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.     

Functional DNAzymes organized into two-dimensional arrays

DNAzymes are catalytically active DNA molecules, which have previously been described in solution. Here, we organize these molecules into a series of two-dimensional (2D) arrays using a periodic arrangement of DNA structures based on the DNA double crossover motif. We demonstrate by means of atomic force microscopy that the DNAzymes are organized according to the design and that they retain their activity when attached in linear strings within the context of the 2D array.     

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.     

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.     

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.     

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.     

Design of a biochemical circuit motif for learning linear functions

Learning and adaptive behaviour are fundamental biological processes. A key goal in the field of bioengineering is to develop biochemical circuit architectures with the ability to adapt to dynamic chemical environments. Here, we present a novel design for a biomolecular circuit capable of supervised learning of linear functions, using a model based on chemical reactions catalysed by DNAzymes. To achieve this, we propose a novel mechanism of maintaining and modifying internal state in biochemical systems, thereby advancing the state of the art in biomolecular circuit architecture. We use simulations to demonstrate that the circuit is capable of learning behaviour and assess its asymptotic learning performance, scalability and robustness to noise. Such circuits show great potential for building autonomous in vivo nanomedical devices. While such a biochemical system can tell us a great deal about the fundamentals of learning in living systems and may have broad applications in biomedicine (e.g. autonomous and adaptive drugs), it also offers some intriguing challenges and surprising behaviours from a machine learning perspective.     

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.     

Unimolecular catalytic DNA biosensor for amplified detection of L-histidine via an enzymatic recycling cleavage strategy

Fluorescence catalytic beacons have emerged as a general platform for sensing applications. However, almost all such sensing systems need covalent modification of the DNAzymes with fluorophore-quencher pairs, which may require elaborate design of the synthetic routes and many heavy and complicated synthetic steps and result in increased cost and lower synthesis yield. Here we report the construction of fluorescent cascadic catalytic beacons. With separation of the molecular recognition module from the signal reporter, this new design both avoids DNAzyme modifications and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification. This allows detection of L-histidine with high sensitivity (LOD = 200 nM) and excellent specificity. The proposed sensing system has also been used for detection of L-histidine in cellular homogenate with satisfactory results.     

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.     

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.     

大跨空间结构风振响应及其计算与试验方法

大跨空间结构体系广泛应用于工业和民用建筑中，并且日趋大型化，多样化，复杂化，风荷载已成为该类结构抗风设计，防灾减灾分析的控制荷载之一，文中针对大跨空间结构，分析了风振形式及其机理，阐述了风振响应计算方法和试验方法。指出随机振动理论，有限单元法和数值分析理论是大跨空间结构风振响应计算的有效理论工具，风洞试验是研究结构风振的主要实验手段，结构风振数值模拟方法具有广阔发展前景。最后，提出值得进一步研究的若干问题。     

Computer-aided modelling of anti-corrosion coatings formulations

Formulation optimisation is one of the more important steps in the development of surface coating systems. Several commercially available computer software packages that can be used to assist the formulation optimisation have been developed, provided that sufficient data are available. For systems having complex behaviour, more flexible techniques are needed. The approach that forms the basis of this paper is based on spline interpolations. Thus, the more complex behaviour of coating systems can be simulated and interpreted. The efficiency and the reliability of the models that have been developed have been evaluated using data that are relevant to anticorrosion coating systems.     

Measuring productive performance using binary and ordinal output variables: the case of the Swedish fire and rescue services

Fire protection is an example of a complex production process. This study measures efficiency by constructing binary and ordinal output variables from information on residential fires in Sweden about how a fire spreads from when the fire and rescue brigade arrives to when a fire is suppressed. The motivations behind this study are that there are only a few studies trying to estimate production efficiency for fire and rescue services, that data on a more detailed level is interesting for some public services, and there is a need to be able to measure efficiency differences even if only a binary or ordinal output variable is available. Using a logit random parameter model, the random effects are interpreted as efficiency differences. The conclusions are that fire and rescue services with a more flexible fire organisation with first response persons, working in collaboration with other municipalities and with larger populations are more efficient.     

