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

Soňa Boháčová,Lenka Poštová Slavětínská,Zuzana Vaníková,Michal Hocek

doi:10.1039/c8ob01106k

Soňa Boháčová, Lenka Poštová Slavětínská + Show 2 more

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https://doi.org/10.1039/c8ob01106k

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Abstract

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.

Highlights

5-(Hydroxymethyl)cytosine (5hmC) is an epigenetic DNA base[1] which is an intermediate in active DNA demethylation[2] and an epigenetic signal regulating gene expression.[3,4] Several protected 5hmC 2′-deoxyribonucleoside building blocks for the phosphoramidite synthesis of modified oligonucleotides (ONs) have been reported,[5] as well as the synthesis and use of the corresponding unprotected 5hmC 2′deoxyribonucleoside triphosphate in the polymerase synthesis of modified DNA.[6]
We have recently reported the use of NB15 or 6-nitropiperonyl (NP)[16] caged 5-hydroxymethyluracil Deoxyribonucleoside triphosphates (dNTPs) for the polymerase synthesis of photocaged DNA that can release 5hmU upon irradiation by UV or visible light
The silylprotected deoxyuridines 1a and 1b were converted to the corresponding deoxycytidines 2a and 2b in two steps consisting of an activation of the oxo group through reaction with 2,4,6-triisopropyl-benzenesulfonyl chloride and DMAP, followed by nucleophilic substitution using ammonia

Summary

Introduction

5-(Hydroxymethyl)cytosine (5hmC) is an epigenetic DNA base[1] which is an intermediate in active DNA demethylation[2] and an epigenetic signal regulating gene expression.[3,4] Several protected 5hmC 2′-deoxyribonucleoside building blocks for the phosphoramidite synthesis of modified oligonucleotides (ONs) have been reported,[5] as well as the synthesis and use of the corresponding unprotected 5hmC 2′deoxyribonucleoside triphosphate (dChmTP) in the polymerase synthesis of modified DNA.[6]. Caged triphosphates dCNBTP and dCNPTP as well as the uncaged dChmTP were tested as substrates for DNA polymerases.[18] The primer extension (PEX) reaction was studied using a 19-mer template (for the sequences of ONs, see Table S2 in the ESI†) encoding for the incorporation of one modified dCR followed by three guanines.

Results

Conclusion