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

This article contains detailed synthetic protocols for the preparation of DNA oligonucleotides containing 8-trifluoromethyl-2'-deoxyguanosine (^CF3 dG) and their application to observe Z-DNA structure in vitro and in living HeLa cells. First, using a catalytic system consisting of FeSO₄ , H₂ SO₄ , and H₂ O₂ in DMSO, we achieved a one-step synthesis of ^CF3 dG through a radical reaction between deoxyguanosine (dG) and CF₃ I, with a yield of 45%. We then obtained the 3'-phosphoramidite of ^CF3 dG through a routine three-step procedure. Next, we employed the ^CF3 dG phosphoramidite monomer in the synthesis of oligonucleotides on a solid-phase DNA synthesizer. Finally, we used the ^CF3 dG-modified DNA oligonucleotides to observe Z-DNA structure in vitro and in living HeLa cells through ¹⁹ F NMR spectroscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of ^CF3 dG phosphoramidites Basic Protocol 2: Preparation of ^CF3 dG-modified DNA oligonucleotides Basic Protocol 3: Evaluation of ^CF3 dG stabilization of Z-DNA structure by CD spectroscopy Basic Protocol 4: Investigation of Z-DNA structure in vitro and in HeLa cells with ^CF3 dG-modified DNA oligonucleotides and ¹⁹ F NMR spectroscopy.