Glucosylated 5-Hydroxymethylpyrimidines as Epigenetic DNA Bases Regulating Transcription and Restriction Cleavage.

5-(ß-d-Glucopyranosyloxymethyl)-2'-deoxyuridine and -cytidine 5'-O-triphosphates were prepared and used for polymerase-mediated (primer extension or PCR) synthesis of DNA containing glucosylated 5-hydroxymethyluracil (5hmU) or 5-hydroxymethyluracil (5hmC). The presence of any glucosylated pyrimidines fully protected DNA from cleavage by type II restriction endonucleases. On the other hand, while the presence of glucosylated 5hmU completely inhibited transcription by bacterial (Escherichia coli) RNA polymerase, the DNA containing the corresponding glucosylated 5hmC allowed a similar level of transcription as natural DNA. This suggests different roles of these hypermodified bases in the epigenetic regulation of transcription in bacteriophages or kinetoplastid parasites. Consequently, enzymatic glucosylation of 5hmC-containing DNA can be used for tuning of transcription activity.

Photocaged 5-(Hydroxymethyl)pyrimidine Nucleoside Phosphoramidites for Specific Photoactivatable Epigenetic Labeling of DNA.

5-Hydroxymethylcytosine and uracil are epigenetic nucleobases, but their biological roles are still unclear. We present the synthesis of 2-nitrobenzyl photocaged 5-hydroxymethyl-2'-deoxycytidine and uridine 3'-O-phosphoramidites and their use in automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as primers for PCR to construct DNA templates modified in the promoter region that allowed switching of transcription through photochemical uncaging.