Stabilization of Z-RNA by chemical bromination and its recognition by anti-Z-DNA antibodies

Limited chemical bromination of poly(r(C-G)) (32% br/sup 8/G, 26% br/sup 5/C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. Raising the ionic strength to 6 M NaBr or NaClO/sub 4/ results in a transition in Br-poly(r(C-G)) to a Z-RNA (Z/sub R/) conformation as judged by CD spectroscopy. /sup 1/H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37/sup 0/C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', AND CH1' resonances in both the A- and Z-forms. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 /sup 0/C. /sup 31/P NMR spectra show six to eight resonances spread over a 1.8 ppm range whose chemical shifts are also consistent with an equilibrium mixture of A- and Z-RNAs. Radioimmunoassay and nitrocellulose filter binding competition experiments were performed to determine the extent of recognition of Br-poly(r(C-G))more » by anti-Z-DNA antibodies. Competition RIA experiments verify the presence of a Z-DNA-like determinant in left-handed Br-poly(r(C-G)) at physiological NaCl concentration. In summary, these spectroscopic and immunochemical studies demonstrate that under conditions of conformational stress (i.e., containing brominated nucleosides) left-handed Z-RNA is stable and is specifically recognized by proteins at physiological temperature and ionic strength.« less