Utilisation of enzymatic labelling with 4-aminophtalimide and 4-hydroxybenzylideneimidazolinone fluorescent derivates for monitoring of DNA-protein interaction

0429324 - ÚOCHB 2015 CZ eng C - Conference Paper (international conference)
Orság, Petr - Pivoňková, Hana - Riedl, Jan - Hocek, Michal - Fojta, Miroslav
Utilisation of enzymatic labelling with 4-aminophtalimide and 4-hydroxybenzylideneimidazolinone fluorescent derivates for monitoring of DNA-protein interaction.
Chemistry of Nucleic Acid Components. 16th Symposium. Praha: Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 340-341. Collection Symposium Series, 14. ISBN 978-80-86241-50-0.
[Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014]
R&D Projects: GA ČR GBP206/12/G151; GA ČR(CZ) GAP301/11/2076
Grant - others:MŠMT(CZ) CZ.1.07/2.3.00/30.0019
Institutional support: RVO:61388963 ; RVO:68081707
Keywords : DNA-protein interaction * API * HBI
Subject RIV: CC - Organic Chemistry; BO - Biophysics (BFU-R)

The 5’-substituted deoxycytosine triphosphates with conjugated solvatochromic derivates of 4-aminophtalimide (API) and derivates of the green fluorescent protein, 4-hydroxybenzylideneimidazolinone (HBI) were synthetized and successfully tested for enzymatic incorporation using primer extension assay. Site specifically labelled oligonucleotide probes were prepared and tested for interaction with p53 and SSB proteins, displaying distinct DNA-binding properties. The incorporation of multiple fluorescent labels did not interfere with natural protein binding and protein interaction leaded in both cases the to the gradual ratiometric increase of the fluorescence intensity moreover accompanied with the changes of the fluorescence emission spectra profile. Neither effect was observed after incubation with BSA, non-DNA binding protein, confirming the specificity of the interaction. Modified nucleoside triphosphates with conjugated fluorescence labels derivates of API and HBI can be used as substrates for preparation of the specific oligonucleotide labelled probes and provide the novel tool for studying and monitoring the DNA-protein interaction.
Permanent Link: http://hdl.handle.net/11104/0234541