Porous Agarose Layered Magnetic Graphene Oxide Nanocomposite for Virus RNA Monitoring in Wastewater.

The detection of virus RNA in wastewater has been established as a valuable method for monitoring Coronavirus disease 2019. Carbon nanomaterials hold potential application in separating virus RNA owing to their effective adsorption and extraction capabilities. However, carbon nanomaterials have limited separability under homogeneous aqueous conditions. Due to the stabilities in their nanostructure, it is a challenge to efficiently immobilize them onto magnetic beads for separation. Here, we develop a porous agarose layered magnetic graphene oxide (GO) nanocomposite that is prepared by agglutinating ferroferric oxide (Fe3O4) beads and GO with agarose into a cohesive whole. With an average porous size of approximately 500 nm, the porous structure enables the unhindered entry of virus RNA, facilitating its interaction with the surface of GO. Upon the application of a magnetic field, the nucleic acid can be separated from the solution within a few minutes, achieving adsorption efficiency and recovery rate exceeding 90% under optimized conditions. The adsorbed nucleic acid can then be preserved against complex sample matrix for 3 days, and quantitatively released for subsequent quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. The developed method was successfully utilized to analyze wastewater samples obtained from a wastewater treatment plant, detecting as few as 10 copies of RNA molecules per sample. The developed aMGO-RT-qPCR provides an efficient approach for monitoring viruses and will contribute to wastewater-based surveillance of community infections.

A Sensitive Technique Unravels the Kinetics of Activation and Trans-Cleavage of CRISPR-Cas Systems.

Activation of the CRISPR-Cas13a system requires the formation of a crRNA-Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR-Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real-time measurements of the dynamically formed active CRISPR-Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants (kon and koff) and dissociation constant (Kd) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real-time trans-cleavage rates of the CRISPR-Cas system and obtaining the real-time concentrations of the active CRISPR-Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7-37 °C) with fast trans-cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g., 25 °C).

Entropy-Driven Three-Dimensional DNA Nanofireworks for Simultaneous Real-Time Imaging of Telomerase and MicroRNA in Living Cells.

Real-time monitoring of different types of intracellular tumor-related biomarkers is of key importance for the identification of tumor cells. However, it is hampered by the low abundance of biomarkers, inefficient free diffusion of reactants, and complex cytoplasmic milieu. Herein, we present a stable and general method for in situ imaging of microRNA-21 and telomerase utilizing simple highly integrated dual tetrahedral DNA nanostructures (TDNs) that can naturally enter cells, which could initiate to form the three-dimensional (3D) higher-order DNA superstructures (DNA nanofireworks, DNFs) through a reliable target-triggered entropy-driven strand displacement reaction in living cells for remarkable signal amplification. Importantly, the excellent biostability, biocompatibility, and sensitivity of this approach benefited from (i) the precise multidirectional arrangement of probes with a pure DNA structure and (ii) the local target concentration enhanced by the spatially confined microdomain inside the DNFs. This strategy provides a pivotal molecular toolbox for broad applications such as biomedical imaging and early precise cancer diagnosis.

Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA.

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

Binding-Mediated Formation of Ribonucleoprotein Corona for Efficient Delivery and Control of CRISPR/Cas9.

Protein coronae formed with nanoparticles confer several useful properties. However, the non-specific nature of protein corona formation makes it difficult to deliver specific proteins for therapeutic applications. Herein, we report on the construction of a new type of protein corona, termed binding-mediated protein corona. This new corona enables the efficient and controllable delivery of functional proteins, which is otherwise challenging for conventional protein coronae. We show the design and delivery of the ribonucleoprotein corona for the CRISPR/Cas9 system. Successful gene editing in human cell lines (Hela and HEK293) demonstrates the efficient delivery, high stability, low cytotoxicity, and well-controlled activity of the Cas9-guide RNA ribonucleoprotein. The binding-mediated protein corona strategy opens up new opportunities for therapeutic protein delivery.

Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology.

We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30-39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.

Molecular Diagnosis of COVID-19: Challenges and Research Needs.

Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.

A Target-Triggered DNAzyme Motor Enabling Homogeneous, Amplified Detection of Proteins.

