Manual Nucleic Acid Research. Future Development

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We can tell you exactly which particle you can use for that," he said. We can produce interferons, but not inflammatory cytokines, for example. This is like a letter or a word, like a text message that we send to the immune system.

The immune system will read your message and text back with the interferon. Materials provided by University of North Carolina at Charlotte. Note: Content may be edited for style and length. Science News. Halman, Ankit B. Shah, Emil F. Khisamutdinov, Marina A. Dobrovolskaia, Kirill A. Nano Letters , ; 18 7 : DOI: ScienceDaily, 26 July University of North Carolina at Charlotte.

Researchers report unraveling the immune recognition of nucleic acid nanoparticles: Particle-specific immune responses may be 'an alphabet for talking to the immune system'. Retrieved September 23, from www. Moreover, structural data on antibody-DNA complexes could be used in the design of antigens with improved specificity, which is of crucial importance to clinical diagnostics 18 , Synthetic antigens could allow establishment of previously unachievable standardization of the a-DNA assays and might open up the exciting possibility of treatment by specific binding and clearance of reactive a-DNAs Recently, other investigators explored rationally designed peptoid antigens for SLE diagnostics Our studies confirm high binding affinity of the new antigens compared to natural DNA.

We show that levels of autoantibodies to particular synthetic nucleic acid antigens in SLE differ among adults and children. The a-dsDNA profiles in SLE also differ from those in patients with another autoimmune disease, ANA-positive polyarticular juvenile idiopathic arthritis, indicating specificity. In addition, using computational methods, we identify specific interactions between dsDNA and corresponding antibodies. The major goal for this study was to develop a sensitive, specific and reproducible test for a-DNA in human samples.

ELISA is a straightforward and well-established assay that allowed us to study the effect of DNA sequence on binding of polyclonal antibodies in a time and cost- effective way 1. After washing, secondary antibodies, specific for human IgG or IgM and conjugated with peroxidase were added. After washing again to remove unbound detection antibodies, TMB substrate was added and the extent of the colorometric reaction was measured and compared among different antigens as a proxy for the amount of bound anti-nucleic acid antibodies; see Fig. Immobilization of antigen and blocking; Step 2.

Incubation with monoclonal antibody or plasma sample; Step 3. Incubation with substrate for color generation; measurement of absorbance. B General approach for the antigen development applied in this study. C Synthetic nucleic acid antigens used in this study. For the sequences of antigens, see Methods. We ensured that antibody binding to the antigens reached the binding equilibrium under the applied incubation conditions 1. S3 in Suppl.

Development of “CLAN” Nanomedicine for Nucleic Acid Therapeutics

Cutoff values for weak positive and positive signals were determined separately for each antigen by an arbitrary statistical method, i. In contrast to pSLE, fewer adults had elevated levels of a-D4 7. ELISA tests of patient samples grouped according to diagnosis. Absorbance values were corrected to total plasma protein determined by Bradford assay, see Methods.

In addition, the hierarchy of sample binding to the synthetic antigens varied. As shown in Supplementary Fig. To explore potential clinical application for a-D5 antibodies, we assessed correlations with clinical parameters, including disease flares and medications, using multi-parameter ordinary least squares OLS in R 28 , as described in Supplementary Information, Suppl.

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Neither a-D4 nor a-D5 levels correlated with medication use. Correlations were determined using OLS for independent groups, as described in Supplementary Information. Changes in disease activity versus changes of corresponding laboratory parameters over time longitudinal assay. All results are based on a total of 32 visits for 8 patients. Delta values were calculated by subtracting values of a defined parameter at each visit from the value from the previous visit. Resulting plots were analyzed in R. Among these, the structure of ED, a ssDNA-binding monoclonal antibody, complexed with the dinucleotide has been solved Taking into account previously reported cross-reactivity of a-ssDNA and a-dsDNA antibodies 30 , we reasoned that both types of complexes could share binding mechanisms.

The simulated system is solvated in a box of water with NaCl ions spheres , as described in Methods. B A close-up view of the dsDNA-antibody binding site, featuring the base pair, dominantly interacting with several amino acid residues labeled through stacking interactions arrows , and hydrogen bonds dashed lines. The error bars indicate the standard deviation computed during the averaging procedure. Note, that the computed values represent the interaction energy of the two nucleic acid side chains with the entire antibody complex.

