Bioorthogonální reakce na DNA pro regulaci transkripce

Chakrapani, Aswathi

This PhD thesis describes the design and synthesis of photocaged or glucosylated derivatives of epigenetic 5-(hydroxymethyl)pyrimidine-modified nucleotides and DNA using chemical and enzymatic methods and the studies on their regulation of gene expression in bacterial (Escherichia coli RNA polymerase) in vitro transcription level. In the first part of the thesis, the design and syntheses of 5-(nitrobenzyloxymethyl)-2'-deoxyuridine (dUNB) and -cytidine (dCNB) phosphoramidites are described. These photocaged nucleoside phosphoramidite building blocks were used in the automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as forward primers in a polymerase chain reaction (PCR) to construct DNA templates modified at specific sites of the promoter region. The specific site photocaged DNA was then irradiated with light to result in the corresponding specific site 5-(hydroxymethyl)-modified DNA. Bacterial in vitro transcription studies of both the specific site photocaged and uncaged DNA were carried out. The incorporation of the photocaged epigenetic pyrimidine nucleotides at the -35 region of the promoter region of the template DNA inhibited transcription partially while the presence of the same outside the -35 region did not have any significant...

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