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

Hardin, Charles C.; Zarling, David A.; Puglisi, Joseph D.; Trulson, Mark O.; Davis, Peter W.; Tinoco, Ignacio

doi:10.1021/bi00390a044

Cited by 33 publications

(56 citation statements)

References 31 publications

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“…Polyclonal sera were obtained from female New Zealand White rabbits or female BALB/c mice inoculated either with 40-60 ,ug of chemically brominated poly[r(C-G)] {referred to as Br-poly[r(C-G)]} or with poly[r(C-G)] of -80 base pairs in length (5). Primary and booster inoculations contained polynucleotides complexed with methylated bovine serum albumin (Sigma) emulsified in Freund's complete adjuvant (Behring, Summerville, NJ).…”

Section: Methodsmentioning

confidence: 99%

“…(5). A detailed report on the extent of bromine modification used to elicit the antibodies used here and its effect on their immunochemical specificities will be published elsewhere (C.C.H., D.A.Z., S. K. Wolk, W. Ross, and I. Tinoco, Jr., unpublished data).…”

Section: Methodsmentioning

confidence: 99%

“…Direct RIA or competition assays with unlabeled polymers or drugs were as described (3)(4)(5) Cytological Preparations, Immunofluorescent Staining, and Photomicroscopy. Crithidia luciliae and Crithidia fasciculata were propagated in culture medium containing 37 mg of brain-heart infusion per ml (Difco) and 25 ,ug of hemin per ml.…”

Section: Methodsmentioning

confidence: 99%

“…The phosphodiester backbone and 2' hydroxyl groups in Z-RNA may be in close enough juxtaposition to specify a 15-to 20-A antibody-binding site (for further discussion, see refs. [3][4][5]. Furthermore, these groups may also be in the vicinity of the exposed portions of the bases on the major convex surface, possibly including the atoms at the 8 position on guanosine and the 5 position on cytosine.…”

Section: Methodsmentioning

confidence: 99%

“…Recently we have shown that chemical bromination of poly[r(C-G)] stabilizes synthetic Z-RNA under physiological conditions (5). Therefore, we sought to test the immunogenicity of Z-RNA with the aim of producing specific immunochemical probes for detecting putative cellular Z-RNA.…”

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confidence: 99%

See 4 more Smart Citations

Cytoplasmic Z-RNA.

Zarling¹,

Calhoun²,

Hardin³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Preor nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or singlestranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.Alternating purine-pyrimidine sequences having a strong potential to adopt stable leftghanded Z-conformations (e.g., in negatively supercoiled DNAs) are repetitive elements in prokaryotic and eukaryotic DNA genomes. Some of these sequences are transcribed into RNA. The in vivo conformational states of these nucleic acids and their functions are not currently known (for reviews, see refs. 1 and 2).Z-DNA sequences exist in deproteinized negatively supercoiled plasmids, viral DNAs, and fixed (protein-deplet- A detailed report on the extent of bromine modification used to elicit the antibodies used here and its effect on their immunochemical specificities will be published elsewhere (C.C.H., D.A.Z., S. K. Wolk, W. Ross, and I. Tinoco, Jr., unpublished data). Polynucleotides were extensively dialyzed against TE buffer (10 mM Tris HCl, pH 7.4/0.1 mM EDTA). Polynucleotide kinase labeling was by the procedure of Silberklang et al. (6).Radioimmunoassay (RIA) and Nitrocellulose Filter Binding Assays. Direct RIA or competition assays with unlabeled polymers or drugs were as described (3-5). The standard RIA buffer was 200 mM NaCl/40 mM Tris HCl, pH 7.5/4 mM EDTA. Reaction mixtures (20 ,ul) containing 20 ng of polynucleotide substrate were equilibrated for 5 min at 37°C before adding 5 ,ul of the first antibody (either rabbit or mouse). Primary reaction mixtures were incubated for 1 hr at 37°C; this was followed by addition of 1 ,ul of the appropriate second antibody, either affinity-purified goat anti-rabbit IgG or goat anti-mouse IgG (2.4 mg/ml). After an additional hour at 37°C, immune complexes were washed (three times) with RIA buffer, centrifuged at 15,000 x g (10°C), resuspended, and then assayed for radioactivity in 5 ml of Aquasol-2 (New England Nuclear) in a liquid scintillation counter (Beckman). A machine background of 15-35 cpm was subtracted. Filter-binding assays were performed using Millititer nitrocellulose filters on a vacuum manifold (Millipore). Reactions were as described for RIA, except for the omission of the second antibody, and were terminated by the...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Cytoplasmic Z-RNA.

Zarling¹,

Calhoun²,

Hardin³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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Effects of nucleotide bromination on the stabilities of Z‐RNA and Z‐DNA: A molecular mechanics/thermodynamic perturbation study

et al. 1989

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The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.

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Z‐RNA: The solution NMR structure of r(CGCGCG)

1990

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Left-handed double-helical Z-RNA has been studied using the ribohexanucleotide pentaphosphate r(CpGpCpGpCpG). One-dimensional and two-dimensional proton nmr experiments were used to probe the structural details of the left-handed helix in concentrated sodium perchlorate solution. In 1M NaClO4 the RNA adopts the normal A-form double helix, and in 6M NaClO4 it is nearly all in the Z form. In 4M NaClO4 it exists as nearly equal parts of A form and Z form. Resonances corresponding to both A and Z form appear in the nmr spectrum, indicating that the duplex exchanges slowly between forms. Spin-spin coupling constants between protons in the ribose rings were used to determine the sugar-pucker conformations of the individual nucleotides. Quantitative nuclear Overhauser experiments were used to determine proton-proton distances within the nucleoside, and from these distances values for the glycosidic torsion angle were determined. The results show that the cytidines adopt C2'-endo sugar puckers (S type) with pseudo-rotation phase values (P) of approximately 165 degrees. The bases are in the anti conformation, with chi values of approximately -140 degrees. The internal guanosines adopt C3'-endo sugar puckers (N type) with P approximately 18 degrees, while the 3'-terminal guanosine ribose exists in an equilibrium between S- and N-type conformations. All three guanosine bases adopt the syn conformation, with chi approximately 70 degrees. The results indicate that the solution structure of Z-RNA is very similar to that of Z-DNA.

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

Cited by 33 publications

References 31 publications

Cytoplasmic Z-RNA.

Cytoplasmic Z-RNA.

Effects of nucleotide bromination on the stabilities of Z‐RNA and Z‐DNA: A molecular mechanics/thermodynamic perturbation study

Z‐RNA: The solution NMR structure of r(CGCGCG)

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