Formation of left-handed helices by C2'-fluorinated nucleic acids under physiological salt conditions.

Recent findings in cell biology have rekindled interest in Z-DNA, the left-handed helical form of DNA. We report here that two minimally modified nucleosides, 2'F-araC and 2'F-riboG, induce the formation of the Z-form under low ionic strength. We show that oligomers entirely made of these two nucleosides exclusively produce left-handed duplexes that bind to the Zα domain of ADAR1. The effect of the two nucleotides is so dramatic that Z-form duplexes are the only species observed in 10 mM sodium phosphate buffer and neutral pH, and no B-form is observed at any temperature. Hence, in contrast to other studies reporting formation of Z/B-form equilibria by a preference for purine glycosidic angles in syn, our NMR and computational work revealed that sequential 2'F…H2N and intramolecular 3'H…N3' interactions stabilize the left-handed helix. The equilibrium between B- and Z- forms is slow in the 19F NMR time scale (≥ms), and each conformation exhibited unprecedented chemical shift differences in the 19F signals. This observation led to a reliable estimation of the relative population of B and Z species and enabled us to monitor B-Z transitions under different conditions. The unique features of 2'F-modified DNA should thus be a valuable addition to existing techniques for specific detection of new Z-binding proteins and ligands.

Telomeric i-motifs and C-strands inhibit parallel G-quadruplex extension by telomerase.

Telomeric C-rich repeated DNA sequences fold into tetrahelical i-motif structures in vitro at acidic pH. While studies have suggested that i-motifs may form in cells, little is known about their potential role in human telomere biology. In this study, we explore the effect of telomeric C-strands and i-motifs on the ability of human telomerase to extend G-rich substrates. To promote i-motif formation at neutral pH, we use telomeric sequences where the cytidines have been substituted with 2'-fluoroarabinocytidine. Using FRET-based studies, we show that the stabilized i-motifs resist hybridization to concomitant parallel G-quadruplexes, implying that both structures could exist simultaneously at telomeric termini. Moreover, through telomerase activity assays, we show that both unstructured telomeric C-strands and telomeric i-motifs can inhibit the activity and processivity of telomerase extension of parallel G-quadruplexes and linear telomeric DNA. The data suggest at least three modes of inhibition by C-strands and i-motifs: direct hybridization to the substrate DNA, hybridization to nascent product DNA resulting in early telomerase dissociation, and interference with the unique mechanism of telomerase unwinding and extension of a G-quadruplex. Overall, this study highlights a potential inhibitory role for the telomeric C-strand in telomere maintenance.