3. Results and Discussion
3.1. Initial Screening for Discrimination between C and 5mC
3.1.2. Screening of modified nucleotides to discriminate 5mC
All modified nucleotides depicted in Figure 10 were tested towards their potential to be used in 5mC detection due to diverging incorporation efficiencies opposite C or the epigenetic marker 5mC by different DNA polymerases. Two DNA polymerases belonging to two different sequence families were chosen for previous screening experiments: the DNA polymerases KlenTaq, belonging to sequence family A and KOD exo-, member of sequence family B.[104] Archeal DNA polymerases (as KOD exo-) are known to have the ability to detect U in DNA by a “read ahead” mechanism.[105] Detection occurs at a site distant from the nucleotide incorporation site. As U differs from T only by an additional methylation in position 5, T can be seen as 5mU. Thus, attempts to identify combinations of modified nucleotides and DNA polymerase with increased discrimination between C and 5mC seem promising.
The unmodified dGTP was tested in comparison to the modified nucleotides 1 - 10 in radioactive single-nucleotide incorporation primer extension experiments employing both mentioned DNA polymerases. Analysis was afterwards performed by denaturing PAGE analysis and visualisation by autoradiography. A non-elongated primer band can be seen as reference on the left side of every gel picture and incorporation of the respective nucleotide by a DNA polymerase can be monitored by the occurrence of a second band, which can be detected above the band resulting from remaining primer due to reduced migration mobility. Incorporation efficiencies were determined by terms of the integrated gel band intensities, as % incorporation = 100*(Iextension)/(Iprimer+Iextension). Discrimination ratios were subsequently determined by calculating the quotient of % incorporation opposite C and
% incorporation opposite 5mC.
Figure 14: Screening of dGTP and modified nucleotides using KlenTaq. a) Partial primer/template sequence used;; b) PAGE analysis of single-nucleotide incorporation primer extension experiments of dGMP and nucleotides 1a - 10 opposite a template containing C in comparison to a template containing 5mC employing the DNA polymerase KlenTaq. 50 µM dGTP/dG*TP and 5 nM KlenTaq were used;; reactions were stopped after indicated time points.
3. Results and Discussion 36
The incorporation of dGMP or the modified nucleotides 1 - 10 by the DNA polymerase KlenTaq does not show promising results (see Figure 14). All modified nucleotides are accepted by this A-family DNA polymerase, but incorporation efficiencies were notably decreased in comparison to the unmodified dGMP (see Figure 14). Those nucleotides, modified in position 8 (2 - 4), are processed with very low efficiencies, compared to the unmodified dGTP. Especially application of nucleotide 4 shows barely any incorporation under the chosen conditions. In addition, no promising differences in incorporation efficiencies between processing of those nucleotides opposite C in comparison to 5mC can be observed. Anyhow, nucleotide 6 shows favoured incorporation opposite C;; but the overall turnover of this nucleotide by the KlenTaq DNA polymerase is very low. Another nucleotide showing some discriminating effects, nucleotide 8, was not further considered as well, since incorporation of this δ-phosphate modified tetraphosphate leads to several different incorporation bands, making interpretation difficult (see Figure 14). We suggest the reason for those multiple incorporation bands to be an additional hydrolysis mechanism of nucleotide 8. If cleavage occurs between ß- and γ-phosphate, instead of cleavage between α- and ß-phosphate, alkylated pyrophosphate would be released during incorporation and the driving force of the incorporation would therefore be maintained.
Hence, I suggest that a second incorporation band derives from incorporation of dGDP in addition to incorporation of dGMP.
In contrast, testing those nucleotides in combination with the B-family DNA polymerase KOD exo-
shows already some discrimination for incorporation of dGMP opposite C and 5mC, as dGTP is processed with slightly higher efficiency opposite C, than opposite 5mC (see Figure 15). This difference in incorporation efficiencies can even be further increased by the application of the modified nucleotides 1 - 10. Again, processing of the modified nucleotides shows decreased efficiencies in comparison to dGTP, but still most modified nucleotides are accepted with good incorporation efficiencies. Again, those nucleotides modified in position 8 (2 - 4) are poorly accepted. For nucleotide 4, as well as for the γ-thiophosphate modified dGTP derivative 6, no notable incorporation opposite C or 5mC can be observed after 60 min under the chosen conditions. Since those nucleotides are very poorly incorporated by both DNA polymerases, they were not considered for further experiments. As already observed in the experiments employing KlenTaq, processing of nucleotide 8 led to an incorporation pattern on the PAGE gels, which is difficult to interpret due to three different extension bands. Despite the decreased incorporation efficiencies for the 8 modified nucleotides 2 and 3, increased discrimination between C and 5mC can be observed for both nucleotides, while incorporation opposite C is favoured over incorporation opposite 5mC. Nucleotide 5 shows decent incorporation efficiencies with slightly favoured incorporation opposite C in comparison to 5mC.
The most promising results can be achieved by processing nucleotides 1a, 7, 9 and 10. All four nucleotides are processed opposite C with remarkably higher efficiencies than opposite 5mC and the incorporation efficiencies opposite C are just slightly decreased compared to the unmodified dGMP.
Therefore, position 6 of dGTP was chosen for further derivatisation. This position seems most promising for the desired application, since nucleotides modified in this position are well accepted by both DNA polymerases and processing of those nucleotides employing the DNA polymerase KOD exo- leads to the most pronounced differences in incorporation efficiencies (see Figure 15).
Figure 15: Screening of dGTP and modified nucleotides using KOD exo-. a) Partial primer / template sequence used;; b) PAGE analysis and quantitative evaluation of single-nucleotide incorporation primer extension experiments of dGMP and nucleotides 1a - 10 opposite a template containing C (black) in comparison to a template containing 5mC (grey) employing the DNA polymerase KOD exo-. 50 µM dGTP or dG*TP and 5 nM KOD exo- were used;; reactions were stopped after indicated time points. Experiments were done at least in triplicates.
3.2. 6 - Modified dGTP Derivatives for the Detection of 5mC
After testing different purine-based 2´-deoxy-nucleotides (see Figure 10) for their ability to sense 5mC in DNA polymerase-catalysed reactions, I found that modification of dGTP in position 6 was most
3. Results and Discussion 38
promising. dGTP analogues, modified at this position are processed with remarkably different efficiencies opposite C than opposite 5mC by the DNA polymerase KOD exo- (see Figure 15). In order to investigate, if this discrimination is more general and extendable to other modifications, I decided to synthesise different dGTP derivatives that are modified at position 6 and explore their potential to enhance the observed discrimination. In addition, modification at position 6 can clarify, if interruption of the Watson-Crick face of dGMP interferes with DNA polymerase-catalysed incorporation opposite C or 5mC.