Complexes of DNA with fluorescent dyes are effective reagents for detection of autoimmune antibodies

Abstract

To date, there are multiple assays developed that detect and quantify antibodies in biofluids. Nevertheless, there is still a lack of simple approaches that specifically detect autoimmune antibodies to double-stranded DNA. Herein we investigate the potential of novel nucleic acid complexes as targets for these antibodies. This is done in a simple, rapid and specific immunofluorescence assay. Specifically, employing 3D nanostructures (DNA origami), we present a new approach in the detection and study of human antibodies to DNA. We demonstrate the detection of anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We tested the most potent non-covalent pairs of DNA and fluorescent dyes. Several complexes showed specific recognition of autoimmune antibodies in human samples of lupus patients using a simple one-step immunofluorescence method. This makes the novel assay developed herein a promising tool for research and point-of-care monitoring of anti-DNA antibodies. Using this method, we for the first time experimentally confirm that the disease-specific autoimmune antibodies are sensitive to the 3D structure of nucleic acids and not only to the nucleotide sequence, as was previously thought.

The Growing Development of DNA Nanostructures for Potential Healthcare‐Related Applications

“…Exploiting a competitive binding assay format, Domljanovic et al used a DNA origami structure for the detection of systemic lupus erythematosus (SLE) . One class of analytes used for the diagnosis of SLE is that of anti‐DNA antibodies which are generated and present in patients .…”

Dihydropyridine Fluorophores Allow for Specific Detection of Human Antibodies in Serum

“…Recent studies show that large well-defined three-dimensional (3D) DNA origami structures are advantageous in terms of brightness in the direct immunofluorescence assay. 10 , 16 , 23 Following this lead, we screened five previously reported origami structures: box, platform, ring, and two prism variants ( Figure 1 b). 10 , 24 − 26 Annealing of origami was done as previously described, 10 and the dye of interest was added during the thermal ramp at 65 °C (see Supporting Information Chapter 4 for details).…”

“…Upon the antibody binding, a fluorophore molecules get released from the DNA origami, providing a simple fluorescence read out for the assay. 16 …”

“…DHP analogues were additionally decorated with planar aromatic moieties and hydrophilic substituents that can provide stacking interactions and hydrogen bonds with large DNA origami. 10 , 16 In this article, we describe the design and synthesis of these new fluorophores and prove their ability to specifically detect human antibodies to DNA in blood samples.…”

Antibodies to synthetic citrullinated peptide epitope correlate with disease activity and flares in rheumatoid arthritis

“…Commercial ELISA kits have been obtained from Abnova, and used following the corresponding manufacturer's protocols (CCP2, Abnova KA4877; RF, Abnova KA2913; ANA, Abnova KA0939). Optical read-out (A450) has been used to monitor binding of autoantibodies to antigens [24][25][26].…”