Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5‐Methylcytosine

Abstract

Abstract5‐Methyl‐2′‐deoxycytosine, the most common epigenetic marker of DNA in eukaryotic cells, plays a key role in gene regulation and affects various cellular processes such as development and carcinogenesis. Therefore, the detection of 5mC can serve as an important biomarker for diagnostics. Here we describe that modified dGTP analogues as well as modified primers are able to sense the presence or absence of a single methylation of C, even though this modification does not interfere directly with Watson–Crick nucleobase pairing. By screening several modified nucleotide scaffolds, O6‐modified 2′‐deoxyguanosine analogues were identified as discriminating between C and 5mC. These modified nucleotides might find application in site‐specific 5mC detection, for example, through real‐time PCR approaches.

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

“…Comparison of the catalytic efficiencies (k cat /K M ) observed for usage of dGTP opposite C or 5mC in the template strand, verified the decreased incorporation efficiencies for dGMP opposite 5mC in comparison to the unmodified C. For the wild-type enzyme no remarkable discrimination could be observed, as already reported before (35). However, when employing the DNA polymerase mutant G245D, the discrimination between C and 5mC increased to a factor of over 2 when comparing the catalytic efficiencies.…”

“…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.…”

“…Therefore, cleared bacterial lysates of the enzymes identified as active (Figure 2) were employed in further studies. Since previous experiments (35) proved that decreased selectivity between C and T could be problematic, we employed three different templates containing C, 5mC or T (see Supplementary Figure S3) in the template position 0 (see Figure 1H). Reactions were stopped after four different time points, analyzed by PAGE and autoradiography and discrimination was calculated by the quotient of % primer extension opposite C divided by % primer extension opposite 5mC.…”

“…DNA polymerases that are widely employed in nucleic acids diagnostics (33,34) fail to discriminate significantly between dGMP incorporation opposite C and 5mC directly (35). This is to be expected since 5-methylation does not affect Watson–Crick base pairing.…”

“…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.…”