Preclinical anticancer activity of DNA-based cleavage molecules

Deoxyribozymes (DNAzymes) are DNA residue-based molecules capable of specific cleavage of complementary mRNA. As such, they are more stable counterparts for the earlier discovered ribozymes. A handful of studies have shown the potential of DNAzymes against cancer both in cell culture and importantly in vivo models. This relatively new molecular entity may progress to clinical trials provided that more extensive testing is carried out at the preclinical stage. While a significant amount of work has gone into chemically stabilizing the molecule, delivery is one area that needs particular attention.     

Preclinical Anticancer Activity of DNA-based Cleavage Molecules

ABSTRACT

Deoxyribozymes (DNAzymes) are DNA residue-based molecules capable of specific cleavage of complementary mRNA. As such, they are more stable counterparts for the earlier discovered ribozymes. A handful of studies have shown the potential of DNAzymes against cancer both in cell culture and importantly in vivo models. This relatively new molecular entity may progress to clinical trials provided that more extensive testing is carried out at the preclinical stage. While a significant amount of work has gone into chemically stabilizing the molecule, delivery is one area that needs particular attention.     

Entrapment of fluorescence signaling DNA enzymes in sol-gel-derived materials for metal ion sensing

Three fluorescence signaling DNA enzymes (deoxyribozymes or DNAzymes) were successfully immobilized within a series of sol-gel-derived matrixes and used for sensing of various metal ions. The DNAzymes are designed such that binding of appropriate metal ions induces the formation of a catalytic site that cleaves a ribonucleotide linkage within a DNA substrate. A fluorophore (fluorescein) and a quencher (DABCYL, [4-(4-dimethylaminophenylazo)benzoic acid]) were placed on the two deoxythymidines flanking the ribonucleotide to allow the generation of fluorescence upon the catalytic cleavage at the RNA linkage. In general, all DNAzymes retained at least partial catalytic function when entrapped in either hydrophilic or hydrophobic silica-based materials, but displayed slower response times and lower overall signal changes relative to solution. Interestingly, it was determined that maximum sensitivity toward metal ions was obtained when DNAzymes were entrapped into composite materials containing approximately 40% of methyltrimethoxysilane (MTMS) and approximately 60% tetramethoxysilane (TMOS). Highly polar materials derived from sodium silicate, diglycerylsilane, or TMOS had relatively low signal enhancements, while materials with very high levels of MTMS showed significant leaching and low signal enhancements. Entrapment into the hybrid silica material also reduced signal interferences that were related to metal-induced quenching; such interferences were a significant problem for solution-based assays and for polar materials. Extension of the solid-phase DNAzyme assay toward a multiplexed assay format for metal detection is demonstrated, and shows that sol-gel technology can provide new opportunities for the development of DNAzyme-based biosensors.     

Six-helix bundles designed from DNA

We present a designed cyclic DNA motif that consists of six DNA double helices that are connected to each other at two crossover sites. DNA double helices with 10.5 nucleotide pairs per turn facilitate the programming of DNA double crossover molecules to form hexagonally symmetric arrangements when the crossover points are separated by seven or fourteen nucleotide pairs. We demonstrate by atomic force microscopy well-formed arrays of hexagonal six-helix bundle motifs both in 1D and in 2D.     

Pyrene-assisted efficient photolysis of disulfide bonds in DNA-based molecular engineering

An efficient pyrene-assisted method has been developed for the photolysis of disulfide bonds, with 77% of disulfides cleaved after only 20 min of irradiation (0.3W) at 350 nm. By employing a DNA framework, it was possible to observe both a distance-dependent cleavage pathway and a radical-forming photoreaction mechanism. To demonstrate the biomedical applications of such pyrene disulfide molecular assemblies, a DNA micelle structure and DNAzyme analog were further studied. Rapid photodriven disassembly of DNA micelles was achieved, allowing the further design of controlled pharmaceutical release at the target region and at a specific time. The DNAzyme analog can carry out multiple turnover reactions that follow the Michaelis-Menten equation, with a kcat of 10.2 min(-1) and a KM of 46.3 μM (0.3W 350 nm light source), comparable to that of common DNAzymes, e.g., 8-17 DNAzyme.     

