摘要：

ConspectusProtein–DNA interactionsare important in replication, transcription,repair, as well as epigenetic modifications of DNA, which involvemethylation and demethylation of DNA resulting in regulation of geneexpression. Understanding of these processes and chemical tools forstudying and perhaps even modulating them could be of great relevanceand importance not only in chemical biology but also in real diagnosticsand treatment of diseases.In the past decade, we have beenworking on development of synthesisof base-modified 2′-deoxyribo- or ribonucleoside triphosphates(dNTPs or NTPs) and their use in enzymatic synthesis of modified nucleicacids using DNA or RNA polymerases. These synthetic and enzymaticmethods are briefly summarized with focus on recent development andoutlining of scope, limitations, and further challenges. The mainfocus of this Account is on applications of base-modified nucleicacids in sensing of protein–DNA interactions, in covalent cross-linkingto DNA-binding proteins ,and in modulation of protein–DNA bindingand transcription. Several environment-sensitive fluorescent nucleotideswere incorporated to DNA probes which responded to protein bindingby light-up, changing of color, or lifetime of fluorescence. Usinga cyclodextrin-peptide transporter, fluorescent nucleotides can betransported through the cell membrane and incorporated to genomicDNA. Several dNTPs bearing reactive groups (i.e., vinylsulfonamideor chloroacetamide) were used for polymerase synthesis of DNA reactiveprobes which cross-link to Cys, His, or Lys in peptides or proteins.An attractive challenge is to use DNA modifications and bioorthogonalreactions in the major groove of DNA for modulation and switchingof protein–DNA interactions. We have systematically exploredthe influence of major-groove modifications on recognition and cleavageof DNA by restriction endonucleases and constructed simple chemicalswitches of DNA cleavage. Systematic study of the influence of major-groovemodifications on transcription with bacterial RNA polymerases revealednot only that some modified bases are tolerated, but also that thepresence of 5-hydroxymethyluracil or -cytosine can even enhance thetranscription (350 or 250% compared to native DNA). Based on theseresults, we have constructed the first chemical switch of transcriptionbased on photocaging of hydroxymethylpyrimidines in DNA by 2-nitrobenzylprotection (transcription off), photochemical deprotection of theDNA (transcription on), and enzymatic phosphorylation (only for 5-hydroxymethyluracil,transcription off). Although it has been so far demonstrated onlyin vitro, it is the proof-of-principle first step toward chemicalepigenetics.

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