We report here the concept of a self-powered, target-triggered DNA motor constructed by engineering a DNAzyme to adapt into binding-induced DNA assembly. An affinity ligand was attached to the DNAzyme motor via a DNA spacer, and a second affinity ligand was conjugated to the gold nanoparticle (AuNP) that was also decorated with hundreds of substrate strands serving as a high-density, three-dimensional track for the DNAzyme motor. Binding of a target molecule to the two ligands induced hybridization between the DNAzyme and its substrate on the AuNP, which are otherwise unable to spontaneously hybridize. The hybridization of DNAzyme with the substrate initiates the cleavage of the substrate and the autonomous movement of the DNAzyme along the AuNP. Each moving step restores the fluorescence of a dye molecule, enabling monitoring of the operation of the DNAzyme motor in real time. A simple addition or depletion of the cofactor Mg2+ allows for fine control of the DNAzyme motor. The motor can translate a single binding event into cleavage of hundreds of substrates, enabling amplified detection of proteins at room temperature without the need for separation.

An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes.

Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (10(16) and 10(14)) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind preferentially to the M11 M-type of S. pyogenes. Estimated binding dissociation constants (Kd) were in the low nanomolar range for the M11 specific sequences; for example, sequence E-CA20 had a Kd of 7±1 nM. These affinities are comparable to those of a monoclonal antibody. The improved bacterial cell-SELEX technique is successful in generating aptamers selective for S. pyogenes and some of its M-types. These aptamers are potentially useful for detecting S. pyogenes, achieving binding profiles of the various M-types, and developing new M-typing technologies for non-specialized laboratories or point-of-care testing.

Methylated Phenylarsenical Metabolites Discovered in Chicken Liver.

We report the discovery of three toxicologically relevant methylated phenylarsenical metabolites in the liver of chickens fed 3-nitro-4-hydroxyphenylarsonic acid (ROX), a feed additive in poultry production that is still in use in several countries. Methyl-3-nitro-4-hydroxyphenylarsonic acid (methyl-ROX), methyl-3-amino-4-hydroxyphenylarsonic acid (methyl-3-AHPAA), and methyl-3-acetamido-4-hydroxyphenylarsonic acid (or methyl-N-acetyl-ROX, methyl-N-AHPAA) were identified in such chicken livers, and the concentration of methyl-ROX was as high as 90 µg kg-1 , even after a five-day clearance period. The formation of these newly discovered methylated metabolites from reactions involving trivalent phenylarsonous acid substrates, S-adenosylmethionine, and the arsenic (+3 oxidation state) methyltransferase enzyme As3MT suggests that these compounds are formed by addition of a methyl group to a trivalent phenylarsenical substrate in an enzymatic process. The IC50 values of the trivalent phenylarsenical compounds were 300-30 000 times lower than those of the pentavalent phenylarsenicals.

Arsenic Metabolites, Including N-Acetyl-4-hydroxy-m-arsanilic Acid, in Chicken Litter from a Roxarsone-Feeding Study Involving 1600 Chickens.

The poultry industry has used organoarsenicals, such as 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, ROX), to prevent disease and to promote growth. Although previous studies have analyzed arsenic species in chicken litter after composting or after application to agricultural lands, it is not clear what arsenic species were excreted by chickens before biotransformation of arsenic species during composting. We describe here the identification and quantitation of arsenic species in chicken litter repeatedly collected on days 14, 24, 28, 30, and 35 of a Roxarsone-feeding study involving 1600 chickens of two strains. High performance liquid chromatography separation with simultaneous detection by both inductively coupled plasma mass spectrometry and electrospray ionization tandem mass spectrometry provided complementary information necessary for the identification and quantitation of arsenic species. A new metabolite, N-acetyl-4-hydroxy-m-arsanilic acid (N-AHAA), was identified, and it accounted for 3-12% of total arsenic. Speciation analyses of litter samples collected from ROX-fed chickens on days 14, 24, 28, 30, and 35 showed the presence of N-AHAA, 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), inorganic arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), and ROX. 3-AHPAA accounted for 3-19% of the total arsenic. Inorganic arsenicals (the sum of As(III) and As(V)) comprised 2-6% (mean 3.5%) of total arsenic. Our results on the detection of inorganic arsenicals, methylarsenicals, 3-AHPAA, and N-AHAA in the chicken litter support recent findings that ROX is actually metabolized by the chicken or its gut microbiome. The presence of the toxic metabolites in chicken litter is environmentally relevant as chicken litter is commonly used as fertilizer.

Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing.

