ArticleNucleic acids research2021
A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.
Article in Nucleic acids research, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 19 citations in OpenAlex.
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- PANDORA-Seq Unveils the Hidden Small Non-Coding RNA Landscape in Hypopharyngeal Carcinoma.International journal of molecular sciences · 2025Article
- MapID-based quantitative mapping of chemical modifications and expression of human transfer RNA.Cell chemical biology · 2025Article
- Immunoactive signatures of circulating tRNA- and rRNA-derived RNAs in chronic obstructive pulmonary disease.Molecular therapy. Nucleic acids · 2024Article
- Analysis of RNA and Its Modifications.Annual review of analytical chemistry (Palo Alto, Calif.) · 2024Review
- Advances in methods for tRNA sequencing and quantification.Trends in genetics : TIG · 2024Review
- General Principles and Limitations for Detection of RNA Modifications by Sequencing.Accounts of chemical research · 2024Article
- Terminal modifications independent cell-free RNA sequencing enables sensitive early cancer detection and classification.Nature communications · 2024Article
- Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.Nature biotechnology · 2024Article
- Article
- Determining RNA Natural Modifications and Nucleoside Analog-Labeled Sites by a Chemical/Enzyme-Induced Base Mutation Principle.Molecules (Basel, Switzerland) · 2023Review
- Making Invisible RNA Visible: Discriminative Sequencing Methods for RNA Molecules with Specific Terminal Formations.Biomolecules · 2022Review
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
Abstract
AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.
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