Reactions Of Nucleic Acids Research Articles

Allosteric stem-loop multicomponent DNAzyme: A versatile stimuli-responsive switch for nanotweezer operations and multifunctional biosensing

DNAzyme, possessing the capabilities of both encoding information and catalyzing chemical reactions, holds significant value in advancing the widespread application of dynamic nanotechnology. Particularly, multicomponent DNAzyme (MNAzyme) has attracted considerable attention due to its remarkable versatility and practicality. However, the inevitable signal leakage of MNAzyme constrains the applications in complex nucleic acid reaction networks. Herein, we present an allosteric stem-loop multicomponent DNAzyme (ASLM) that incorporates a blocking domain to mitigate leakage resulting from secondary structures of MNAzyme while simultaneously stabilizing binding affinity during complex formation. As the sequence of blocking domain serves as an extension independent of the functional region in MNAzyme, this modulation structure can be flexibly assembled with conformation-switchable nanotweezer to form a time-responsive molecular device, enabling dynamic and dissipative operations through nucleic acid and RNase H. More importantly, the ASLM structure can be directly incorporated into DNA catalytic circuits for detecting multiple biomolecules (nucleic acid, protease, and small molecule). With its scalability and versatility, this effector-activated allosteric regulator will serve as a universal stimuli-responsive switch in complex and hierarchical DNA networks, expanding the scope of DNA nanotechnology in molecular device construction and diagnostic biosensing.

Temperature control technology for PCR

AbstractIn order to solve the obvious nonlinear problem of temperature and complex structure of PCR instrument during nucleic acid amplification. In this paper, a new nucleic acid amplification device and temperature control algorithm were proposed. In the device, in order to improve the rise and fall rate and make the whole reaction device smaller and simpler, this paper uses a microfluidic chip for nucleic acid reaction. At the same time, in the warming and cooling module, the temperature is controlled by the semiconductor chilling plate, the air‐cooled cooling device and the heat sink structure, which greatly improves the speed of nucleic acid amplification. In the algorithm, a hybrid algorithm is designed, using Particle Swarm Optimization (PSO) to optimize PID algorithm parameters, and then based on fuzzy theory, according to the temperature control requirements of nucleic acid amplification, fuzzy rules are analyzed and fuzzy reasoning is carried out, and then combined with PID to achieve rapid response and overshooting control of temperature control. Finally, the measurement noise is filtered by Kalman filter. Finally, COMSOL and MATLAB software are used to simulate and compare, and it is proved that the device has a certain heat dissipation effect in the process of nucleic acid amplification. This algorithm can improve the accuracy and robustness of the control system, improve the response speed, reduce the overshoot, shorten the adjustment time, and restrain the interference.

Highly sensitive and label-free immunoassay of chloramphenicol based on poly-adenine-mediated spherical nucleic acid and hybridization chain reaction amplification

In this paper, a label-free fluorescent immunoassay for chloramphenicol was established based on a DNA-assisted signal amplification strategy. The poly-adenine mediated spherical nucleic acid was firstly employed as the substrate of antibody to construct the immunonanoprobes. Enzyme-free signal amplification techniques, hybridization chain reaction and strand displacement reaction, were utilized to develop the dual amplification strategy. The output signal was expressed through the use of G-quadruplex to achieve label free fluorescence changes. Under optimized conditions, the detection limit of this method was reduced to 0.0044 ng/L. The method exhibited a high accuracy to the detection of chloramphenicol in milk samples with a recovery rate from 94.5% to 118.1%. However, the limitations of this method probably lied in the overlong detection time and the possible interference from other compounds present in food samples. The developed label-free fluorescent immunoassay could provide an innovative view for the reliable and ultrasensitive detection of chloramphenicol, which is conducive to the food safety.

Nanopore sensitization based on a double loop hybridization chain reaction and G-quadruplex.

The widespread implementation of solid-state nanopores faces challenges such as lower resolution and increased electrical noise when compared to biological nanopores. Incorporating specific nucleic acid reactions can enhance resolution. In this study, we've developed a nucleic acid amplifier to enhance the sensitivity of solid-state nanopores, utilizing a G-rich sequence and hybridization chain reaction. This amplifier improves target concentration and volume amplification, showing promise in nanopore sensitivity tests.

High sensitivity and specificity rates of cobas® HPV test as a primary screening test for cervical intraepithelial lesions in a real-world setting.

