TY  - JOUR
A1  - Liaqat, Anam
A1  - Sednev, Maksim V.
A1  - Stiller, Carina
A1  - Höbartner, Claudia
T1  - RNA-cleaving deoxyribozymes differentiate methylated cytidine isomers in RNA
JF  - Angewandte Chemie International Edition
N2  - 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.
KW  - organic chemistry
KW  - site-specific RNA cleavage
KW  - deoxyribozymes
KW  - epitranscriptomics
KW  - in vitro selection
KW  - RNA modification
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256519
VL  - 60
ER  - 
TY  - JOUR
A1  - Kabinger, Florian
A1  - Stiller, Carina
A1  - Schmitzová, Jana
A1  - Dienemann, Christian
A1  - Kokic, Goran
A1  - Hillen, Hauke S.
A1  - Höbartner, Claudia
A1  - Cramer, Patrick
T1  - Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis
JF  - Nature Structural & Molecular Biology
N2  - 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.
KW  - Molnupiravir
KW  - RNA-Dependent RNA Polymerase
KW  - SARS-CoV2 Replication Impairment
KW  - Molnupiravir-Induced RNA Mutagenesis Mechanism
KW  - Cryoelectron Microscopy
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254603
VL  - 28
ER  - 
TY  - JOUR
A1  - Liaqat, Anam
A1  - Sednev, Maksim V.
A1  - Stiller, Carina
A1  - Höbartner, Claudia
T1  - RNA-Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA
JF  - Angewandte Chemie International Edition
N2  - 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.
KW  - Deoxyribozymes
KW  - Epitranscriptomics
KW  - RNA Modification
KW  - Site-Specific RNA Cleavage
KW  - in vitro Selection
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254544
VL  - 60
IS  - 35
ER  -