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

Abstract

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) 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. More extensive bromination of poly[r(C-G)] (> 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37.degree. C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1'', and CH1'' resonances in both the A- and Z-forms. GH8 .dblarw. GH1'' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. 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.degree. C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., and Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York]. 31P 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)] by anti-Z-DNA antibodies. The polyclonal rabbit anti-Br-poly[d(C-G)] IgG preparations T4 and Z6 [Zarling, D. A., Arndt-Jovin, D. J., Robert-Nicoud, M., McIntosh, L. P., Thomae, R., and Jovin, T. M. (1984a) J. Mol. Biol. 176, 369-415; Zarling, D. A., Arndt-Jovin, D. J., McIntosh, L. P., Robert-Nicoud, M., and Jovin, T. M. (1984b) J. Biomol. Struct. Dyn. 1, 1081-1107] specifically recognize the Z-form of Br-poly[r(C-G)], although the binding affinities are lower for Z-RNA than for various forms of Z-DNA. Competition RIA experiments verify the presence of a Z-DNA-like determinant in left-handed Br-poly[r(C-G)] at physiological NaCl concentgration. The phosphate anion specifically inhibits recognition of Z-RNA by anti-Z-DNA IgGs, consistent with recognition of a phosphodiester backbone determinant. 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.