TY  - CHAP
A1  - Liaqat, Anam
A1  - Sednev, Maksim V.
A1  - Höbartner, Claudia
T1  - In Vitro Selection of Deoxyribozymes for the Detection of RNA Modifications
T2  - Ribosome Biogenesis: Methods and Protocols
N2  - 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.
KW  - RNA
KW  - deoxyribozymes
KW  - modified RNA nucleotides
KW  - catalytic DNA
KW  - epitranscriptomics
KW  - in vitro selection
KW  - RNA cleavage
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-279208
SN  - 978-1-0716-2501-9
PB  - Humana Press
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 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  - 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  -