Point-of-care nucleic acid tests: assays and devices.

The COVID-19 pandemic (caused by the SARS_CoV_2 virus) has emphasized the need for quick, easy-to-operate, reliable, and affordable diagnostic tests and devices at the Point-of-Care (POC) for homes/fields/clinics. Such tests and devices will contribute significantly to the fight against the COVID-19 pandemic and any future infectious disease epidemic. Often, academic research studies and those from industry lack knowledge of each other's developments. Here, we introduced DNA Polymerase Chain Reaction (PCR) and isothermal amplification reactions and reviewed the current commercially available POC nucleic acid diagnostic devices. In addition, we reviewed the history and the recent advancements in an effort to develop reliable, quick, portable, cost-effective, and automatic point-of-care nucleic acid diagnostic devices, from sample to result. The purpose of this paper is to bridge the gap between academia and industry and to share important knowledge on this subject.

RNA Nanostructures: From Structure to Function.

Nucleic-acid nanostructures, which have been designed and constructed with atomic precision, have been used as scaffolds for different molecules and proteins, as nanomachines, as computational components, and more. In particular, RNA has garnered tremendous interest as a building block for the self-assembly of sophisticated and functional nanostructures by virtue of its ease of synthesis by in vivo or in vitro transcription, its superior mechanical and thermodynamic properties, and its functional roles in nature. In this Topical Review, we describe recent developments in the use of RNA for the design and construction of nanostructures. We discuss the differences between RNA and DNA that make RNA attractive as a building block for the construction of nucleic-acid nanostructures, and we present the uses of different nanostructures─RNA alone, RNA-DNA, and functional RNA nanostructures.

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

DNA nanotechnology.

The base sequence encoded in nucleic acids yields significant structural and functional properties into the biopolymer. The resulting nucleic acid nanostructures provide the basis for the rapidly developing area of DNA nanotechnology. Advances in this field will be exemplified by discussing the following topics: (i) Hemin/G-quadruplex DNA nanostructures exhibit unique electrocatalytic, chemiluminescence and photophysical properties. Their integration with electrode surfaces or semiconductor quantum dots enables the development of new electrochemical or optical bioanalytical platforms for sensing DNA. (ii) The encoding of structural information into DNA enables the activation of autonomous replication processes that enable the ultrasensitive detection of DNA. (iii) By the appropriate design of DNA nanostructures, functional DNA machines, acting as "tweezers", "walkers" and "stepper" systems, can be tailored. (iv) The self-assembly of nucleic acid nanostructures (nanowires, strips, nanotubes) allows the programmed positioning of proteins on the DNA templates and the activation of enzyme cascades.

Self-assembly of DNA nanotubes with controllable diameters.

The synthesis of DNA nanotubes is an important area in nanobiotechnology. Different methods to assemble DNA nanotubes have been reported, and control over the width of the nanotubes has been achieved by programmed subunits of DNA tiles. Here we report the self-assembly of DNA nanotubes with controllable diameters. The DNA nanotubes are formed by the self-organization of single-stranded DNAs, exhibiting appropriate complementarities that yield hexagon (small or large) and tetragon geometries. In the presence of rolling circle amplification strands, that exhibit partial complementarities to the edges of the hexagon- or tetragon-building units, non-bundled DNA nanotubes of controlled diameters can be formed. The formation of the DNA tubes, and the control over the diameters of the generated nanotubes, are attributed to the thermodynamically favoured unidirectional growth of the sheets of the respective subunits, followed subjected to the folding of sheets by elastic-energy penalties that are compensated by favoured binding energies.

Generation of photocurrents by bis-aniline-cross-linked Pt nanoparticle/photosystem I composites on electrodes.