Cervical carcinoma (CC) is the fourth most common malignancy among women. Screening with Papanicolau smear is linked to a reduction in CC incidence rates when screening programs have been developed. However, this technique has several limitations, including moderate sensitivity rates for detection of cervical preneoplastic HPV-related lesions. In this real-world study, we proposed to evaluate the sensitivity and specificity rates of cobas® test, which amplifies target DNA fragments by polymerase chain reaction and hybridization of nucleic acids for the detection of 14 HR-HPV types in a single analysis) used as primary screening test for CC and preneoplastic lesions in women aged 25-65 years in a large University Hospital in Buenos Aires. A total of 1044 patients were included in the sample (median age: 46 years); sensitivity and specificity rates for the HR-HPV test used as primary screening test were 98.66% (95% confidence interval [95CI]: 97.67-99.3%) and 87.15% (95CI: 84.93-89.15%), respectively. The positive predictive value was 88.47% (95CI: 86.54%-90.42%) and the negative predictive value was 98.48% (95CI: 97.75%-99.23%). The cobas® HR-HPV testing was highly sensitive and specific for the detection of CC and preneoplastic lesions in real practice.

The Photophysics of Nucleic Acids: Consequences for the Emergence of Life

AbstractAbsorption of ultraviolet (UV) radiation can trigger a variety of photophysical and photochemical reactions in nucleic acids. In the prebiotic era, on the surface of the early Earth, UV light could have played a major role in the selection of the building blocks of life via a balance between synthetic and destructive pathways. As nucleic acid monomers assembled into polymers, their survival and facility for non‐enzymatic replication hinged on their photostability and the ability for self‐repair of lesions, e. g., by UV‐induced charge transfer. Such photoprocesses are known to be sequence‐dependent and could have led to an additional prebiotic selection of the genetic sequence pools available to the earliest life forms. This review summarizes the photophysical processes in nucleic acids upon the absorption of a UV photon and their implications for chemical and genetic selection at the emergence of life and the origin of translation.

Recent advances in PCR-free nucleic acid detection for SARS-COV-2.

As the outbreak of Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory disease coronavirus 2 (SARS-COV-2), fast, accurate, and economic detection of viral infection has become crucial for stopping the spread. Polymerase chain reaction (PCR) of viral nucleic acids has been the gold standard method for SARS-COV-2 detection, which, however, generally requires sophisticated facilities and laboratory space, and is time consuming. This review presents recent advances in PCR-free nucleic acid detection methods for SARS-CoV-2, including emerging methods of isothermal amplification, nucleic acid enzymes, electrochemistry and CRISPR.

Recent advances of using personal glucose meter as a biosensor readout for non-glucose targets.

Personal glucose meter (PGM) has become the most successful biosensor in past decades due to its advantages of small size, convenient operation, and low cost. To take advantage of many years of research and development of PGMs, new signal transduction methods has been developed to expand the PGM from simple monitoring blood glucose to detection of numerous non-glucose targets. This review summarizes recent advance of PGM-based biosensors for non-glucose targets including signal transduction, signal amplification and target molecule recognition and analysis. Current challenges and future directions are also discussed. PGM can be used as biosensor readout to detect various non-glucose targets from metal ion, small molecule to protein and even living organisms such as bacteria and other pathogens by using different signal transduction elements such as invertase and amylase, and different signal amplification methods such as nanomaterials, nucleic acid reaction, liposome encapsulation, hydrogel trapping, DNAzyme amplification and biotin-streptavidin reaction.

Dissipative DNA nanotechnology.

DNA nanotechnology has emerged as a powerful tool to precisely design and control molecular circuits, machines and nanostructures. A major goal in this field is to build devices with life-like properties, such as directional motion, transport, communication and adaptation. Here we provide an overview of the nascent field of dissipative DNA nanotechnology, which aims at developing life-like systems by combining programmable nucleic-acid reactions with energy-dissipating processes. We first delineate the notions, terminology and characteristic features of dissipative DNA-based systems and then we survey DNA-based circuits, devices and materials whose functions are controlled by chemical fuels. We emphasize how energy consumption enables these systems to perform work and cyclical tasks, in contrast with DNA devices that operate without dissipative processes. The ability to take advantage of chemical fuel molecules brings dissipative DNA systems closer to the active molecular devices that exist in nature.

Dissipative biocatalytic cascades and gated transient biocatalytic cascades driven by nucleic acid networks.

