Guanine polynucleotides are self-antigens for human natural autoantibodies and are significantly reduced in the human genome.

In the course of investigating anti-DNA autoantibodies, we examined IgM and IgG antibodies to poly-G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20-mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo-nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti-G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170,000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain-associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti-G20 antibodies; so natural anti-G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti-G20 antibodies in human health and disease and of runs of G20 in the human genome.

Enhanced translocation of poly(dt)45 through an α-hemolysin nanopore by binding with antibody.

The translocation time of poly(dT)(45) through an α-hemolysin pore was reduced in the presence of a DNA-binding Fab fragment. The Fab acts as a rudder to steer the DNA into the pore.

A five-amino-acid motif in the undefined region of the TLR8 ectodomain is required for species-specific ligand recognition.

Toll-like receptors play important roles in regulating immunity against microbial infections. Toll-like receptor 8 (TLR8) belongs to a subfamily comprising TLR7, TLR8 and TLR9. Human TLR8 mediates anti-viral immunity by recognizing ssRNA viruses, and triggers potent anti-viral and antitumor immune responses upon ligation by synthetic small molecular weight ligands. Interestingly, distinct from human TLR8, mouse TLR8 was not responsive to ligand stimulation in the absence of polyT-oligodeoxynucleotides (polyT-ODN). The molecular basis for this distinct ligand recognition is still unclear. In the present study, we compared the activation of TLR8 from different species including mouse, rat, human, bovine, porcine, horse, sheep, and cat by ligand ligations. Only the TLR8s from the rodent species (i.e., mouse and rat TLR8s) failed to respond to ligand stimulation in the absence of polyT-ODN. Multiple sequence alignment analysis suggested that these two rodent TLR8s lack a five-amino-acid motif that is conserved in the non-rodent species with varied sequence. This small motif is located in an undefined region of the hTLR8 ectodomain, immediately following LRR-14. Deletion mutation analysis suggested that this motif is essential for the species-specific ligand recognition of hTLR8, whereas it is not required for self-dimerization and intracellular localization of this receptor.

Quantification of UVR-induced DNA damage: global- versus gene-specific levels of thymine dimers.

The induction and repair of DNA damage has been shown to occur heterogeneously throughout the mammalian genome. As a consequence, analysis of these parameters at a global genome level may not reflect important gene-level events. Few techniques have been established to explore quantitatively gene-specific DNA damage and repair. Most of these are polymerase chain reaction (PCR)-based assays and are relatively insensitive, relying on decreased PCR amplification arising from damage in template DNA. We have developed a quantitative assay that combines specific immunocapture of damaged DNA by an antiserum specific for thymine dimers (IgG479), with PCR amplification of a 149 bp fragment of the human H-ras proto-oncogene. Quantification of DNA damage was based upon proportionality between the amount of the PCR product and the initial amount of damage. Detection of thymine dimers was possible with nanogram amounts of genomic DNA and increased in a linear, dose-responsive manner. Using this assay, gene-level induction of thymine dimers was shown to be directly proportional to levels induced in the global genome of ultraviolet radiation (UVR)-exposed, extracted DNA as measured by gas chromatography-mass spectrometry (GC-MS). This result suggests that global damage assessments do indeed reflect gene-level events although we predict that this relationship may not be maintained when applied to a cellular system. These findings demonstrate the suitability of this approach to the detection of UVR-induced DNA damage at the level of individual genes.

Evaluation of antibodies to thymine ROS-modified DNA poly(dT), in patients with immunologic disorders: relationships to anti n-DNA and anti ENA autoantibodies.

