Switching transcription with bacterial RNA polymerase through photocaging, photorelease and phosphorylation reactions in the major groove of DNA.

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.

Protected 2'-deoxyribonucleoside triphosphate building blocks for the photocaging of epigenetic 5-(hydroxymethyl)cytosine in DNA.

2'-Deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)cytosine (5hmC) protected with photocleavable groups (2-nitrobenzyl or 6-nitropiperonyl) were prepared and studied as substrates for the enzymatic synthesis of oligonucleotides and DNA containing a photocaged epigenetic 5hmC base. DNA probes containing photocaged or free 5hmC in the recognition sequence of restriction endonucleases were prepared and used for the study of the photorelease of caged DNA by UV or visible light at different wavelengths. The nitrobenzyl-protected dNTP was a slightly better substrate for DNA polymerases in primer extension or PCR, whereas the nitropiperonyl-protected nucleotide underwent slightly faster photorelease at 400 nm. However, both photocaged building blocks can be used in polymerase synthesis and the photorelease of 5hmC in DNA.

Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA.

Nucleosides, nucleotides and 2'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside monophosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).

5-(Hydroxymethyl)uracil and -cytosine as potential epigenetic marks enhancing or inhibiting transcription with bacterial RNA polymerase.

DNA templates containing 5-hydroxymethyluracil or 5-hydroxymethylcytosine were used in an in vitro transcription assay with RNA polymerase from Escherichia coli. A strong enhancement of transcription was observed from DNA containing the Pveg promoter whereas a decrease was observed from DNA containing the rrnB P1 promoter, suggesting that they may act as epigenetic marks.

Polymerase synthesis of photocaged DNA resistant against cleavage by restriction endonucleases.

5-[(2-Nitrobenzyl)oxymethyl]-2'-deoxyuridine 5'-O-triphosphate was used for polymerase (primer extension or PCR) synthesis of photocaged DNA that is resistant to the cleavage by restriction endonucleases. Photodeprotection of the caged DNA released 5-hydroxymethyluracil-modified nucleic acids, which were fully recognized and cleaved by restriction enzymes.