Living systems consist of complex transient cellular networks guiding structural, catalytic, and switchable functions driven by auxiliary triggers, such as chemical or light energy inputs. We introduce two different transient, dissipative, biocatalytic cascades, the coupled glucose oxidase (GOx)/horseradish peroxidase (HRP) glucose–driven oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the radical anion (ABTS•−) and the lactate dehydrogenase (LDH)/nicotinamide adenine dinucleotide (NAD+) lactate–driven reduction of NAD+ to NADH. The transient biocatalytic systems are driven by nucleic acid reaction modules using a nucleic acid fuel strand L1′ and a nicking enzyme, Nt.BbvCI, as fuel-degrading catalyst, leading to the dynamic spatiotemporal transient formation of structurally proximate biocatalysts activating the biocatalytic cascades and transient coupled processes, including the generation of chemiluminescence and the synthesis of alanine. Subjecting the mixture of biocatalysts to selective inhibitors allows the gated transient operation of the biocatalysts. The kinetics of transient biocatalytic cascades are accompanied by kinetic models and computational simulations.

Rapid Nucleic Acid Reaction Circuits for Point-of-care Diagnosis of Diseases.

An urgent need exists for a rapid, cost-effective, facile, and reliable nucleic acid assay for mass screening to control and prevent the spread of emerging pandemic diseases. This urgent need is not fully met by current diagnostic tools. In this review, we summarize the current state-of-the-art research in novel nucleic acid amplification and detection that could be applied to point-of-care (POC) diagnosis and mass screening of diseases. The critical technological breakthroughs will be discussed for their advantages and disadvantages. Finally, we will discuss the future challenges of developing nucleic acid-based POC diagnosis.

Faradaic-free electrokinetic nucleic acid amplification (E-NAAMP) using localized on-chip high frequency Joule heating.

We present a novel Faradaic reaction-free nucleic acid amplification (NAA) method for use with microscale liquid samples. Unlike previous Joule heating methods where the electrodes produce electrolysis gaseous by-products and require both the electrodes be isolated from a sample and the venting of produced electrolysis gas, our electrokinetic Nucleic Acid Amplification (E-NAAMP) method alleviates these issues using a radio frequency (RF) alternating current electric field. In this approach, a pair of microscale thin film gold electrodes are placed directly in contact with a nucleic acid reaction mixture. A high frequency (10-40 MHz) RF potential is then applied across the electrode pair to induce a local Ohmic current within the sample and drive the sample temperature to increase by Joule heating. The temperature increase is sustainable in that it can be generated for several hours of constant use without generating any pH change to the buffer or any microscopically observable gaseous electrolysis by-products. Using this RF Joule heating approach, we demonstrate successful direct thermal amplification using two popular NAA biochemical reactions: loop-mediated isothermal amplification and polymerase chain reaction. Our results demonstrate that a simple microscale electrode structure can be used for thermal regulation for NAA reactions without observable electrolytic reactions, minimal enzyme activity loss and sustained (>50 h use per device) continuous operations without electrode delamination. As such, E-NAAMP offers substantial miniaturization of the heating elements for use in microfluidic or miniaturized NAA reaction systems.

The Limitation of the Combination of Transition State Theory and Thermodynamics for the Reactions of Proteins and Nucleic Acids.

When a reaction is accompanied by a change with the speed close to or slower than the reaction rate, a circulating reaction flow can exist among the reaction states in the macroscopic stationary state. If the accompanying change were at equilibrium in the timescale of the relevant reaction, the transition-state theory would hold to eliminate the flow.

A portable centrifugal genetic analyzer for multiplex detection of feline upper respiratory tract disease pathogens

We present a portable genetic analyzer with an integrated centrifugal disc which is equipped with a glass-filter extraction column for purifying nucleic acid (NA) and multiple reaction chambers for analyzing major feline upper respiratory tract disease (FURTD) pathogens. We targeted four kinds of FURTD including Feline herpesvirus 1 (FHV), Mycoplasma felis (MPF), Bordetella bronchiseptica (BDB), and Chlamydophila felis (CDF). The portable genetic analyzer consists of a spinning motor, two pairs of Peltier heaters, two Minco heater, fluorescent optics, a touchscreen, and software for data analysis, so loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) can be performed. The overall size of the genetic analyzer was 28 cm × 28 cm × 26 cm and the weight was 10 kg, which was deliverable for point-of-care testing (POCT). Owing to the sophisticated microchannel design and spinning program, the serial injection of the sample solution, the washing solution, and the elution solution was executed through a glass filter membrane for nucleic acid (NA) extraction, and then the cocktail with the purified genome was aliquoted into 9 reaction chambers for LAMP or PCR. The whole process for the LAMP reaction or the PCR was completed within 1.5 h. The fluorescence profiles by a scanning mode showed the matched results between the LAMP and the PCR.

