DNA modifications on pyrimidine nucleobases play diverse roles in biology such as protection of bacteriophage DNA from enzymatic cleavage, however, their role in the regulation of transcription is underexplored. We have designed and synthesized a series of uracil 2'-deoxyribonucleosides and 5'-O-triphosphates (dNTPs) bearing diverse modifications at position 5 of nucleobase, including natural nucleotides occurring in bacteriophages, α-putrescinylthymine, α-glutaminylthymine, 5-dihydroxypentyluracil, and methylated or non-methylated 5-aminomethyluracil, and non-natural 5-sulfanylmethyl- and 5-cyanomethyluracil. The dNTPs bearing basic substituents were moderate to poor substrates for DNA polymerases, but still useful in primer extension synthesis of modified DNA. Together with previously reported epigenetic pyrimidine nucleotides, they were used for the synthesis of diverse DNA templates containing a T7 promoter modified in the sense, antisense or in both strands. A systematic study of the in vitro transcription with T7 RNA polymerase showed a moderate positive effect of most of the uracil modifications in the non-template strand and some either positive or negative influence of modifications in the template strand. The most interesting modification was the non-natural 5-cyanomethyluracil which showed significant positive effect in transcription.

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Homologues of natural epigenetic pyrimidine nucleosides and nucleotides were designed and synthesized. They included 5-ethyl-, 5-propyl-, 5-(1-hydroxyethyl)-, 5-(1-hydroxypropyl)- and 5-acetyl- and 5-propionylcytosine and -uracil 2'-deoxyribonucleosides and their corresponding 5'-O-triphosphates (dNXTPs). The epimers of 5-(1-hydroxyethyl)- and 5-(1-hydroxypropyl)pyrimidine nucleosides were separated and their absolute configuration was determined by a combination of X-ray and NMR analysis. The modified dNXTPs were used as substrates for PCR synthesis of modified DNA templates used for the study of transcription with bacterial RNA polymerase. Fundamental differences in transcription efficiency were observed, depending on the various modifications. The most notable effects included pronounced stimulation of transcription from 5-ethyluracil-bearing templates (200% transcription yield compared to natural thymine) and an enhancing effect of 5-acetylcytosine versus inhibiting effect of 5-acetyluracil. In summary, these results reveal that RNA polymerase copes with dramatically altered DNA structure and suggest that these nucleobases could potentially play roles as artificial epigenetic DNA nucleobases.

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We report proof of principle biomimetic switching of transcription in vitro through non-natural chemical reactions in the major groove of DNA templates. Photocaged DNA templates containing nitrobenzyl-protected 5-hydroxymethyluracil or - cytosine permitted no transcription with E. coli RNA polymerase (OFF state). Their irradiation with 400 nm light resulted in DNA templates containing hydroxymethylpyrimidines, which switched transcription ON with a higher yield (250-350%) compared to non-modified DNA. Phosphorylation of templates containing 5-hydroxymethyluracil (but not 5-hydroxymethylcytosine) then turned transcription OFF again. It is the first step towards artificial bioorthogonal chemical epigenetics.

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