BACKGROUND: Hydroxyl radical, one of the most potent of all reactive oxygen species, is known to modify adenine and thymine in cellular DNA, producing some modified DNA fragments (ROS-DNA) with different antigenic properties. Despite several in vitro studies about ROS-DNA, data regarding their clinical significance are scanty. The aim of our study was to seek out the presence and clinical significance of the anti poly(dT) auto-antibodies in a group of patients suspected of autoimmune disease. METHODS: We initially evaluated more than 900 consecutive sera of hospitalized patients (range age from 6 to 70 yrs) referred to our laboratory during 18 months. Anti n-DNA, anti-ENA and poly(dT) autoantibodies were performed on 158 ANA positive sera and 28 ANA negative sera from patients strongly suspected of rheumatic diseases or affected by HCV infection. RESULTS: Anti poly (dT) were found in 22 out of 186 sera. As regards the clinical evaluation, 8 patients were affected by SLE, 5 by Scleroderma, 3 by HCV-related chronic hepatitis, 4 by recurrent abortions (without presence of the anti-cardiolipin antibodies and other clinical symptoms). In two patients the ACR criteria and the clinical aspects did not allow a definite diagnosis. Anti-Histones were detected in 18 out of 22 poly (dT) positive patients. CONCLUSIONS: Our data suggest that anti poly(dT) autoantibodies are sensitive markers of various autoimmune diseases, but with a minor specificity as compared to anti n-DNA for the diagnosis of SLE.

Monoclonal antibody against DNA adducts with osmium structural probes.

Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure. A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice. OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins. The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid). The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy. The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen). A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane.

Reactive oxygen species modified thymine and poly(dT) present unique epitope for human anti-DNA autoantibodies.

Hydroxyl radical, one of the most potent of all reactive oxygen species has been implicated in many human degenerative diseases and is known to modify adenine and thymine in cellular DNA. In the present studies, adenine, thymine and their synthetic homopolymers poly(dA), poly(dT) were ROS-modified and subsequently used as inhibitors of native DNA binding to human anti-DNA autoantibodies. Besides nDNA, modified thymine and poly(dT) were effective inhibitors of DNA-anti-DNA antibody interaction. The relative affinity of ROS-modified poly(dT) was better than that of native DNA. Visual detection of modified thymine and poly(dT) binding to affinity purified anti-DNA IgG by an indirect band shift assay support competition inhibition data. The enhanced recognition of ROS-DNA by anti-DNA autoantibodies, as reported earlier, could be due to the ROS-induced modification of thymine.

Sequence and structure specific antibodies from phage display libraries.

A large combinatorial phage display library was panned against five nucleic acid antigens, calf thymus DNA, poly[d(GC)], poly[d(AT)], poly(dA) x poly(dT) and poly(rA) x poly(dT). After the third and fourth rounds of panning, many positive clones were selected against poly[d(GC)], poly(dA) x poly(dT) and poly(rA) x poly(dT). The specificity of these antibodies was tested by both direct and competitive solid phase radioimmune assays. All the clones derived from panning with poly[d(GC)] were non-specific and bound to all nucleic acids. The poly(rA) x poly(dT) derived clones were specific for single-stranded nucleic acids, with some sequence preferences, and the poly(dA) x poly(dT) derived clones showed considerable specificity for this antigen. The sequences of these phage-derived antibodies showed no similarities with DNA-binding antibodies from other sources. Even after six rounds of panning no positive clones were detected which bound to poly[d(AT)] and after seven rounds only two were derived from panning with calf thymus DNA. Therefore, sequence- and structure specific antibodies can be recovered from phage display libraries but not all sequences may be represented in the repertoire.

Monoclonal antibodies targeted to alpha-oligonucleotides. Characterisation and application in nucleic acid detection.