High throughput quantification of short nucleic acid samples by capillary electrophoresis with automated data processing

Analysis of catalytic activity of nucleic acid enzymes is crucial for many applications, ranging from biotechnology to the search for antiviral drugs. Commonly used analytical methods for quantifying DNA and RNA reaction products based on slab-gel electrophoresis are limited in throughput, speed, and accuracy. Here we report the optimization of high throughput methods to separate and quantify short nucleic acid reaction products using DNA sequencing instruments based on capillary electrophoresis with fluorescence detection. These methods afford single base resolution without requiring extensive sample preparation. Additionally, we show that the utility of our system extends to quantifying RNA products. The efficiency and reliability of modern instruments offers a large increase in throughput but complications due to variations in migration times between capillaries required us to develop a computer program to normalize the data and quantify the products for automated kinetic analysis. The methods presented here greatly increase sample throughput and accuracy and should be applicable to many nucleic acid enzymes.

Multiplexed and single-cell detection of microRNA with plasmonic nanoparticle assemblies

We present a label-free, low cost, and miniatured biosensing platform based on the disassembly of core-satellite plasmonic nanoparticle assemblies. The rapid and selective detection of an exemplary nucleic acid biomarker, has-miRNA-210-3p, was achieved via the strand displacement nucleic acid reaction. Target binding leads to dehybridization of the DNA linkers and changes in the scattering properties of nanostructures as monitored by darkfield microscopy. We demonstrate the ability to detect microRNA expunged from single cells and the potential to multiplex discrete assemblies to enable diverse biological applicability. The work may help translate the applicability of microRNA as diagnostic biomarkers, quantitate their abundance in the microenvironment, and facilitate the study of their correlation or causation to other biomolecules at the single-cell level.

Quadruplex-Templated and Catalyzed Ligation of Nucleic Acids.

Template-guided chemical reactions between nucleic acid strands are an important process in biomedical research. However, almost all of these reactions employ an oligonucleotide-templated approach that is based on the double-helix alignment. The moderate stability of the double helix makes this approach unsuitable for many chemical reactions, so alternative nucleic acid alignment mechanisms, demonstrating higher thermal and chemical stability, are desirable. Earlier, we described a noncovalent coupling mechanism between DNA strands through a quadruplex-and-Mg2+ connection (QMC). QMC is based on G-quadruplexes and allows unusually stable and specific interactions. Herein, a novel catalytic nucleic acid reaction, based on QMC, is described. This approach uses G-tetrads as a structural and recognition element without employing Watson-Crick complementarity rules at any stage of substrate/catalyst formation or interaction between them. Quadruplex-templated ligation can be achieved through the self-ligation of two nucleic acid strands, or through a quadruplex catalyst, which forms a G-triplex and specifically connects the strands. The process is extraordinarily robust and efficient. For instance, the ligation of carbodiimide-activated substrates can proceed in boiling solutions, and complete ligation is demonstrated within a minute. The quadruplex-templated and catalyzed reactions will create new opportunities for chemical reactions requiring harsh experimental conditions.

New Promises from an Old Friend: Iodine-Rich Compounds as Prospective Energetic Biocidal Agents.

For a very long time, frequent occurrences of biocrises have wreaked havoc on human beings, animals, and the environment. As a result, it is necessary to develop biocidal agents to destroy or neutralize active agents by releasing large amounts of strong biocides which are obtained upon detonation. Iodine is an efficient biocidal agent for bacteria, fungi, yeasts, viruses, spores, and protozoan parasites, and it is the sole element in the periodic table that can destroy microbes without contaminating the environment. Based on chemical biology, the mechanism of iodine as a bactericide may arise from oxidation and iodination reactions of cellular proteins and nucleic acids. However, because of the high vapor pressure causing elemental iodine to sublime readily at room temperature, it is inconvenient to use this material in its normal solid state directly as a biocidal agent under ambient conditions. Iodine-rich compounds where iodine is firmly bonded in molecules as a C-I or I-O moiety have been observed to be among the most promising energetic biocidal compounds. Gaseous products comprised of large amounts of iodine or iodine-containing components as strong biocides are released in the decomposition or explosion of iodine-rich compounds. Because of the detonation pressure, the iodine species are distributed over a large area greatly improving the efficacy of the system and requiring considerably less effort compared to traditional biocidal methods. The commercially available tetraiodomethane and tetraiodoethene, which possess superb iodine content also have the disadvantages of volatility, light sensitivity, and chemically reactivity, and therefore, are not suitable for use directly as biocidal agents. It is absolutely critical to synthesize new iodine-rich compounds with good thermal and chemical stabilities.In this Account, we describe our strategies for the syntheses of energetic iodine-rich compounds while maintaining the maximum iodine content with concomitant stability and routes for the synthesis of oxygen-containing iodine-rich compounds to improve the oxygen balance and achieve both high-energy and high-iodine content. In the other work, which involves cocrystals, iodine-containing polymers were also summarized. It is hoped that this Account will provide guidelines for the design and syntheses of new iodine-rich compounds and a route for the development of inexpensive, more efficient, and safer iodine-rich antibiological warfare agents of the future.

