CRISPR/Cas9-Mediated Disruption of ZNF543 Gene: An Approach Toward Discovering Its Relation to TRIM28 Gene in Parkinson's Disease.

Genetic studies of familial forms of Parkinson's disease (PD) have shown that the ZNF543 gene is a candidate gene that operates relevant to this disease. However, until now, there is no evidence for ZNF543 gene function in PD, and mechanisms resulting from its mutation have not been elucidated. Given the same genetic location of the ZNF543 gene with TRIM28 and their effects on PD pathogenesis, we surmised that ZNF543 might act as a transcription factor for TRIM28 gene expression. By knocking out the ZNF543 gene via the CRISPR/Cas9 editing platform, we assessed the functional effect of loss of expression of this gene on TRIM28 gene expression. Four sgRNAs with different PAM sequences were designed against two parts of the regulatory region of ZNF543 gene, and highly efficient disruption of ZNF543 expression in human neuroblastoma cell line was evaluated by polymerase chain reaction and T7 endonuclease assay. Moreover, evaluation of TRIM28 gene expression in ZNF543-knocked-out cells indicated a significant increase in TRIM28 gene expression, suggesting that ZNF543 probably regulates the expression of TRIM28. This approach offers a window into pinpointing the mechanism by which ZNF543 gene mutations mediate PD pathogenicity.

Role of the Probe Sequence/Structure in Developing an Ultra-Efficient Label-Free COVID-19 Detection Method Based on Competitive Dual-Emission Ratiometric DNA-Templated Silver Nanoclusters as Single Fluorescent Probes.

We report the development of a label-, antibody-, enzyme-, and amplification-free ratiometric fluorescent biosensor for low-cost and rapid (less than 12 min) diagnosis of COVID-19 from isolated RNA samples. The biosensor is designed on the basis of cytosine-modified antisense oligonucleotides specific for either N gene or RdRP gene that can form silver nanoclusters (AgNCs) with both green and red emission on an oligonucleotide via a one-step synthesis process. The presence of the target RNA sequence of SARS-CoV-2 causes a dual-emission ratiometric signal transduction, resulting in a limit of detection of 0.30 to 10.0 nM and appropriate linear ranges with no need for any further amplification, fluorophore, or design with a special DNA fragment. With this strategy, five different ratiometric fluorescent probes are designed, and how the T/C ratio, the length of the stem region, and the number of cytosines in the loop structure and at the 3' end of the cluster-stabilizing template can affect the biosensor sensitivity is investigated. Furthermore, the effect of graphene oxide (GO) on the ratiometric behavior of nanoclusters is demonstrated and the concentration-/time-dependent new competitive mechanism between aggregation-caused quenching (ACQ) and aggregation-induced emission enhancement (AIE) for the developed ssDNA-AgNCs/GO nanohybrids is proposed. Finally, the performance of the designed ratiometric biosensor has been validated using the RNA extract obtained from more than 150 clinical samples, and the results have been confirmed by the FDA-approved reverse transcription-polymerase chain reaction (RT-PCR) diagnostic method. The diagnostic sensitivity and specificity of the best probe is more than >90%, with an area under the receiver operating characteristic (ROC) curve of 0.978.

Split-luciferase complementary assay of NLRP3 PYD-PYD interaction indicates inflammasome formation during inflammation.

The NLRP3 inflammasome is a key macromolecular complex of the innate immune system that activates the inflammatory signalling cascade in response to a wide range of stimuli. Structural studies have shown that the intracellular cytosolic receptor NLRP3 oligomerizes upon stimulation and serves as a scaffold to form the ASC filaments necessary for procaspase-1 activation. Despite the abundant structural evidences on NLRP3 inflammasome, the interactions of the NLRP3 Pyrin domain and its functional relevance are poorly understood. In this study, the split luciferase complementation assay is used as an alternative approach to investigate NLRP3PYD-NLRP3PYD interactions during inflammasome formation. Since the homotypic NLRP3 interaction is mainly based on electrostatic interactions, a phosphomimetic residue (S5) at the interface of the NLRP3PYDs interactions has been mutated to show a disruptive effect on luciferase activity. According to the results presented, the designed biosensor was able to monitor the NLRP3PYD-NLRP3PYD interaction in vitro. The current reporter assay not only provides a specific NLRP3PYD-NLRP3PYD assay to study the PYD-PYD interaction in vitro, but also provides a suitable system for screening chemicals and drugs to identify activators and inhibitors of NLRP3.

Dual catalytic DNA circuit-induced gold nanoparticle aggregation: An enzyme-free and colorimetric strategy for amplified detection of nucleic acids.