Pt nanocrystals are implanted into photosystem I (PSI) by a photochemical reaction. The PSI with the associated Pt nanoclusters was modified with thioaniline and electropolymerized with thioaniline-functionalized Pt nanoparticles (NPs) to yield a bis-aniline-cross-linked PSI/Pt NPs composite. The alignment of the PSI with respect to the Pt NPs leads to effective charge separation and to generation of a photocurrent, ϕ (λ = 420 nm) = 2.6%, IPCE ∼ 0.35%. The bis-aniline units cross-linking the PSI/Pt NPs composite exhibit quasireversible redox features (E(0)' = 0.05 V vs Ag/AgCl, at pH = 7.4). Biasing the electrode potential, E > 0.1 V vs SCE, results in the formation of the oxidized quinoid bis-aniline state that acts as an electron acceptor. At this applied potential, the bridges mediate the electron transfer to the electrode, resulting in a ∼10-fold higher photocurrent, as compared to the system that includes the reduced bis-aniline bridging units. Furthermore, the ferredoxin (Fd) electron relay was modified with thioaniline units and incorporated into the PSI/Pt NP composite during the electropolymerization process. The Fd bound to the matrix mediates the electron transfer toward the electrode and facilitates charge separation that results in enhanced generation of the photocurrent, ϕ (λ = 420 nm) = 3.8%, IPCE ∼ 0.5%.

Covalently linked DNA nanotubes.

The present study introduces an approach to prepare covalently linked DNA nanotubes. A circular DNA that includes at its opposite poles thiol and amine functionalities acts as the building block for the construction of the DNA nanotubes. The circular DNA is cross-linked with a bis-amide-modified nucleic acid to yield DNA nanowires, and these are subsequently cross-linked by a bis-thiolated nucleic acid to yield the DNA nanotubes. Alternatively, a circular DNA that includes four amine functionalities on its poles is cross-linked in one-step by the bis-thiolated nucleic acid to yield the nanotubes. The resulting nanostructures are stable and nonseparable upon heating.

Self-assembly of aptamer-circular DNA nanostructures for controlled biocatalysis.

Two kinds of circular DNA components are generated by the hybridization of short nucleic acids with the 3' and 5' ends of single-stranded DNA chains. The circular DNA components include, each, complementary domains for the anticocaine aptamer subunits, and sequence-specific domains for the auxiliary hybridization of programmed nucleic acid-functionalized proteins. The circular DNA components are self-assembled, in the presence of cocaine, into DNA nanowires (micrometer-long nanowires exhibiting heights of ca. 1.6-3.0 nm). Nucleic acids functionalized with glucose oxidase (GOx) and horseradish peroxidase (HRP) are hybridized with the circular DNA components to yield nanostructures consisting of HRP and GOx on the DNA scaffold. A biocatalytic cascade, where the GOx-catalyzed oxidization of glucose by O(2) yields H(2)O(2), and the resulting H(2)O(2) oxidizes 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS(2-)), in the presence of HRP, is activated by the system. The biocatalyzed oxidization of ABTS(2-) on the DNA scaffold is 6-fold enhanced as compared to a nonbridged homogeneous system of the two biocatalysts. The enhanced biocatalytic cascade on the DNA scaffold is attributed to high local concentrations of the reactive components in the vicinity of biocatalysts.

Control of bioelectrocatalytic transformations on DNA scaffolds.

The spatial organization of biomolecules on a DNA scaffold linked to an electrode leads to programmed biocatalytic transformations. This is exemplified by the electrical contacting of glucose oxidase (GOx) linked to the DNA scaffold with the electrode. A nucleic acid functionalized with a ferrocene relay unit was hybridized with the DNA scaffold at a position adjacent to the electrode, and GOx functionalized with nucleic acid units complementary to the specific domain of the DNA template was hybridized with the DNA scaffold in a position remote from the electrode. Under these conditions, ferrocene-mediated oxidation of the redox center of GOx occurred, and the effective bioelectrocatalytic oxidation of glucose was activated. Exchange of the position of GOx and the electron-mediator groups prohibited the bioelectrocatalytic oxidation of glucose. In another system, a nucleic acid-functionalized microperoxidase-11 (MP-11) and the nucleic acid-modified GOx were hybridized with the adjacent and remote sites, respectively, on the DNA scaffold associated with the electrode. In this configuration, effective MP-11-catalyzed reduction of H(2)O(2) generated by the GOx-catalyzed oxidation of glucose occurred, and the resulting bioelectrocatalytic cathodic currents were controlled by the concentration of glucose. Exchanging the positions of MP-11 and GOx on the DNA scaffold eliminated the MP-11-electrocatalyzed reduction of H(2)O(2).