Efficacy and Safety of First-Line Therapeutic Drugs with Potential for the Treatment of Novel Coronavirus Pneumonia: A Meta-Analysis

Background: Novel coronavirus pneumonia is spreading all over the world. While countries are actively dealing with the epidemic situation, the lack of specific drugs also indirectly makes the situation hard to control. At present, the internationally controversial first-line treatment drugs cover Hydroxychloroquine, Favipiravir, Remdesivir and so on. Methods: To evaluate the efficacy and safety of first-line treatment drugs for novel coronavirus pneumonia, this study investigated the turning-negative rate in the viral nucleic acid, survival rate and adverse reactions rate of novel coronavirus pneumonia patients under treated with Hydroxychloroquine, Azithromycin, Favipiravir and Remdesivir by performing meta-analysis. Findings: The result showed that hydroxychloroquine could not improve the turning-negative rate in the viral nucleic acid detection of novel coronavirus pneumonia patients compared with conventional therapy (RR=0·925, 95% CI (0·685, 1·250), P=0·61), and at the same time, it also associated with high adverse reactions rate up to 21·6%. Hydroxychloroquine combined with azithromycin can significantly increase the turning-negative rate to almost 62%, but patients’ survival rate is reduced for serious adverse reaction. The efficacy of favipiravir is stable (RR=2·70, 95% CI (1·25, 5·85), P=0·012) and its adverse reaction is under control (ES=6·10%, 95% CI (0·000, 0·121)). Remdesivir shows its therapeutic potential, but the high rate of adverse reactions (ES=58·20%, 95% CI (0·316, 0·848)) is also worthy of attention. More high-quality clinical trial data is required to investigate the efficacy and safety of first-line treatment drugs in further research. Interpretation: In summary, this article found that the clinical medication regimen for patients with mild and common COVID-19 is hydroxychloroquine 400mg/day or favipiravir 1200mg/day, and could be appropriately combined with conventional treatment, such as oxygen theraphy and carrimyclin, abidol, etc. For patients with severe COVID-19, daily use of remdesivir 100~200mg has the best effect, the treatment of hydroxychloroquine combined with azithromycin is regarded as the a last resort because of its severe adverse reaction and it can be combined with oxygen therapy together with other antibacterial drugs. And we suggest FDA to approve the favipiravir to use for patients and decrease the use of hydroxychloroquine combined with azithromycin. Funding Statement: This research work was supported by the National Natural Science Foundation of China (Grant No. 81973180 and No. 81673298), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China for Excellent Young Talents (Grant No. BK20180077) and the Drug Innovation Major Project (2019ZX09301-142.) Declaration of Interests: The authors declare no competing interests.

Catalytic and redox activity of nucleic acids at mercury electrodes: Roles of nucleobase residues

Using a series of specifically designed oligonucleotides we have identified adenine and cytosine nucleobases as residues involved in catalytic hydrogen evolution reaction (CHER) of nucleic acids at the hanging mercury drop electrode (HMDE). Due to the CHER, nucleic acids yield catalytic peak HNA allowing their label-free and reagent-less analysis at low concentrations. Additionally, our results suggest that presence of the electroactive bases (adenine and cytosine) facilitates guanine reduction which is for the first time linked to a signal measurable at the HMDE — the peak P. We assume that the peak P is connected with reduction of guanine to 7,8-dihydroguanine, of which reoxidation to guanine is detected at the electrode by the earlier described anodic peak G. Processes connected with the above mentioned signals were studied using cyclic voltammetry by inspection of dependences on experimental parameters such as negative vertex potential and pH.