The occurrence of a large number of diverse arsenic species in the environment and in biological systems makes it important to compare their relative toxicity. The toxicity of arsenic species has been examined in various cell lines using different assays, making comparison difficult. We report real-time cell sensing of two human cell lines to examine the cytotoxicity of fourteen arsenic species: arsenite (AsIII), monomethylarsonous acid (MMAIII) originating from the oxide and iodide forms, dimethylarsinous acid (DMAIII), dimethylarsinic glutathione (DMAGIII), phenylarsine oxide (PAOIII), arsenate (AsV), monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV), monomethyltrithioarsonate (MMTTAV), dimethylmonothioarsinate (DMMTAV), dimethyldithioarsinate (DMDTAV), 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox), and 4-aminobenzenearsenic acid (p-arsanilic acid, p-ASA). Cellular responses were measured in real time for 72hr in human lung (A549) and bladder (T24) cells. IC50 values for the arsenicals were determined continuously over the exposure time, giving rise to IC50 histograms and unique cell response profiles. Arsenic accumulation and speciation were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS). On the basis of the 24-hr IC50 values, the relative cytotoxicity of the tested arsenicals was in the following decreasing order: PAOIII≫MMAIII≥DMAIII≥DMAGIII≈DMMTAV≥AsIII≫MMTTAV>AsV>DMDTAV>DMAV>MMAV≥Rox≥p-ASA. Stepwise shapes of cell response profiles for DMAIII, DMAGIII, and DMMTAV coincided with the conversion of these arsenicals to the less toxic pentavalent DMAV. Dynamic monitoring of real-time cellular responses to fourteen arsenicals provided useful information for comparison of their relative cytotoxicity.

Methylated and thiolated arsenic species for environmental and health research - A review on synthesis and characterization.

Hundreds of millions of people around the world are exposed to elevated concentrations of inorganic and organic arsenic compounds, increasing the risk of a wide range of health effects. Studies of the environmental fate and human health effects of arsenic require authentic arsenic compounds. We summarize here the synthesis and characterization of more than a dozen methylated and thiolated arsenic compounds that are not commercially available. We discuss the methods of synthesis for the following 14 trivalent (III) and pentavalent (V) arsenic compounds: monomethylarsonous acid (MMAIII), dicysteinylmethyldithioarsenite (MMAIII(Cys)2), monomethylarsonic acid (MMAV), monomethylmonothioarsonic acid (MMMTAV) or monothio-MMAV, monomethyldithioarsonic acid (MMDTAV) or dithio-MMAV, monomethyltrithioarsonate (MMTTAV) or trithio-MMAV, dimethylarsinous acid (DMAIII), dimethylarsino-glutathione (DMAIII(SG)), dimethylarsinic acid (DMAV), dimethylmonothioarsinic acid (DMMTAV) or monothio-DMAV, dimethyldithioarsinic acid (DMDTAV) or dithio-DMAV, trimethylarsine oxide (TMAOV), arsenobetaine (AsB), and an arsenicin-A model compound. We have reviewed and compared the available methods, synthesized the arsenic compounds in our laboratories, and provided characterization information. On the basis of reaction yield, ease of synthesis and purification of product, safety considerations, and our experience, we recommend a method for the synthesis of each of these arsenic compounds.

p-Azidophenylarsenoxide: An Arsenical "Bait" for the In Situ Capture and Identification of Cellular Arsenic-Binding Proteins.

Identification of arsenic-binding proteins is important for understanding arsenic health effects and for developing arsenic-based therapeutics. We report here a strategy for the capture and identification of arsenic-binding proteins in living cells. We designed an azide-labeled arsenical, p-azidophenylarsenoxide (PAzPAO), to serve bio-orthogonal functions: the trivalent arsenical group binds to cellular proteins in situ, and the azide group facilitates click chemistry with dibenzylcyclooctyne. The selective and efficient capture of arsenic-binding proteins enables subsequent enrichment and identification by shotgun proteomics. Applications of the technique are demonstrated using the A549 human lung carcinoma cells and two in vitro model systems. The technique enables the capture and identification of 48 arsenic-binding proteins in A549 cells incubated with PAzPAO. Among the identified proteins are a series of antioxidant proteins (e.g., thioredoxin, peroxiredoxin, peroxide reductase, glutathione reductase, and protein disulfide isomerase) and glyceraldehyde-3-phosphate dehydrogenase. Identification of these functional proteins, along with studies of arsenic binding and enzymatic inhibition, points to these proteins as potential molecular targets that play important roles in arsenic-induced health effects and in cancer treatment.

