Tipos de trabajo de riesgo

plasmid pGhmC was confirmed by identification of the [M +H]+ _{ions of 5-ghmdC (m/z 420.2). Identification of} [M +H]+ ions of 5-ghmC (m/z 304.2) is attributed to the elimination of a 2-deoxyribose moiety from 5-ghmdC caused by cleavage of the N-glycosidic linkage.

Coupling of functional groups to 5-hmC containing plasmid DNA

To label plasmid DNA with a fluorophore, selective tagging by copper-free click chemistry was used (Baskin et al., 2007). T4 β-glucosyltransferase was used to catalyze the transfer of an azido-sugar from chemically synthesized UDP-6-deoxy-6-azido-glucose (6-N3-UDPG) to the allylic hydroxyl group of 5-hmC. The plasmids

were then fluorescently labeled with dibenzocyclooctyne-Cy5 (DCBO-Cy5) and Cy5 fluorescence emission at 670 nm was used to assess the degree of labelling. Based on the relative fluorescence units (RFU) of the spectrum between 590 and 720 nm, we concluded that on average there are 4-5 fluorophores per plasmid molecule (Fig S3). To assess the functionality of the labeling, we immobilized the plasmid DNA on glass slides and imaged the samples using TIRF microscopy. By depositing samples of purified and washed pHmC-Cy5 on glass slides, single fluorescent spots could be visualized. By analysing the point-spread function, information about the number of fluorophores and their intensities could be compared to pMK0-Cy5 and pGhmC-Cy5 negative control plasmids (Fig 6). The pHmC-Cy5 sample contained approximately 2.1*102 _fluorescent

molecules per mm2_{, which is approximately 20 times higher than either the pMK0-Cy5and approximately 9}

times higher than pGhmC-Cy5 samples. This demonstrates successful labelling of the 5-hmC-containing plasmid

DNA (Fig 6). The low number of localizations when imaging pGhmC-Cy5 plasmids suggests that almost all 5- hmC nucleobases are glucosylated in vivo by β-glucosyltransferase.

Figure 6. Fluorescence microscopy imaging of plasmids labeled with Cy5. Labeling of plasmid DNA with Cy5 is specific to pHmC.

Single plasmid photobleaching analysis

Illumination of Cy5 with the excitation laser of 642 nm causes photobleaching and switching to the non- fluorescent OFF-state of the fluorophores. Time traces of individual fluorescent spots show the step-wise decrease or increase of fluorescence intensity (Fig 7). The number of steps corresponds to the number of fluorophores labeled in the plasmid. To confirm that the image signal originates from Cy5, we used recovery to the fluorescent ON-state using a 405 nm laser. Fluorescence of the spots was recovered indicating the presence of Cy5.

Figure 7. Step-wise photobleaching and recovery of Cy5 fluorophores labeled to pHmC DNA of two individual time traces. Recovery and bleaching of individual Cy5 fluorophores is indicated by the red lines.

Discussion

In this work we present the introduction the of 5-hmC and 5-ghmC synthesis pathway from T4 into E. coli and we demonstrate its utility proof of concept by covalent coupling of functional groups to plasmid DNA. The introduction of 5-hmC and 5-ghmC synthesis pathway from T4 may provide insights into the effect of 5-hmC and 5-ghmC on regulatory processes in E. coli. The relatively modest levels of incorporation are beneficial when only low levels of labels are desirable, for example when immobilizing a plasmid on a surface. The levels of substitution of cytosine by 5-hmC can potentially be increased by increasing the expression levels of the introduced T4 genes. While this work was in progress, Mehta and colleagues published a similar approach of introducing the 5-ghmC synthesis pathway in E. coli (Mehta et al., 2016). Achieved substitution levels in plasmid DNA were approximately 71% and 45% for 5-hmC and 5-ghmC, respectively. The higher substitution levels, compared to our results, can likely be attributed to the use of codon-optimized T4 genes and higher plasmid copy numbers.

Labeling of DNA with fluorophores is commonly used to visualize DNA molecules, however available methods are often restricted to short oligonucleotides. In contrast our method can be used for labeling DNA molecules of any size, including genomic DNA. Further steps are also easy to implement, making our design a generally applicable method for DNA labeling. In addition, covalent coupling of azide groups to DNA molecules allows

labeling of any click-chemistry compatible functional group. Labeling of 5-hmC-azide with biotin could be used as a method to immobilize DNA. Alternatively, DNA may be labeled with peptides, non-fluorescent dyes, magnetic beads, or gold particles. This work creates a modular method for functionalization of DNA and opens up new DNA-labeling approaches.

6

Figure 7. Step-wise photobleaching and recovery of Cy5 fluorophores labeled to pHmC DNA of two individual time traces. Recovery and bleaching of individual Cy5 fluorophores is indicated by the red lines.

Discussion

In this work we present the introduction the of 5-hmC and 5-ghmC synthesis pathway from T4 into E. coli and we demonstrate its utility proof of concept by covalent coupling of functional groups to plasmid DNA. The introduction of 5-hmC and 5-ghmC synthesis pathway from T4 may provide insights into the effect of 5-hmC and 5-ghmC on regulatory processes in E. coli. The relatively modest levels of incorporation are beneficial when only low levels of labels are desirable, for example when immobilizing a plasmid on a surface. The levels of substitution of cytosine by 5-hmC can potentially be increased by increasing the expression levels of the introduced T4 genes. While this work was in progress, Mehta and colleagues published a similar approach of introducing the 5-ghmC synthesis pathway in E. coli (Mehta et al., 2016). Achieved substitution levels in plasmid DNA were approximately 71% and 45% for 5-hmC and 5-ghmC, respectively. The higher substitution levels, compared to our results, can likely be attributed to the use of codon-optimized T4 genes and higher plasmid copy numbers.

Labeling of DNA with fluorophores is commonly used to visualize DNA molecules, however available methods are often restricted to short oligonucleotides. In contrast our method can be used for labeling DNA molecules of any size, including genomic DNA. Further steps are also easy to implement, making our design a generally applicable method for DNA labeling. In addition, covalent coupling of azide groups to DNA molecules allows

labeling of any click-chemistry compatible functional group. Labeling of 5-hmC-azide with biotin could be used as a method to immobilize DNA. Alternatively, DNA may be labeled with peptides, non-fluorescent dyes, magnetic beads, or gold particles. This work creates a modular method for functionalization of DNA and opens up new DNA-labeling approaches.