Cooperative enhancement of deoxyribozyme activity by chemical modification and added cationic copolymer

Deoxyribozymes (DNAzymes) having RNA-cleaving activity have widely been explored as tools for therapeutic and diagnostic purposes. Both the chemical cleaving step and the turnover step should be improved for enhancing overall activity of DNAzymes. We have shown that cationic copolymer enhanced DNAzyme activity by increasing turnover efficacy. In this paper, effectｓ of the copolymer on DNAzymes modified with locked nucleic acids (LNA) or 2′-O-methylated (2′-OMe) nucleic acids were studied. The copolymer increased activity of these chemically modified DNAzymes. More than 30-fold enhancement in multiple-turnover catalytic activity was observed with 2′-OMe-modified DNAzyme in the presence of the copolymer. DNAzyme catalytic activity was successfully enhanced by cooperation of the added copolymer and chemical modification of DNAzyme.     

生物分子模板法制备低维金属纳米材料研究进展（Ⅱ）

本文概述了蛋白质、DNA等生物分子模板表面化学沉积制备低维金属纳米材料的最新研究进展。蛋白质可自组装形成不同层次的纳米结构,而DNA分子可自组装形成纳米线和网状结构。这些不同的纳米结构可作为模板,化学沉积,制备金属纳米管、纳米线等一维或二维纳米材料。金属纳米管和纳米线具有导电、导热、磁性和具有特殊的量子效应,在电磁活性复合材料,可控缓释系统、纳米器件和纳米电路等领域有重要的应用前景。     

Artificial Engineered Natural Killer Cells Combined with Antiheat Endurance as a Powerful Strategy for Enhancing Photothermal‐Immunotherapy Efficiency of Solid Tumors

Although photothermal therapy (PTT) is preclinically applied in solid tumor treatment, incomplete tumor removal of PTT and heat endurance of tumor cells induces significant tumor relapse after treatment, therefore lowering the therapeutic efficiency of PTT. Herein, a programmable therapeutic strategy that integrates photothermal therapeutic agents (PTAs), DNAzymes, and artificial engineered natural killer (A‐NK) cells for immunotherapy of hepatocellular carcinoma (HCC) is designed. The novel PTAs, termed as Mn‐CONASHs, with 2D structure are synthesized by the coordination of tetrahydroxyanthraquinone and Mn²⁺ ions. By further adsorbing polyetherimide/DNAzymes on the surface, the DNAzymes@Mn‐CONASHs exhibit excellent light‐to‐heat conversion ability, tumor microenvironment enhanced T₁‐MRI guiding ability, and antiheat endurance ability. Furthermore, the artificial engineered NK cells with HCC specific targeting TLS11a‐aptamer decoration are constructed for specifically eliminating any possible residual tumor cells after PTT, to systematically enhance the therapeutic efficacy of PTT and avoid tumor relapse. Taken together, the potential of A‐NK cells combined with antiheat endurance as a powerful strategy for immuno‐enhancing photothermal therapy efficiency of solid tumors is highlighted, and the current strategy might provide promising prospects for cancer therapy.     

Antisense technology as a potential strategy for the treatment of coronaviruses infection: With focus on COVID‐19

After the outbreak of coronavirus disease 2019 (COVID‐19) in December 2019 and the increasing number of SARS‐CoV‐2 infections all over the world, researchers are struggling to investigate effective therapeutic strategies for the treatment of this infection. Targeting viral small molecules that are involved in the process of infection is a promising strategy. Since many host factors are also used by SARS‐CoV‐2 during various stages of infection, down‐regulating or silencing these factors can serve as an effective therapeutic tool. Several nucleic acid‐based technologies including short interfering RNAs, antisense oligonucleotides, aptamers, DNAzymes, and ribozymes have been suggested for the control of SARS‐CoV‐2 as well as other respiratory viruses. The antisense technology also plays an indispensable role in the treatment of many other diseases including cancer, influenza, and acquired immunodeficiency syndrome. In this review, we summarised the potential applications of antisense technology for the treatment of coronaviruses and specifically COVID‐19 infection.     

Highly Conductive Thin Uniform Gold‐Coated DNA Nanowires

Over the past decades, DNA, the carrier of genetic information, has been used by researchers as a structural template material. Watson‐Crick base pairing enables the formation of complex 2D and 3D structures from DNA through self‐assembly. Various methods have been developed to functionalize these structures for numerous utilities. Metallization of DNA has attracted much attention as a means of forming conductive nanostructures. Nevertheless, most of the metallized DNA wires reported so far suffer from irregularity and lack of end‐to‐end electrical connectivity. An effective technique for formation of thin gold‐coated DNA wires that overcomes these drawbacks is developed and presented here. A conductive atomic force microscopy setup, which is suitable for measuring tens to thousands of nanometer long molecules and wires, is used to characterize these DNA‐based nanowires. The wires reported here are the narrowest gold‐coated DNA wires that display long‐range conductivity. The measurements presented show that the conductivity is limited by defects, and that thicker gold coating reduces the number of defects and increases the conductive length. This preparation method enables the formation of molecular wires with dimensions and uniformity that are much more suitable for DNA‐based molecular electronics.     

