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 use of nucleic acid, DNA and RNA, based strategies to disrupt gene expression as a therapeutic is quickly emerging. Indeed, synthetic oligonucleotides represent a major component of emerging gene therapeutics. However, the efficiency and specificity of intracellular uptake for non-modified oligonucleotides is rather poor. Utilizing RNA based oligonucleotides as therapeutics is even more challenging to deliver, due to extremely fast enzymatic degradation of the RNAs. Like unmodified oligonucleotides, RNAs also get rapidly degraded in vivo and demonstrate large off-target binding events when they can reach and enter the desired target cells. One approach that holds much promise is the utilization of “click chemistry” to conjugate receptor or cell specific targeting molecules directly to the effector oligonucleotides. We discuss here the applications of the breakthrough technology of CuAAC “click-chemistry” and the immense potential in utilizing “click chemistry” in the development of new age targeted oligonucleotide therapeutics.

Cancer is a major health risk in the modern society that requires rapid, reliable, and inexpensive diagnostics. Because of the low abundance of cancer DNA in biofluids, current detection methods require DNA amplification. The amplification can be challenging; it provides only relative quantification and extends time and cost of an assay. Herein, we report a new oligonucleotide hybridization platform for amplification-free detection of human cancer DNA. Using a large PEG-capture probe allows rapid separation of the bound (mutant) versus unbound (wild type) DNA. Next, a supramolecular hydrogel forming peptide attached to a detection oligonucleotide probe serves as a signal amplification tool. Having screened multiple short peptides and fluorophores, we identified the system P1 + cyanine 3.5 that allows for sensitive quantitative detection of mutation L858R in EGFR oncogene. The peptide–fluorophore-based assay provides absolute target DNA quantification at the detection limit of 20 ng cancer DNA versus >500 ng for Cy3.5-labeled oligonucleotide in only 1 hour.