6‐Substituted 2‐Aminopurine‐2′‐deoxyribonucleoside 5′‐Triphosphates that Trace Cytosine Methylation

Watzdorf, Janina von; Marx, Andreas

doi:10.1002/cbic.201600245

Cited by 7 publications

(5 citation statements)

References 55 publications

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“…In addition, we could ensure selectivity for this incorporation opposite C in comparison to the other nucleobases T, A and G. Taken together, by employing a systematic approach to mutate KOD exo- DNA polymerase and subsequent screening, we identified mutants at one site of KOD exo- DNA polymerase that are capable to discriminate between C and 5mC. Interestingly, the herein developed system keeps the selectivity for incorporation according to the Watson–Crick rule which is in stark contrast to earlier approaches that exploit modified nucleotides (35,41). A multiple sequence alignment of several family-B DNA polymerases (Supplementary Figure S7) shows that the glycine identified in this study is conserved among several family B DNA polymerases.…”

Section: Discussionmentioning

confidence: 88%

“…We have engineered a variant of Thermococcus kodakaraensis (KOD) exo- DNA polymerase that enables the direct discrimination between C and 5mC at single sites when primer extension is performed from mismatched primer termini while matched primer template complexes failed to show significant discrimination (40). Furthermore, we explored the potential of modified nucleotides to be employed in site specific 5mC detection (35,41). We found that O 6 -alkylated dGTP-analogues are processed opposite C and 5mC with different efficiencies by KOD exo- DNA polymerase.…”

Section: Introductionmentioning

confidence: 99%

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5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

Huber¹,

Watzdorf²,

Marx³

2016

Nucleic Acids Res

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DNA methylation of cytosine in eukaryotic cells is a common epigenetic modification, which plays an important role in gene expression and thus affects various cellular processes like development and carcinogenesis. The occurrence of 5-methyl-2′-deoxycytosine (5mC) as well as the distribution pattern of this epigenetic marker were shown to be crucial for gene regulation and can serve as important biomarkers for diagnostics. DNA polymerases distinguish little, if any, between incorporation opposite C and 5mC, which is not surprising since the site of methylation is not involved in Watson–Crick recognition. Here, we describe the development of a DNA polymerase variant that incorporates the canonical 2′-deoxyguanosine 5′-monophosphate (dGMP) opposite C with higher efficiency compared to 5mC. The variant of Thermococcus kodakaraensis (KOD) exo- DNA polymerase was discovered by screening mutant libraries that were built by rational design. We discovered that an amino acid substitution at a single site that does not directly interact with the templating nucleobase, may alter the ability of the DNA polymerase in processing C in comparison to 5mC. Employing these findings in combination with a nucleotide, which is fluorescently labeled at the terminal phosphate, indicates the potential use of the mutant DNA polymerase in the detection of 5mC.

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Section: Discussionmentioning

confidence: 88%

Section: Introductionmentioning

confidence: 99%

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

Huber¹,

Watzdorf²,

Marx³

2016

Nucleic Acids Res

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“…bisulfite sequencing of m 5 C, or SHAPE-MaP 52 ) or unnatural bases (e.g. insertion of artificial nucleotides opposite m 5 C or O 6 -BnG 53–56 ) opposite modified nucleotides or arrest of the polymerase activity, if it cannot efficiently insert a base opposite the modification site (e.g. adenosine methylation by DMS or older versions of SHAPE probing 57, 58 ).…”

Section: Introductionmentioning

confidence: 99%

“…Category 3 consists of primer extension assays utilizing either incorporation of natural (e.g., bisulfite sequencing of m 5 C, or SHAPE-MaP) or unnatural bases (e.g., insertion of artificial nucleotides opposite m 5 C or O 6 -BnG − ) opposite modified nucleotides or arrest of the polymerase activity, if it cannot efficiently insert a base opposite the modification site (e.g., adenosine methylation by DMS or older versions of SHAPE probing , ). Currently, there are no known artificial bases that can be inserted opposite OG or the hydantoin lesions with the required specificity to map exclusively these sites .…”

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confidence: 99%

Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers

2017

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Reactive oxygen species, both endogenous and exogenous, can damage nucleobases of RNA and DNA. Among the nucleobases, guanine has the lowest redox potential making it a major target of oxidation. Although, RNA is more prone to oxidation than DNA, oxidation of guanine in RNA has been studied to a significantly lesser extent. One of the reasons for this is that many tools that were previously developed to study oxidation of DNA cannot be used on RNA. In the current study, the lack of a method for seeking sites of modification in RNA where oxidation occurs is addressed. For this purpose, reverse transcription of RNA containing major products of guanine oxidation was used. Extension of a DNA primer annealed to an RNA template containing 8-oxo-7,8-dihydroguanine (OG), 5-guanidinohydantoin (Gh), or the R and S diastereomers of spiroiminodihydantoin (Sp) was studied under standing start conditions. SuperScript III reverse transcriptase is capable of bypassing these lesions in RNA inserting predominantly A opposite OG, predominantly G opposite Gh, and almost an equal mixture of A and G opposite the Sp diastereomers. These data should allow RNA sequencing of guanine oxidation products by following characteristic mutation signatures formed by the reverse transcriptase during primer elongation past G oxidation sites in the template RNA strand.

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“…This fidelity data foreshadows an important limitation in enzymatic XNA synthesis that will likely need to be addressed as the field moves forward. On the other hand, the promiscuous behavior of polymerases has been leveraged to read and record the presence of epigenetic DNA and RNA modifications via misincorporation "signatures" at the modifications [162][163][164][165][166][167][168][169][170]. In addition, the first instance of direct sequencing of an XNA was recently reported; FANA was sequenced using nanopore technology, albeit with relatively short read lengths [171].…”

Section: Analysis Of Xnas and Xna Polymerasesmentioning

confidence: 99%

Modified nucleic acids: replication, evolution, and next-generation therapeutics

2020

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Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs.

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6‐Substituted 2‐Aminopurine‐2′‐deoxyribonucleoside 5′‐Triphosphates that Trace Cytosine Methylation

Cited by 7 publications

References 55 publications

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers

Modified nucleic acids: replication, evolution, and next-generation therapeutics

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