Nucleic acid amplification of individual molecules in a microfluidic device

A microfluidic device was developed that enabled rapid polymerase chain reaction (PCR) analysis of individual DNA molecules. The device combined a means for accessing samples serially from a microtiter plate, channels for assembling eight parallel PCR reactions, and integrated resistive heaters for rapid thermocycling (>5 degrees C/s heating, >7 degrees C/s cooling) of samples as they flowed continuously through PCR channels. Amplification was monitored by fluorescence detection of Taqman probes. The long, narrow channels (10 microm x 180 microm x 40 mm) allowed sufficient separation between neighboring DNA templates to enable amplification of discreet DNA molecules. The functionality of the device was demonstrated by reproducibly amplifying a 2D6.6 CYP450 template and distinguishing between wild-type and mutant sequences using Taqman probes. A comparison of the rate of individual amplification events to the expected Poisson distribution confirmed that the device could reliably analyze individual DNA molecules. This work establishes the feasibility of rapid, single-molecule interrogation of nucleic acids.     

Disassembly of 2D Vertical Heterostructures

As one of the most widely discussed fields, the assembly of nanomaterials has always been extensively studied. However, its inverse process, namely disassembly, is still limited in the ambit of biomolecules. Specifically, in the emerging 2D research field, disassembly still remains unexplored. Inspired by the disassembly of DNA molecules via breaking intermolecular hydrogen bonds, the disassembly of 2D vertical heterostructures (2DVHs) is first achieved through the weakening of the interlayer van der Waals interactions. As a demonstration, ReS₂/WS₂ VHs is successfully disassembled into individual building blocks. Density functional theory calculations are performed to study the disassembly of the 2DVHs, which simulate that 2DVHs are first activated by the disassembly promoters and then disassembled with weakened interlayer van der Waals interactions. Such a disassembly process demonstrates that it has great potential to be expanded as a general strategy to achieve the disassembly of other 2D superstructures.     

DNA as a template in self-assembly of Au nano-structure

In this study, an alkanethiol-assisted self-assembly of Au nano-particles and its size tunable technique were confirmed. To fabricate a one-dimensional (1D) template, -DNA was first laid on mica substrate by dropping diluted -DNA solution, 12.6?ng/[micro sign]l, on freshly cleaved mica. By dropping colloidal gold solution on mica surface with the DNA templates laid on it, the -DNA was then pulled straight via capillary force by applying solvent absorbing tissue at outer circumference of the mica substrate. Moreover, it fixed on mica via gravity force and Van der Waals force between mica surface and the DNA. Au nano-particles would be arrayed along the straight DNA molecules to form 1D Au arrays. Then based on the synthesis of 1D nano-structure via DNA template and Au nano-particles, the simple 2D nano-structure, a ring, would also be studied.     

DNAzyme-based colorimetric sensing of lead (Pb(2+)) using unmodified gold nanoparticle probes

Novel functional oligonucleotides, especially DNAzymes with RNA-cleavage activity, have been intensively studied due to their potential applications in therapeutics and sensors. Taking advantage of the high specificity of 17E DNAzyme for Pb(2+), highly sensitive and selective fluorescent, electrochemical and colorimetric sensors have been developed for Pb(2+). In this work, we report a simple, sensitive and label-free 17E DNAzyme-based sensor for Pb(2+) detection using unmodified gold nanoparticles (GNPs) based on the fact that unfolded single-stranded DNA could be adsorbed on the citrate protected GNPs while double-stranded DNA could not. By our method the substrate cleavage by the 17E DNAzyme in the presence of Pb(2+) could be monitored by color change of GNPs, thereby Pb(2+) detection was realized. The detection of Pb(2+) could be realized within 20?min, with a detection limit of 500?nM. The selectivity of our sensor has been investigated by challenging the sensing system with other divalent metal ions. Since common steps such as modification and separation could be successfully avoided, the sensor developed here could provide a simple, cost-effective yet rapid and sensitive measurement tool for Pb(2+) detection and may prove useful in the development of sensors for clinical toxicology and environmental monitoring in the future.     