低温流体空化特性的数值计算研究

采用数值计算的方法研究了液氮和液氢的空化流动特性。为了考虑温度影响,控制方程采用了连续方程、动量方程及能量方程,并应用二次开发方法在商业软件中引入Merkle 空化模型及物质属性,物性参数随流场温度变化而不断更新。分别对液氮和液氢几个工况进行了计算,并与实验结果进行了对比。结果发现,在液氮和液氢中,当流体温度接近临界点时,热力学效应表现显著。热力学效应显著主要表现在空穴变短、水蒸汽含量减少和汽液界面变的模糊。由于密度比、饱和蒸汽压随温度变化梯度等物质属性的不同,相对液氮,液氢的热力学效应更加明显。     

Turbulent energy transfer in a nonlinear isotropic fluid

In this paper we consider a hypothetical non-Newtonian fluid, in which the usual Newtonian constitutive relationship is perturbed by the presence of a small quadratic term. It is argued that this model is worth studying because (a) such non-linearities are believed to lead to anomalous turbulent effects, (b) it is a necessary preliminary to a full treatment of a more realistic model, which takes account of fluid anisotropy. The equations of motion are derived in the Fourier wavenumber-time domain and specialised to the case of isotropic turbulence. Recent developments in the theory of turbulence are used to obtain a closed equation for the energy spectrum and this is solved for the dissipation-range energy spectrum. It is concluded that the inertial transfer of turbulent energy is reduced in such a way that the spectrum contains more energy, but falls off more rapidly, than in the Newtonian case.     

某大型商贸中心的人员疏散性能化分析

新型建筑不断涌现使得火灾安全性能化设计被广泛应用，人员疏散是其中的关键环节。结合某大型商贸中心的火灾安全性能化设计方案，利用CAFE模型分析了人员疏散性能。结果表明，由于该模型量化了人员疏散过程中人与人、人与环境之间的作用力，因此计算得到的人员疏散时间比同类软件更加保守，分析结果具有更高的可靠性。     

The risks to healthy tissues from the use of existing and emerging techniques for radiation therapy

As radical radiotherapy treatments become more effective, more and more cancer patients are becoming cured of their disease and surviving for decades. Damage to exposed healthy tissues that becomes manifest in the medium-to-long-term is becoming a more significant factor in the choice of individual treatment plans and treatment modality. However, currently there are no reliable objective methods for predicting in an individual patient the occurrence of normal tissue complications, or second cancers caused by radiation. This is especially needed as new competing techniques and modalities become available, such as IMRT, protons, carbon ions, etc., all advancing the ability to focus the radiation dose on the target while sparing normal tissue. ALLEGRO is a Euratom-funded project that is currently investigating the current state of knowledge, and attempting to define the priority research areas. Preliminary considerations of the problems to be solved and research priorities are presented.     

离散分布式动力吸振器的设计及在船舶工程中的应用

用动力吸振器控制设备的振动,对质量比有一定的要求,在工程实践中(如潜艇,飞机,车辆等),由于空间的限制,安装一个大型动力吸振器十分困难。将一个动力吸振器改为若干个较小的动力吸振器,构成所谓的“离散分布式”动力吸振器(MTMD)则容易得多。本文讨论离散分布式动力吸振器的抑振特性。分析结果说明,在小范围内符合特定非线性间隔调谐频率的离散分布式动力吸振器不仅能更好地适应空间的限制条件,而且具有更宽的消振频带和更稳定的抑振效果,在失谐条件下的减振效果也比单个动力吸振器要好,并给出了分布式动力吸振器的优化设计方法及船舶工程应用实例。     

Improved method of estimating the product quality after multiple inspections

A complex product is often inspected more than once in a sequential manner to ensure the product’s quality. Based on the number of defects discovered during each round of inspection process, we can estimate the number of defects still remaining in the product. For each defect, the probability that the defect will be detected during each inspection cycle is usually assumed to be a known ‘constant’. However, in many practical situations, some defects are easily detected, while others are much more difficult to identify. In this paper, we propose a ‘beta-geometric’ inspection model in which the heterogeneity in detection probability is described by a beta distribution. In a numerical study, we show that our more realistic inspection model clearly outperforms traditional estimation methods that are based on the assumption of a constant detection probability.