DNA origami book biosensor for multiplex detection of cancer-associated nucleic acids.

DNA nanotechnology provides a promising approach for the development of biomedical point-of-care diagnostic nanoscale devices that are easy to use and cost-effective, highly sensitive and thus constitute an alternative to expensive, complex diagnostic devices. Moreover, DNA nanotechnology-based devices are particularly advantageous for applications in oncology, owing to being ideally suited for the detection of cancer-associated nucleic acids, including circulating tumor-derived DNA fragments (ctDNAs), circulating microRNAs (miRNAs) and other RNA species. Here, we present a dynamic DNA origami book biosensor that is precisely decorated with arrays of fluorophores acting as donors and acceptors and also fluorescence quenchers that produce a strong optical readout upon exposure to external stimuli for the single or dual detection of target oligonucleotides and miRNAs. This biosensor allowed the detection of target molecules either through the decrease of Förster resonance energy transfer (FRET) or an increase in the fluorescence intensity profile owing to a rotation of the constituent top layer of the structure. Single-DNA origami experiments showed that detection of two targets can be achieved simultaneously within 10 min with a limit of detection in the range of 1-10 pM. Overall, our DNA origami book biosensor design showed sensitive and specific detection of synthetic target oligonucleotides and natural miRNAs extracted from cancer cells. Based on these results, we foresee that our DNA origami biosensor may be developed into a cost-effective point-of-care diagnostic strategy for the specific and sensitive detection of a variety of DNAs and RNAs, such as ctDNAs, miRNAs, mRNAs, and viral DNA/RNAs in human samples.

Natural and Modified Oligonucleotide Sequences Show Distinct Strand Displacement Kinetics and These Are Affected Further by Molecular Crowders.

DNA and RNA strand exchange is a process of fundamental importance in biology. Herein, we used a FRET-based assay to investigate, for the first time, the stand exchange kinetics of natural DNA, natural RNA, and locked nucleic acid (LNA)-modified DNA sequences in vitro in PBS in the absence or presence of molecular additives and macromolecular crowders such as diethylene glycol dimethyl ether (deg), polyethylene glycol (peg), and polyvinylpyrrolidone (pvp). The results show that the kinetics of strand exchange mediated by DNA, RNA, and LNA-DNA oligonucleotide sequences are different. Different molecular crowders further affect the strand displacement kinetics, highlighting the complexity of the process of nucleic acid strand exchange as it occurs in vivo. In a peg-containing buffer, the rate constant of displacement was slightly increased for the DNA displacement strand, while it was slightly decreased for the RNA and the LNA-DNA strands compared with displacement in pure PBS. When we used a deg-containing buffer, the rate constants of displacement for all three sequences were drastically increased compared with displacement in PBS. Overall, we show that interactions of the additives with the duplex strands have a significant effect on the strand displacement kinetics and this effect can exceed the one exerted by the chemical nature of the displacement strand itself.

DNA Origami as Emerging Technology for the Engineering of Fluorescent and Plasmonic-Based Biosensors.

DNA nanotechnology is a powerful and promising tool for the development of nanoscale devices for numerous and diverse applications. One of the greatest potential fields of application for DNA nanotechnology is in biomedicine, in particular biosensing. Thanks to the control over their size, shape, and fabrication, DNA origami represents a unique opportunity to assemble dynamic and complex devices with precise and predictable structural characteristics. Combined with the addressability and flexibility of the chemistry for DNA functionalization, DNA origami allows the precise design of sensors capable of detecting a large range of different targets, encompassing RNA, DNA, proteins, small molecules, or changes in physico-chemical parameters, that could serve as diagnostic tools. Here, we review some recent, salient developments in DNA origami-based sensors centered on optical detection methods (readout) with a special emphasis on the sensitivity, the selectivity, and response time. We also discuss challenges that still need to be addressed before this approach can be translated into robust diagnostic devices for bio-medical applications.

Optical and theoretical study of strand recognition by nucleic acid probes.

