Observation of Z-DNA Structure via the Synthesis of Oligonucleotide DNA Containing 8-Trifluoromethyl-2-Deoxyguanosine

Abstract

This article contains detailed synthetic protocols for the preparation of DNA oligonucleotides containing 8-trifluoromethyl-2′-deoxyguanosine (CF3dG) and their application to observe Z-DNA structure in vitro and in living HeLa cells. First, using a catalytic system consisting of FeSO4, H2SO4, and H2O2 in DMSO, we achieved a one-step synthesis of CF3dG through a radical reaction between deoxyguanosine (dG) and CF3I, with a yield of 45%. We then obtained the 3′-phosphoramidite of CF3dG through a routine three-step procedure. Next, we employed the CF3dG phosphoramidite monomer in the synthesis of oligonucleotides on a solid-phase DNA synthesizer. Finally, we used the CF3dG-modified DNA oligonucleotides to observe Z-DNA structure in vitro and in living HeLa cells through 19F NMR spectroscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of CF3dG phosphoramidites. Basic Protocol 2: Preparation of CF3dG-modified DNA oligonucleotides. Basic Protocol 3: Evaluation of CF3dG stabilization of Z-DNA structure by CD spectroscopy. Basic Protocol 4: Investigation of Z-DNA structure in vitro and in HeLa cells with CF3dG-modified DNA oligonucleotides and 19F NMR spectroscopy.