The aim of the present study was to test the antigenicity of alpha-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize alpha-deoxyribonucleotides. Two were raised against a poly(alpha-dT) sequence and specifically recognized the alpha-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as alpha-nucleotides and, surprisingly, only recognized the alpha-dG nucleotide. For all four Mabs, no cross reactivity was observed with beta-oligonucleotides. These Mabs were reactive with alpha-oligonucleotide sequences whether these sequences were single-stranded or hybridized to DNA or RNA. The four Mabs were tested in a sandwich hybridization assay that consisted of an alpha-oligonucleotide (for target sequence recognition), one of the four Mabs (for recognition of the hybridized alpha-oligonucleotide), and goat anti-mouse antibody conjugated to horse radish peroxidase (HRP) (for detection). One of the monoclonal antibodies, Mab 2E11D7, was directly conjugated to HRP and used in sandwich hybridization to detect PCR fragments of HPV 18 DNA. The sensitivity of this reaction was 1 pg of plasmid DNA containing the HPV 18 fragment. The specificity of the detection was demonstrated using HPV 6/11 and 16 DNA sequences.

Elucidation of anti-ssDNA autoantibody BV 04-01 binding interactions with homooligonucleotides.

Binding interactions of various synthetic oligohomonucleotides with anti-ssDNA autoantibody BV 04-01 (IgG2b, kappa) and the corresponding single-chain antibody (SCA) 04-01/212 were studied. Oligonucleotide binding to IgG or SCA resulted in quenching of the protein's tryptophan fluorescence permitting direct assessment of ligand binding under equilibrium conditions. The effect of oligothymidylate length, (dT)n, on tryptophan quenching was evaluated. The equilibrium dissociation constants (Kd) for the binding of (dT)6 and (dT)8 were the same [(1.3 +/- 0.02) x 10(-7) M], while decreasing the length of the oligothymidylate to (dT)3 increased the Kd an order of magnitude. To assess base specificity, the comparative binding of other hexahomonucleotides was examined. Neither (dA)6 nor (dC)6 showed measurable binding, while the dissociation constant for (dG)6 was (7.1 +/- 0.3) x 10(-7) M. Fluorescence lifetime quenching data correlated with the steady-state binding results and indicated that the quenching process contains both dynamic and static components. The ability of BV 04-01 to bind (dT)6 and (dG)6 nucleotides was further supported by fluorescence anisotrophy studies with fluorescein-labeled hexadeoxynucleotides. Various levels of tryptophan fluorescence quenching upon titration with oligothymidylates of different length, as well as the similar affinities for (dT)6 and (dG)6, supported the concept that the groove-type binding pocket in BV 04-01 consists of binding subsites that cooperatively adapt for efficient binding of oligonucleotides.

The relationship between autoantibodies and intrauterine growth retardation in hypertensive disorders of pregnancy.

Abnormal levels of autoantibodies have recently been demonstrated in patients with hypertensive disorders of pregnancy and patients whose fetuses have intrauterine growth retardation. We determined total immunoglobulin levels (immunoglobulin G, immunoglobulin M, and immunoglobulin A) and a broad panel of autoantibodies (six antiphospholipid, four antihistone, and four antipolynucleotide antibodies) in 50 normotensive pregnant females, 19 patients with preeclampsia, and 18 patients with chronic hypertension to examine the relationship to intrauterine growth retardation. Mothers who were delivered of infants with intrauterine growth retardation demonstrated significantly more autoantibody abnormalities in the two hypertensive groups and in the normotensive control group as compared with patients delivered of appropriately grown infants. The most frequently observed autoantibody abnormalities were antiphospholipid antibodies and the most frequently observed among those were immunoglobulin G isotypes. Total immunoglobulin levels in both hypertensive and normotensive groups were identical. These results suggest a close association between the degree of B-cell activation and both severity of hypertensive diseases and development of intrauterine growth retardation in their offspring.

Specific binding to methylated polynucleotides in monoclonal anti-poly(dT) antibodies from autoimmune mice.

