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1.
Angew Chem Int Ed Engl ; 57(2): 417-421, 2018 01 08.
Article in English | MEDLINE | ID: mdl-29115744

ABSTRACT

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N6 -methyladenosine (m6 A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m6 A-containing RNA prior to sequencing, since m6 A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6 A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N6 -methylation. We identified a mutant that exhibits increased misincorporation opposite m6 A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m6 A sites directly from the sequencing data of untreated RNA samples.


Subject(s)
Adenosine/analogs & derivatives , DNA-Directed DNA Polymerase/metabolism , Adenosine/genetics , DNA Methylation , High-Throughput Nucleotide Sequencing , Protein Engineering , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
2.
Curr Opin Biotechnol ; 48: 187-195, 2017 12.
Article in English | MEDLINE | ID: mdl-28618333

ABSTRACT

A multitude of biotechnological techniques used in basic research as well as in clinical diagnostics on an everyday basis depend on DNA polymerases and their intrinsic capability to replicate DNA strands with astoundingly high fidelity. Applications with fundamental importance to modern molecular biology, including the polymerase chain reaction and DNA sequencing, would not be feasible without the advances made in characterizing these enzymes over the course of the last 60 years. Nonetheless, the still growing application scope of DNA polymerases necessitates the identification of novel enzymes with tailor-made properties. In the recent past, DNA polymerases optimized for diverse PCR and sequencing applications as well as enzymes that accept a variety of unnatural substrates for the synthesis and reverse transcription of modified nucleic acids have been developed.


Subject(s)
Biotechnology/methods , DNA-Directed DNA Polymerase/metabolism , Nucleotides/metabolism , Polymerase Chain Reaction , Sequence Analysis, DNA , Substrate Specificity
3.
Nucleic Acids Res ; 44(8): 3495-502, 2016 05 05.
Article in English | MEDLINE | ID: mdl-27016740

ABSTRACT

Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR.


Subject(s)
DNA-Directed DNA Polymerase/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Ribosomal, 18S/metabolism , RNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , Humans , Methylation , Polymerase Chain Reaction/methods , RNA, Ribosomal, 18S/chemistry , RNA-Directed DNA Polymerase/genetics
4.
Bioorg Med Chem ; 24(5): 995-1001, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26827138

ABSTRACT

The Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has become increasingly important in the conjugation chemistry of biomolecules. For example, it is an efficient and convenient method to generate defined ubiquitin-protein conjugates. Here, we investigate the effect of surfactants on the efficiency of CuAAC for chemical protein ubiquitylation. We found that anionic surfactants enhance conjugate formation by up to 10-fold resulting in high yields even at low (i.e., micromolar) concentrations of the reactants. Notably, the herein investigated conjugates are functional and thus properly folded.


Subject(s)
Alkynes/chemistry , Azides/chemistry , Copper/chemistry , DNA Polymerase beta/chemistry , Surface-Active Agents/chemistry , Ubiquitin/chemistry , Catalysis , Click Chemistry , Cycloaddition Reaction , Humans , Models, Molecular , Ubiquitination
5.
Angew Chem Int Ed Engl ; 53(31): 8154-8, 2014 Jul 28.
Article in English | MEDLINE | ID: mdl-24923910

ABSTRACT

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3'-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.


Subject(s)
5-Methylcytosine/analysis , Polymerase Chain Reaction/methods , HeLa Cells , Humans
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