Enzyme cascades activated on topologically programmed DNA scaffolds.

The ability of DNA to self-assemble into one-, two- and three-dimensional nanostructures, combined with the precision that is now possible when positioning nanoparticles or proteins on DNA scaffolds, provide a promising approach for the self-organization of composite nanostructures. Predicting and controlling the functions that emerge in self-organized biomolecular nanostructures is a major challenge in systems biology, and although a number of innovative examples have been reported, the emergent properties of systems in which enzymes are coupled together have not been fully explored. Here, we report the self-assembly of a DNA scaffold made of DNA strips that include 'hinges' to which biomolecules can be tethered. We attach either two enzymes or a cofactor-enzyme pair to the scaffold, and show that enzyme cascades or cofactor-mediated biocatalysis can proceed effectively; similar processes are not observed in diffusion-controlled homogeneous mixtures of the same components. Furthermore, because the relative position of the two enzymes or the cofactor-enzyme pair is determined by the topology of the DNA scaffold, it is possible to control the reactivity of the system through the design of the individual DNA strips. This method could lead to the self-organization of complex multi-enzyme cascades.

Self-assembly of enzymes on DNA scaffolds: en route to biocatalytic cascades and the synthesis of metallic nanowires.

DNA strands consisting of programmed sequence-specific domains were synthesized by the rolling circle amplification (RCA) process. The spatial positioning of glucose oxidase (GOx) and of horseradish peroxidase (HRP) on the RCA-synthesized DNA template via hybridization enabled the activation of the bienzyme cascade. The GOx-catalyzed oxidation of glucose yielded gluconic acid and H(2)O(2), and the resulting H(2)O(2) oxidized 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic-acid] (ABTS(2-)) in the presence of HRP. The enzyme cascade could not be activated in the absence of the organizing DNA template or in the presence of a foreign DNA. Also, Au NPs-functionalized GOx was hybridized with the RCA-synthesized single-stranded DNA. The biocatalytic growth of the NPs through the oxidation of glucose, in the presence of AuCl(4)(-), enabled the synthesis of 1-5 microm long Au wires, exhibiting a width of ca. 150 nm.

Supramolecular aptamer-thrombin linear and branched nanostructures.

Alpha and beta conjugated bis-aptamers against thrombin act as bidentate "glue" for the self-assembly of thrombin nanowires; mixing the bidentate aptamer with a tripodal tridentate alpha aptamer construct yields branched thrombin nanowire structures.

Probing kinase activities by electrochemistry, contact-angle measurements, and molecular-force interactions.

Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistance in the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5 degrees , phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8 degrees. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3 degrees . The third method to characterize the CK2 system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6+/-2 pN), significant rupture forces (multiples of 120+/-20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region.

A polycatenated DNA scaffold for the one-step assembly of hierarchical nanostructures.

A unique DNA scaffold was prepared for the one-step self-assembly of hierarchical nanostructures onto which multiple proteins or nanoparticles are positioned on a single template with precise relative spatial orientation. The architecture is a topologically complex ladder-shaped polycatenane in which the "rungs" of the ladder are used to bring together the individual rings of the mechanically interlocked structure, and the "rails" are available for hierarchical assembly, whose effectiveness has been demonstrated with proteins, complementary DNA, and gold nanoparticles. The ability of this template to form from linear monomers and simultaneously bind two proteins was demonstrated by chemical force microscopy, transmission electron microscopy, and confocal fluorescence microscopy. Finally, fluorescence resonance energy transfer between adjacent fluorophores confirmed the programmed spatial arrangement between two different nanomaterials. DNA templates that bring together multiple nanostructures with precise spatial control have applications in catalysis, biosensing, and nanomaterials design.