Six monoclonal antibodies (mAbs) reactive to synthetic polynucleotide, poly(dT), were established from spontaneous autoimmune MRL/MpJ-lpr/lpr mice and male BXSB mice by spleen cell hybridization method, and were analyzed for cross-reactivity with polydeoxy-5-methylcytidylic acid [poly(dmC)] in comparison with its unmethylated counterpart poly(dC). By direct binding tests, these mAbs, all of which had preponderant binding activity to poly(dT) relative to poly(dU), were divided into two groups: (i) four mAbs showing reactivity to poly(dmC) as well as to natural DNA preparations and (ii) two mAbs with limited reactivity to poly(dT) but no binding to poly(dmC) or natural DNAs. Inhibition binding tests with these synthetic polynucleotides demonstrated that one mAb (TP-A9) in the first group reacted specifically to poly(dmC), as well as to poly(dT). In the second group, one mAb (TP-B5) showed highly specific reactivity to poly(dT) that could not be inhibited by poly(dU), poly(dC), or poly(dmC). However, another mAb (TP-C8) in the second group showed reactivity to poly(dT) that could be inhibited by poly(dU) as well as by poly(dT). Thus, these findings indicate that monoclonal anti-poly(dT) antibodies can cross-react with poly(dmC) with different specificities and suggest that the methylated base may be one of the major antigenic sites of DNA molecules recognized by anti-DNA antibodies spontaneously produced in autoimmune mice.

Cross-reactions of nucleic acids with monoclonal antibodies to phosphatidylinositol phosphate and cholesterol.

Four monoclonal IgM antibodies to phosphatidylinositol phosphate (PIP), four antibodies to cholesterol and one antibody to liposomes containing phosphatidylcholine, cholesterol and dicetyl phosphate were tested for reactivity with denatured DNA. Three of four antibodies to PIP cross-reacted strongly with denatured DNA. The other antibodies did react with denatured DNA but only very weakly. The binding to DNA was competed by synthetic polynucleotides. In competitive assays, one of the anti-PIP antibodies was particularly reactive with poly(dT) and another with poly(I) and poly(dG). Binding of an anti-cholesterol antibody to ssDNA was also inhibited by poly(I) and poly(dG). Two of the anti-PIP antibodies were also reactive with mononucleotides, and all four bound inositol hexaphosphate. High concns of nucleosides did not compete for binding, indicating that phosphate is involved in the binding site. Phospholipids, particularly those containing inositol phosphate, also competed for binding to DNA, but to varying extents, indicating a variable overlap in the antibody binding site for DNA and phospholipid determinants. These antibodies, induced by immunization with liposomes, showed cross-reactivity characteristics often found with certain types of autoantibodies, but they did not bear the H130 idiotype, which was identified on IgM anti-DNA autoantibodies from MRL-lpr/lpr mice.

Nucleic acid-binding specificity and idiotypic expression of canine anti-DNA antibodies.

Serum samples collected from eleven lupus dogs during an active phase of the disease all bound native and denatured DNA, poly(dT), poly(I) and poly(G). Nine bound poly(C); 10 bound poly(U); and 3 bound poly(A). Sera from 22 normal dogs were negative with all of these antigens. The canine sera were also probed for the presence of three idiotypic markers, one related to human lupus anti-DNA antibodies and two related to murine lupus antibodies. One canine lupus serum expressed idiotopes related to murine anti-DNA idiotype (Id) termed H130: (a) the canine serum bound to anti-H130 anti-Id; (b) it inhibited the binding of anti-H130 Id to its homologous Id; and (c) the anti-H130 Id inhibited the binding of the canine serum to DNA. These findings suggest that anti-DNA variable regions exhibit interspecies similarities, probably reflecting the conservation of the encoding gene segments through evolution.

Application of the molecular replacement method to multidomain proteins. 2. Comparison of various methods for positioning an oriented fragment in the unit cell.

The capabilities of several different methods to determine the correct translation of a model for the application of the molecular replacement method of structure determination to multidomain proteins have been analyzed. The structure of the Fab fragment of the autoimmune anti-poly(dT)-specific antibody HED10 was determined using molecular replacement and provides an example for comparing different methods of determining the correct translation of the model and for evaluating the importance of the parameters used. Expansion to space group P1 and phasing with a correctly oriented randomly positioned model was found to be superior to either the Crowther-Blow translation function [Crowther & Blow (1967). Acta Cryst. 23, 544-548] or a brute-force search when only a small part of the molecule was used as a model.

