PMC

Depiction of the known cytosine modifications mC and hmC and of the putative oxidative “demethylation” intermediates fC and caC. The base excision repair (BER) pathway is a second possible demethylation pathway via the intermediate hmU.

A) Values of hmC in % of dG. B) Values of mC in % of dG. A)+B) Data represent values for each tissue of at least two mice with standard deviation (SD) (See for details). Light-colored bars represent data from our earlier study .

High magnification images of hmC immunoreactivity in the dentate gyrus (DG) and the hilus of mouse hippocampus. A) Signal for anti-hmC (green) and Hoechst 33342 nuclear dye (blue) B) Competition of anti-hmC with 2 µM hmC-DNA. The scale bar marks 20 µm.

Scale bar: 200 µM. Left column: mouse tissues stained with anti-hmC (green). Middle column: mouse tissues stained with anti-hmC (green) and Hoechst 33342 (blue) for nuclear staining. Right column: Bright field pictures of corresponding tissue. In every second row 2 µM hmC-DNA were added to compete the anti-hmC staining signal out.

A) HPLC-chromatogram of the synthesized cytosine and uracil modifications caC, hmC, hmU, mC, and fC as 2′-deoxynucleosides showing excellent separation of the compounds. B) Detected values of the potential intermediates as example in olfactory bulb. The red line indicates the detection limits of the modified nucleosides in enzymatically digested DNA samples.

a) CO, PPh₃, Pd₂(dba)₃·CHCl₃, Bu₃SnH, 97%, with Bu₃SnD 49%, b) HF pyridine, 75%, c) NaBD₄, CeCl₃·7H₂O, 21%, d) TBAF, 51%, e) CO, TMS-Et-OH, DIPEA, Pd₂Cl₂(MeCN)₂, 52%, f) TBAF, 69%, g) Reference h) HF·pyridine, 70%. All reactions could also be carried out in a protective group free manner but resulted in reduced yields and tedious workups (see for details).

DNA is extracted from any kind of tissue and subsequently enzymatically digested to the nucleosides. Subsequently, a known amount of the stable isotope-labeled standard nucleoside is added. In the HPLC-MS analysis one signal for the natural (light) and one for the synthetic (heavy) compound is detected in each experiment. Quantification is performed by comparing the integrals of the specific high resolution ion current of the natural compound (amount to be determined) with their corresponding heavy atom labeled derivative (known amount).