An enzyme-free dual catalytic DNA circuit for amplified detection of nucleic acids has been developed. The system functions based on a cyclic self-assembly of two auxiliary hairpins (H1 and H2) and three biotinylated hairpin oligonucleotides (H3, H4 and H5), in the format of two molecular circuits. In the upstream circuit, a target initiator (I) besides H1 and H2 hairpins constructs H1-H2 duplexes that trigger the operation of a subsequent circuit. In the downstream circuit, the H1-H2 duplex initiates cascaded self-assembly reactions, produces triplex H3-H4-H5, as sensing system, and releases the H1-H2 duplex as the catalyst for the self-assembly of additional hairpins. The H3-H4-H5 triplex acts as the scaffolds for assembling and orienting the streptavidin-functionalized gold nanoparticles (SA-AuNPs) into a lattice-like arrangement that generates a DNA-SA-AuNP cross-linked network, resulting in a dramatic pale red-to-blue color change. By ingeniously engaging two catalytic circuits with feedback amplification capabilities, the system can detect the target nucleic acid with an LOD value of 5 femtomolar and unambiguously discriminate spurious targets (i.e. targets containing substitution, insertion, and deletion nucleotides) without instrumentation. Simple and convenient operation of the assay makes the DNA circuit appropriate for point-of-care monitoring in resource-constrained settings.

DNAzyme-embedded hyperbranched DNA dendrimers as signal amplifiers for colorimetric determination of nucleic acids.

A DNAzyme-embedded hyperbranched DNA dendrimer is used as a colorimetric signal amplifier in an ultrasensitive detection scheme for nucleic acids. The hyperbranched DNA dendrimers were constructed by single-step autonomous self-assembly of three structure-free DNA monomers. A cascade of self-assembly reactions between the first and second strands leads to the formation of linear DNA concatemers containing overhang flank fragments. The third strand, which bears a peroxidase-mimicking DNAzyme domain, serves as a bridge to trigger self-assembly between the first and second strands across the side chain direction. This results in a chain branching growth of the DNAzyme-embedded DNA dendrimer. This signal amplifier was incorporated into the streptavidin-biotin detection system which comprises an adaptor oligonucleotide and a biotinylated capture probe. The resulting platform is capable of detecting a nucleic acid target with an LOD as low as 0.8 fM. Such sensitivity is comparable if not superior to most of the reported enzyme-free (and even enzyme-assisted) signal amplification strategies. The DNA dendrimer based method is expected to provide a universal platform for extraordinary signal enhancement in detecting other nucleic acid biomarkers by altering the respective sequences of adaptor and capture probe. Graphical abstract Schematic of an assembly of a DNAzyme-embedded hyperbranched DNA dendrimer which operates as a signal amplifier for nucleic acids detection. The nanostructure is constructed by autonomous self-assembly of three DNA monomers. Colored letters represent each domain, and complementary domains are marked by asterisk. Domain d represents the DNAzyme sequence.

DNA Domino-Based Nanoscale Logic Circuit: A Versatile Strategy for Ultrasensitive Multiplexed Analysis of Nucleic Acids.

In recent years, the analytical application of logical nanodevices has attracted much attention for making accurate decisions on molecular diagnosis. Herein, a DNA domino-based nanoscale logic circuit has been constructed by integrating three logic gates (AND-AND-YES) for simultaneous analysis of multiple nucleic acid biomarkers. In the first AND gate, a chimeric target DNA comprising of four biomarkers was hybridized to three biomarker-specific oligonucleotides (TRs) via their 5'-end regions and to a capture probe-magnetic microparticle. After harvesting the complex, 3' overhang regions of the TRs were labeled with three distinct monolayer double-stranded (ds) DNA-gold nanoparticles (DNA-AuNPs). Upon gleaning the complex and addition of initiator oligonucleotide, a series of toehold-mediated strand displacement reactions, which are reminiscent of a domino chain, spontaneously occurred between the confined dsDNAs on the nanoparticles' surface in the second AND gate. The output of the second gate entered into the last gate and triggered an exponential hairpin assembly to form four-way junction nanostructures. The resulting nanostructures bear split parts of DNAzyme at each end of the four arms which, in the presence of hemin, form catalytic hemin/G-quadruplex DNAzymes with peroxidase activity. The smart biosensor has exhibited a turn-on signal when all biomarkers are present in the sample. In fact, should any of the biomarkers be nonexistent, the signal remains turned-off. The biosensor can detect the biomarkers with a LOD value of 100 aM and a noticeable capability to discriminate single-nucleotide substitutions.

A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7.

E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10(3) CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories.

Analysis of yeh Fimbrial Gene Cluster in Escherichia coli O157:H7 in Order to Find a Genetic Marker for this Serotype.

yeh fimbrial gene cluster encodes a type of putative fimbrial complex belonging to chaperone-usher assembly pathway. Recent studies have shown that yeh fimbrial gene cluster is present in 94 % of Escherichia coli isolates and responsible for adhesion to some abiotic surfaces. Our preliminary comparative genomic analysis of 96 complete genomes of different E. coli strains revealed that the major region of this gene cluster is unique to E. coli O157:H7 strains. To investigate the detail of the analysis, we BLAST the sequence of this gene cluster against the existing complete and draft genome sequences of different E. coli strains and other genera belonging to Enterobacteriaceae family in NCBI database. The results showed that this gene cluster is properly unique to E. coli O157:H7 strains and could be used as a stable and specific genetic signature for the identification of this serotype. In this respect, we also experimentally validated the specificity of this gene cluster for the identification of E. coli O157:H7 strains by loop-mediated isothermal amplification method.