentiate modified RNA substrates from their unmodified counterpart.
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.
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.