Nucleic Acid Enzyme-Activated CRISPR-Cas12a With Circular CRISPR RNA for Biosensing

clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as “NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C).” Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.     

Control of self-assembled 2D nanostructures by methylation of guanine

Methylation of DNA nucleobases is an important control mechanism in biology applied, for example, in the regulation of gene expression. The effect of methylation on the intermolecular interactions between guanine molecules is studied through an interplay between scanning tunneling microscopy (STM) and density functional theory with empirical dispersion correction (DFT-D). The present STM and DFT-D results show that methylation of guanine can have subtle effects on the hydrogen-bond strength with a strong dependence on the position of methylation. It is demonstrated that the methylation of DNA nucleobases is a precise means to tune intermolecular interactions and consequently enables very specific recognition of DNA methylation by enzymes. This scheme is used to generate four different types of artificial 2D nanostructures from methylated guanine. For instance, a 2D guanine windmill motif that is stabilized by cooperative hydrogen bonding is revealed. It forms by self-assembly on a graphite surface under ambient conditions at the liquid-solid interface when the hydrogen-bonding donor at the N1 site of guanine is blocked by a methyl group.     

Ultrasensitive coincidence fluorescence detection of single DNA molecules

We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.     

Charge Transport in 2D DNA Tunnel Junction Diodes

Recently, deoxyribonucleic acid (DNA) is studied for electronics due to its intrinsic benefits such as its natural plenitude, biodegradability, biofunctionality, and low‐cost. However, its applications are limited to passive components because of inherent insulating properties. In this report, a metal–insulator–metal tunnel diode with Au/DNA/NiO_x junctions is presented. Through the self‐aligning process of DNA molecules, a 2D DNA nanosheet is synthesized and used as a tunneling barrier, and semitransparent conducting oxide (NiO_x) is applied as a top electrode for resolving metal penetration issues. This molecular device successfully operates as a nonresonant tunneling diode, and temperature‐variable current–voltage analysis proves that Fowler–Nordheim tunneling is a dominant conduction mechanism at the junctions. DNA‐based tunneling devices appear to be promising prototypes for nanoelectronics using biomolecules.     

Sizing of single globular DNA molecules by using a circular acceleration technique with laser trapping

We describe a method for in situ sizing individual huge DNA molecules by laser trapping. Single DNA molecules are reversibly transformed, without mechanical fragmentation of fragile huge-sized DNA, from their random coil state into their globular state induced by condensing agents poly(ethylene glycol) and Mg(2+). With the use of a globular DNA molecule folded by condensation, the critical velocity of the circularly accelerated single globular DNA molecule by laser trapping was found to be proportional to the size of the DNA. Yeast, Saccharomyces cerevisiae, chromosome III (285 kbp) was successfully sized (281 +/- 40 kbp) from a calibration curve scaled using lambda, T4, and yeast chromosome VI (48.5, 166, and 385 kbp, respectively). The use of critical velocity as a sizing parameter makes it possible to size single DNA molecules without prior conformational information, i.e., the radius of a single globular huge DNA molecule as a nanoparticle. A sized single globular DNA molecule could be trapped again for subsequent manipulation, such as transportation of it anywhere. We also investigated a possibility of reusing the globular DNA molecules condensed by PEG and Mg(2+) for PCR and found that PCR efficiency was not deteriorated in the presence of the condensation agents.     

Label-free detection of DNA hybridization using pyrene-functionalized single-walled carbon nanotubes: effect of chemical structures of pyrene molecules on DNA sensing performance

We investigate the effect of functional groups of pyrene molecules on the electrical sensing performance of single-walled carbon nanotubes (SWNTs) based DNA biosensor, in which pyrenes with three different functional groups of carboxylic acid (Py-COOH), aldehyde (Py-CHO) and amine (Py-NH2) are used as linker molecules to immobilize DNA on the SWNT films. UV/Visible absorption spectra results show that all of the pyrene molecules are successfully immobilized on the SWNT surface via pi-pi stacking interaction. Based on fluorescence analysis, we show that the amide bonding of amine terminated DNA via pyrene containing carboxylic groups is the most efficient to immobilize DNA on the nanotube film. The electrical detection results show that the conductance of Py-COOH modified SWNT film is increased upon DNA immobilization, followed by further increase after hybridization of target DNAs. It indicates that the pyrene molecules with carboxylic acid groups play an important role to achieve highly efficient label-free detection by nondestructive and specific immobilization of DNAs.