To study binding specificity of the antibody for DNA, we mutated the initial base pair in the antigen to alternative variants in total, 40 variants were tested. Based on molecular modeling, the bound nucleotides adopted a conformation in which the nucleobase was twisted away from the sugar moiety. The predominant binding arises from the stacking interactions between thymidine and the W50 and W95 amino acid residues, the cytidine and Y32 arrow in the figure. The hydrogen bonds between thymidine and N95, and cytidine and K50 dashed lines in Fig. The average interaction energies of the antibody with the diverse base pairs are shown in Fig.

The time-dependence of energies in the three simulations are shown in Fig. S11 , which illustrates that the energy fluctuates steadily around some average value. Based on the results of ELISA and molecular modeling, we concluded that antigen D5 was the most reactive in binding DNA in pediatric and adult SLE, and that the structural basis for the recognition involved both the stacking interactions and hydrogen bonding between TC dinucleotide repeat of D5 and amino acids in antibodies. In our present work, we took a next step towards better understanding of autoantibodies to nucleic acids and towards an improved assay using novel synthetic DNA molecules.

The new antigens were also stable upon storage as individual molecules and after immobilization on microtiter plates data not shown.

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The major advantages of applying synthetic antigens are high homogeneity, controlled purity and most importantly, known sequence According to our studies, SLE patients had overall higher titer of antibodies toward sequence specific antigens, and only few patients had antibodies toward ATCG-mixed ds analogues without a distinguished pattern. Dose-response curves and studies of 21mer antigens additionally confirmed that target binding by a-DNA was sensitive to the nucleotide sequence of applied antigens.

Based on our results, it is possible that antibody reactivity toward D5 is a distinctive feature of SLE, with the highest activity in pediatric disease. One possible explanation for this could be the overexpression of D5 in SLE. However, the biological role of D5 and other sequence-controlled antigens requires more investigation. A combination of the methods described herein and of modern genomic technologies could be an exciting next step towards better understanding of a-DNA and their role in SLE.

This could be caused by coiling of the ss antigen into 3D shapes that may interact non-specifically Currently, there are conflicting reports on correlation between a-dsDNA and other ANA with clinical phenotypes of autoimmune diseases 9 , Most consistently reported associations are lupus nephritis, total disease activity index and flares in SLE, and chronic uveitis in oligoarticular JIA 9 , 33 — In this study, we hypothesized that sequence specific antibodies might correlate with a different subset of clinical phenotypes and help determine subgroups of patients based on their a-DNA status.

We focused on several aspects of increased antibody titers: correlation with other biomarkers or treatment at a single time-point disease onset , and correlation with flares during the treatment course. However, we found no correlation with other biomarkers including ANA, complement or anti-Smith antibodies. A recent report describes molecular subgroups in pSLE according to transcriptional analysis We speculate that a larger panel of synthetic nucleic acids might allow higher resolution of molecular subsets in a clinically practical way.

In most pSLE patients, upcoming exacerbations of disease were not clearly predicted by changes in common serological tests, including ANA. Clinically tested a-dsDNA levels decreased along with the treatment, but they did not rise prior to flares. However, increased titers of IgG antibodies to antigens were detected in all a-DNA positive patients used in this study prior to flares. Computational analysis of binding between short dsDNA antigens and monoclonal antibody shed light on the molecular basis of recognition.


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Overall, synthetic antigens described herein demonstrate high specificity, sensitivity and reproducibility in detection of a-DNAs in SLE, a disease known to be associated with a-DNAs, and for the first time enable a detailed structural study of sequence specificity of these autoantibodies. Other important advantages of the new synthetic antigens compared to natural heterogeneous molecules are: 1 Known specificity, including easily controlled sequence-specific binding of a-DNA antibodies; 2 Potential to determine individual antibody profiles which may have clinical implications; and 3 Potential to determine the biological role of a-DNA in SLE.

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Thus, rationally designed nucleic acids might become a basis for development of standard clinical and scientific assays for SLE and other autoimmune conditions where ANAs have been detected, such as mixed connective tissue disease and scleroderma. Detection of sequence-specific a-DNA cannot be applied alone; however, these assays could become a valuable supplement to existing laboratory tests and analysis of clinical manifestations, with the aim of improving diagnostics and treatment of autoimmune diseases.

The samples were characterized by clinical data, common serological biomarkers, treatment intensity and disease activity, see Supplementary Information. The methods were carried out in accordance with the relevant guidelines and regulations as stated in the Act on Processing of Personal Data adopted by the Danish Data Protection Agency on June 2 nd Personal data of patients was not used in this work. Therefore the informed consent from the individuals was not needed.

Nucleic Acid Research

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