The Effects of SELEX Conditions on the Resultant Aptamer Pools in the Selection of Aptamers Binding to Bacterial Cells.

Aptamers of high affinity and specificity have a wide range of analytic and clinical applications. Selection of DNA or RNA aptamer molecules usually involves systematic evolution of ligands via exponential enrichment (SELEX), in which a random DNA or RNA library is incubated with a target molecule, and the oligonucleotides that bind the target are then separated from the nonbinders, PCR amplified, and used as refined libraries in the next round of selection. Conventional SELEX methodologies require the use of purified target molecules and their immobilization onto a solid support. However, purified targets from cells are not always available, and fixing the target to a support may alter its conformation. To overcome these problems, we have developed a SELEX technique using live bacterial cells in suspension as targets, for selecting DNA aptamers specific to cell-surface molecules. Through the selection of aptamers binding to Lactobacillus acidophilus and Streptococcus pyogenes, we report here optimization of this technique and show how varying selection conditions impact the characteristics of resultant aptamer pools, including the binding affinity, selectivity, and the secondary structures. We found that the use of larger starting library sequence diversity, gel purification of the subsequent pools, and the introduction of counter-selection resulted in a more efficient SELEX process and more selective aptamers. A SELEX protocol with lower starting sequence diversity, the use of heat denaturation, and the absence of counter-selection still resulted in high-affinity aptamer sequences specific to the target cell types; however, the SELEX process was inefficient, requiring 20 rounds, and the aptamers were not specific to the strain of the bacterial cells. Strikingly, two different SELEX methodologies yielded the same sequence that bound strongly to the target S. pyogenes cells, suggesting the robustness of the bacterial cell-SELEX technique.

Binding-Induced DNA Nanomachines Triggered by Proteins and Nucleic Acids.

We introduce the concept and operation of a binding-induced DNA nanomachine that can be activated by proteins and nucleic acids. This new type of nanomachine harnesses specific target binding to trigger assembly of separate DNA components that are otherwise unable to spontaneously assemble. Three-dimensional DNA tracks of high density are constructed on gold nanoparticles functionalized with hundreds of single-stranded oligonucleotides and tens of an affinity ligand. A DNA swing arm, free in solution, is linked to a second affinity ligand. Binding of a target molecule to the two ligands brings the swing arm to AuNP and initiates autonomous, stepwise movement of the swing arm around the AuNP surface. The movement of the swing arm, powered by enzymatic cleavage of conjugated oligonucleotides, cleaves hundreds of oligonucleotides in response to a single binding event. We demonstrate three nanomachines that are specifically activated by streptavidin, platelet-derived growth factor, and the Smallpox gene. Substituting the ligands enables the nanomachine to respond to other molecules. The new nanomachines have several unique and advantageous features over DNA nanomachines that rely on DNA self-assembly.

DNAzyme motor systems and logic gates facilitated by toehold exchange translators.

DNAzyme motor systems using gold nanoparticles (AuNPs) as scaffolds are useful for biosensing and in situ amplification because these systems are free of protein enzymes, isothermal, homogeneous, and sensitive. However, detecting different targets using the available DNAzyme motor techniques requires redesigns of the DNAzyme motor. We report here a toehold-exchange translator and the translator-mediated DNAzyme motor systems, which enable sensitive responses to various nucleic acid targets using the same DNAzyme motor without requiring redesign. The translator is able to efficiently convert different nucleic acid targets into a specific output DNA that further activates the pre-silenced DNAzyme motor and consequently initiates the autonomous walking of the DNAzyme motor. Simply adjusting the target-binding region of the translator enables the same DNAzyme motor system to respond to various nucleic acid targets. The translator-mediated DNAzyme motor system is able to detect as low as 2.5 pM microRNA-10b and microRNA-21 under room temperature without the need of separation or washing. We further demonstrate the versatility of the translator and the DNAzyme motor by successful construction and operation of four logic gates, including OR, AND, NOR, and NAND logic gates. These logic gates use two microRNA targets as inputs and generate amplified fluorescence signals from the operation of the same DNAzyme motor. Incorporation of the toehold-exchange translator into the DNAzyme motor technology improves the biosensing applications of DNA motors to diverse nucleic acid targets.