Application of the molecular replacement method to multidomain proteins. 1. Determination of the orientation of an immunoglobulin Fab fragment.

Multidomain proteins provide special problems in the application of the molecular replacement method of structure determination. The structure of the Fab fragment from the autoimmune poly(dT)-specific antibody HED10 has been determined using molecular replacement. An analysis of the effects of varying the model and the parameters used in the rotation function indicates that dividing the molecule into individual relatively rigid domains simplifies interpretation of the results, and that the optimal parameters depend on the molecule under study.

Antigen inhibition of the interaction between murine monoclonal anti-idiotypic antibodies and human monoclonal anti-DNA antibodies.

Monoclonal anti-idiotypic (Id) antibodies to human monoclonal anti-DNA antibodies were obtained by a somatic cell hybridization. One, termed as D1E2, was directed to Id of anti-single-stranded (ss) DNA antibody (0-81) and the other, 1F5, to anti-double-stranded (ds) DNA antibody (NE-1). Each anti-Id antibody behaved like a mirror image of the corresponding antigens, when determined by competitive inhibition radioimmunoassay. Therefore, D1E2 and 1F5 are regarded as Ab2 beta or Ab2 gamma. These antibodies will make useful reagents to understand and manipulate the autoantibody production in human.

Immunochemical approaches to gene probe assays.

Antibodies specific for helical nucleic acids can be applied to assays for hybridized DNA and/or RNA. The assays can use either radioactive or nonradioactive detection systems. Antibodies specific for RNA-DNA hybrids are applicable to assays for measuring hybrid helices that are immobilized on plastic or nitrocellulose, whether the helices are preformed in solution or are formed on the solid-phase support. Alternatively, anti-RNA-DNA hybrid antibodies can be immobilized and used to capture hybrids formed in solution, resulting in an assay with a high signal-to-noise ratio. It has been applied to a test for the presence of ribosomal RNA of Campylobacter jejuni in biological samples.

The production of antibodies to DNA in normal mice following immunization with poly(ADP-ribose).

Monoclonal antibodies were prepared from C57/black mice which had been immunized with poly(ADP-ribose). As expected, some of these antibodies were very specific for poly(ADP-ribose) but, surprisingly, several bound to DNA as well. Analysis of the sera of these mice also showed elevated levels of antibodies to DNA. Monoclonal antibodies against poly(ADP-ribose) were also recovered from unimmunized NZB/W mice which provided an animal model for systemic lupus erythematosus(SLE). These antibodies also cross-reacted with DNA. Comparison of the specificities of the monoclonal antibodies from the two groups of mice showed some striking similarities. In particular, three out of 11 antibodies from the C57/black mice preferred poly(dT) as judged by a solid phase radioimmunoassay. Similarly, 10 out of 17 antibodies from the NZB/W group showed the same type of specificity pattern. These results demonstrate that anti-DNA antibodies can be induced by poly(ADP-ribose) and that some of the autoimmune DNA-binding antibodies found in SLE may result from exposure to poly(ADP-ribose).

Multiple serologic reactions and their relationship to clinical activity in systemic lupus erythematosus.

Analysis of the binding of serum from 56 systemic lupus erythematosus patients to native DNA (nDNA), denatured DNA (dDNA), poly I, poly(dT), RNA, and cardiolipin revealed multiple antigen binding in many of the sera. Raised levels of antibodies (IgG and/or IgM) to denatured DNA were found in the highest percentage (68%) of patients. A small subset of patients with multiple raised IgM antibodies, renal disease, and vasculitic skin rash was identified. No correlation between multiple serologic activity and clinical disease was found.