Detection of nucleic acids is crucial to the study of their basic properties and consequently to applying this knowledge to the determination of pathologies such as cancer. In this work, our goal is to determine new trends for creating diagnostic tools for cancer driver mutations. Herein, we study a library of natural and modified oligonucleotide duplexes by a combination of optical and theoretical methods. We report a profound effect of additives on the duplexes, including nucleic acids as an active crowder. Unpredictably and inconsistent with DNA+LNA/RNA duplexes, locked nucleic acids contribute poorly to mismatch discrimination in the DNA+LNA/DNA duplexes. We develop a theoretical framework that explains poor mismatch discrimination in KRAS oncogene. We implement our findings in a bead-bait genotyping assay to detect mutated human cancer RNA. The performance of rationally designed probes in this assay is superior to the LNA-primer polymerase chain reaction, and it agrees with sequencing data.

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay.

Nucleic acid hybridisation plays a key role in many biological processes, including transcription, translation, and regulation of gene expression. Several sophisticated applications rely on this fundamental interaction, including the polymerase chain reaction, sequencing, and gene therapy. To target a nucleic acid sequence specifically, synthetic oligonucleotides with a suitable affinity and specificity towards the target need to be designed. The affinity of potential probes or therapeutic agents to their target sequence is generally investigated by melting experiments, which break the hydrogen-bonding and stacking interactions that stabilise the double helix resulting in the formation of two single strands. In this paper, we report a comparative study of hybridisation for short fluorescent oligonucleotides labelled with cyanine and ATTO dyes, performed by the currently used UV melting assay and by a more sensitive fluorescence melting experiment. Using different oligonucleotide sequences in the concentration range of 5 nm to 2 µm, we observed a stabilising effect of the fluorophores on the duplexes, especially at low concentrations. We paid particular attention to the effect of polycations and to molecular crowding as major parameters that define the stability and the geometry of nucleic acid duplexes in biological samples. We also demonstrated how the fluorometry-based melting data could aid the design of a probe targeting a human BRAF gene fragment to reduce the off-target binding by a factor of seven.

Dihydropyridine Fluorophores Allow for Specific Detection of Human Antibodies in Serum.

Antigen recognition by antibodies plays an important role in human biology and in the development of diseases. This interaction provides a basis for multiple diagnostic assays and is a guide for treatments. We have developed dihydropyridine-based fluorophores that form stable complexes with double-stranded DNA and upon recognition of the antibodies to DNA (anti-DNA) provide an optical response. The fluorophores described herein have advantageous optical properties compared to those of the currently available dyes making them valuable for research and clinical diagnostics. By studying a series of novel fluorophores, crucial parameters for the design were established, providing the required sensitivity and specificity in the detection of antibodies. Using these DNA-fluorophore complexes in a direct immunofluorescence assay, antibodies to DNA are specifically detected in 80 patients diagnosed with an autoimmune disease, systemic lupus erythematosus. Positivity indicated by emission change of α-(4'-O-methoxyphenyl)-2-furyl dihydropyridine strongly correlates with other disease biomarkers and autoimmune arthritis.

Studies of Impending Oligonucleotide Therapeutics in Simulated Biofluids.

Synthetic oligonucleotides, their complexes and conjugates with other biomolecules represent valuable research tools and therapeutic agents. In spite of growing applications in basic research and clinical science, only few studies have addressed the issue of such compounds' stability in biological media. Herein, we studied the stability of two therapeutically relevant oligonucleotide probes in simulated biofluids; the 21 nucleotide-long DNA/locked nucleic acid oligonucleotide ON targeted toward cancer-associated BRAF V600E mutation, and a longer DNA analog (TTC) originating from BRAF gene. We found that stability of peptide-oligonucleotide conjugates (POCs) in human serum (HS) was superior compared with the naked or complexed 21mer oligonucleotide, whereas stability of POCs in simulated gastric juice (GJ) was dependent on the peptide sequence. Addition of pepstatin A in general increased the stability of oligonucleotides after 24 h digestion in HS and simulated GJ. Similarly, complexation with optimal amounts of histone proteins was found to rescue oligonucleotide stability after 24 h digestion in hydrochloric acid.

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

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.