Topologically-Interlocked Minicircles as Probes of DNA Topology and DNA-Protein Interactions

DNA minicircles exist in biological contexts, such as kinetoplast DNA, and are promising components for creating functional nanodevices. They have been used to mimic the topological features of nucleosomal DNA and to probe DNA-protein interactions such as HIV-1 and PFV integrases, and DNA gyrase. Here, we synthesized the topologically-interlocked minicircle rotaxane and catenane inside a frame-shaped DNA origami. These minicircles are 183 bp in length, constitute six individual single-stranded DNAs that are ligated to realize duplex interlocking, and adopt temporary base pairing of single strands for interlocking. To probe the DNA-protein interactions, restriction reactions were carried out on DNAs with different topologies such as free linear duplex or duplex constrained inside origami and free or topologically-interlocked minicircles. Except the free linear duplex, all tested structures were resistant to restriction digestion, indicating that the topological features of DNA, such as flexibility, curvature, and groove orientation, play a major role in DNA-protein interactions.     

Monovalent cation localization in DNA A-tracts with different sequences

The free solution mobilities of 26-base pair (bp) DNA oligomers containing A-tracts with and without internal ApT steps have been measured by capillary electrophoresis, using the mobility of a 26-bp random-sequence oligomer as a reference. The background electrolytes (BGEs) contained mixtures of Li⁺ and tetrapropylammonium (TPA⁺) ions, keeping the total cation concentration constant at 0.3 M. The mobility ratios equaled 1.00 in 0.3 M TPA⁺, indicating that the A-tract and reference oligomers had the same B-form conformation in this BGE. With increasing [Li⁺], the mobility ratio decreased as Li⁺ ions became localized in the A-tract minor groove, suggesting that the A-tract was now in the B* conformation. If the A-tract contained an internal ApT step and the oligomer contained less than ∼50% A + T, the mobility ratio reached a reduced plateau value that remained constant as the [Li⁺] increased to 0.3 M. However, for A-tracts without an internal ApT step and for A-tracts embedded in oligomers containing more than 50% A + T, the mobility ratios increased again at high [Li⁺], eventually reaching a plateau value of 1.00. Hence, DNA A-tracts in solution appear to exist as mixtures of the B and B* conformations, with the fractional concentration of each conformer depending on the [Li⁺], the A-tract sequence, and the total A + T content of the oligomer.     

Electrochemical Affinity Sensors Using Field Effect Transducer Devices for Chemical Analysis

The review presents advances and main challenges of the affinity sensors based on field- effect transistors published during the last five years. The different nanomaterial-based field-effect transistors are classified according to the nature of the nanomaterials, beginning by silicon, the “gold-standard” semiconductor, the gallium nitride semiconductor, the organic semiconductors, the silicon nanowires, the inorganic nanomaterials, the carbon nanotubes and the graphene. Due to its exceptional electrical properties, the main works are devoted to graphene. The obtained analytical performances for the detection of biomarkers, of DNA sequences and of miRNA are listed. The relation between the operational conditions - nature of the nanomaterials, procedure of preparation, choice of the receptor molecule, method of immobilization – and the analytical performance are discussed. The perspective of industrialization of these affinity sensors based on field-effect transistors is discussed.     

Selective In Situ Analysis of Mature microRNAs in Extracellular Vesicles Using a DNA Cage-Based Thermophoretic Assay

Mature microRNAs (miRNAs) in extracellular vesicles (EVs) are involved in different stages of cancer progression, yet it remains challenging to precisely detect mature miRNAs in EVs due to the presence of interfering RNAs (such as longer precursor miRNAs, pre-miRNAs) and the low abundance of tumor-associated miRNAs. By leveraging the size-selective ability of DNA cages and polyethylene glycol (PEG)-enhanced thermophoretic accumulation of EVs, we devised a DNA cage-based thermophoretic assay for highly sensitive, selective, and in situ detection of mature miRNAs in EVs with a low limit of detection (LoD) of 2.05 fM. Our assay can profile EV mature miRNAs directly in serum samples without the interference of pre-miRNAs and the need for ultracentrifugation. A clinical study showed that EV miR-21 or miR-155 had an overall accuracy of 90 % for discrimination between breast cancer patients and healthy donors, which outperformed conventional molecular probes detecting both mature miRNAs and pre-miRNAs. We envision that our assay can advance EV miRNA-based diagnosis of cancer.     

Real-time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA-Incorporated Thiazole Orange-Modified Nucleotides

A modified 2′-deoxycytidine triphosphate derivative ( dC^TOTP ) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dC^TOTP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dC^TOTP . When dC^TOTP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics.     

A DNA Origami-based Bactericide for Efficient Healing of Infected Wounds

Bacteria infection is a significant obstacle in the clinical treatment of exposed wounds facing widespread pathogens. Herein, we report a DNA origami-based bactericide for efficient anti-infection therapy of infected wounds in vivo. In our design, abundant DNAzymes (G4/hemin) can be precisely organized on the DNA origami for controllable generation of reactive oxygen species (ROS) to break bacterial membranes. After the destruction of the membrane, broad-spectrum antibiotic levofloxacin (LEV, loaded in the DNA origami through interaction with DNA duplex) can be easily delivered into the bacteria for successful sterilization. With the incorporation of DNA aptamer targeting bacterial peptidoglycan, the DNA origami-based bactericide can achieve targeted and combined antibacterial therapy for efficiently promoting the healing of infected wounds. This tailored DNA origami-based nanoplatform provides a new strategy for the treatment of infectious diseases in vivo.     

Light-Activated Assembly of DNA Origami into Dissipative Fibrils

Hierarchical DNA nanostructures offer programmable functions at scale, but making these structures dynamic, while keeping individual components intact, is challenging. Here we show that the DNA A-motif—protonated, self-complementary poly(adenine) sequences—can propagate DNA origami into one-dimensional, micron-length fibrils. When coupled to a small molecule pH regulator, visible light can activate the hierarchical assembly of our DNA origami into dissipative fibrils. This system is recyclable and does not require DNA modification. By employing a modular and waste-free strategy to assemble and disassemble hierarchical structures built from DNA origami, we offer a facile and accessible route to developing well-defined, dynamic, and large DNA assemblies with temporal control. As a general tool, we envision that coupling the A-motif to cycles of dissipative protonation will allow the transient construction of diverse DNA nanostructures, finding broad applications in dynamic and non-equilibrium nanotechnology.     

Decorated DNA-Based Scaffolds as Lateral Flow Biosensors

Here we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA-based structures decorated with reporter tags and recognition elements. We have rationally re-engineered tile-based DNA tubular structures that can act as scaffolds and can be decorated with recognition elements of different nature (i.e. antigens, aptamers or proteins) and with orthogonal fluorescent dyes. As a proof-of-principle we have developed sandwich and competitive multiplex lateral flow platforms for the detection of several targets, ranging from small molecules (digoxigenin, Dig and dinitrophenol, DNP), to antibodies (Anti-Dig, Anti-DNP and Anti-MUC1/EGFR bispecific antibodies) and proteins (thrombin). Coupling the advantages of functional DNA-based scaffolds together with the simplicity of LFAs, our approach offers the opportunity to detect a wide range of targets with nanomolar sensitivity and high specificity.