An approach for state differentiation in nucleic acid circuits: Application to diagnostic DNA computing.

BACKGROUND: Differentiating between different states in nucleic acid circuits is crucial for various biological applications. One approach, there is a requirement for complicated sequential summation, which can be excessive for practical purposes. By selectively labeling biologically significant states, this study tackles the issue and presents a more cost-effective and streamlined solution. The challenge is to efficiently distinguish between different states in a nucleic acid circuit. RESULTS: An innovative method is introduced in this study to distinguish between states in a nucleic acid circuit, emphasizing the biologically relevant ones. The circuit comprises four DNA logic gates and two detection modules, one for determining fetal gender and the other for diagnosing X-linked genetic disorders. The primary module generates a G-quadruplex DNAzyme when activated by specific biomarkers, which leads to a distinct colorimetric signal. The secondary module responds to hemophilia and choroideremia biomarkers, generating one or two DNAzymes. The absence of female fetus indicators results in no DNAzyme or color change. The circuit can differentiate various fetal states by producing one to four active DNAzymes in response to male fetus biomarkers. A single-color solution for state differentiation is provided by this approach, which promises significant advancements in DNA computing and diagnostic applications. SIGNIFICANCE: The innovative approach used in this study to distinguish states in nucleic acid circuits holds great significance. By selectively labeling biologically relevant states, circuit design is simplified and complexity is reduced. This advancement enables cost-effective and efficient diagnostic applications and contributes to DNA computing, providing a valuable solution to a fundamental problem.

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.

A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay.

Background: The current COVID-19 pandemic has highlighted the need for faster and more cost-effective diagnostic methods. The RNA extraction step in current diagnostic methods, such as real-time qPCR, increases the cost and time required for testing. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a promising technique for developing diagnostic tests with desired sensitivity and specificity without the need for RNA extraction. Materials and Methods: An RT-LAMP assay was developed to detect SARS-CoV-2 with a sensitivity of 0.5 copies of positive control plasmid per microliter in 40 min. Several rapid RNA extraction protocols were evaluated using different reagents, including bovine serum albumin, Triton X-100, Tween 20, proteinase K, guanidine hydrochloride, guanidinium isothiocyanate (GITC), and thermal treatment. Finally, the sensitivity and specificity of the developed direct RT-LAMP were determined using 150 upper respiratory tract samples. Results: Method 10 was selected as the most efficient protocol for the RNA extraction step. The sensitivity and specificity of the developed direct RT-LAMP assay with clinical samples were estimated at 98.4% and 88.8%, respectively. Conclusion: These results suggest that the combination of GITC and Triton X-100 detergent is a highly efficient method for RNA extraction and direct RT-LAMP detection of SARS-CoV-2 in clinical samples, providing a valuable tool for the rapid and cost-effective diagnosis of COVID-19.

An intelligent DNA nanorobot for detection of MiRNAs cancer biomarkers using molecular programming to fabricate a logic-responsive hybrid nanostructure.

Herein, we designed a DNA framework-based intelligent nanorobot using toehold-mediated strand displacement reaction-based molecular programming and logic gate operation for the selective and synchronous detection of miR21 and miR125b, which are known as significant cancer biomarkers. Moreover, to investigate the applicability of our design, DNA nanorobots were implemented as capping agents onto the pores of MSNs. These agents can develop a logic-responsive hybrid nanostructure capable of specific drug release in the presence of both targets. The prosperous synthesis steps were verified by FTIR, XRD, BET, UV-visible, FESEM-EDX mapping, and HRTEM analyses. Finally, the proper release of the drug in the presence of both target microRNAs was studied. This Hybrid DNA Nanostructure was designed with the possibility to respond to any target oligonucleotides with 22 nucleotides length.

Binary detection of protein and nucleic acid enabled cancer diagnosis through branched hybridization chain reaction.

Developing isothermal bio-analyzers for amplified detection of multi-factor diseases like cancer biomarkers (nucleic acid and protein) has facilitated the early diagnosis and clinical theranostics. In light of that, a sensitive detection system was developed assisted by the recognition capability of a functional aptamer followed by cyclic self-assembly of three auxiliary hairpins via branched hybridization chain reaction (b-HCR) performance. In the downstream process, in the presence of hemin, split sequences of a DNAzyme brought in close proximity to facilitate the color alteration of the solution to a green appearance. By ingenious exerting multi-level amplification, the assay empowered sensitive detection of miR-21 and PDGF-BB related cancer biomarkers with LOD values of 10 pM and 40 nM, respectively. Taken together, simplicity, enzyme-free nature, excellent generality, affordable cost without any washing steps and immobilization makes the presented system a promising analytical tool in synthetic biology, designing nanomachines and point-of-care detection in resource-constrained settings.

Borderline Boolean states improve the biosensing applications of DNA circuits.

Molecular circuits have been used in a wide range of diagnosis applications, from the detection of chemical molecules in solution to the complex processing of cell surface receptors. One of the most important challenges of these systems is the lack of distinguishability between different circuit states when each circuit state represents a specific disease. In this work, we designed a molecular amplification circuit with borderline Boolean states that each state can be distinguished with different color intensity. For this purpose, two DNA complexes and four DNA hairpin structures were designed to detect miR-218 and miR-215 biomarkers. One of the designed DNA complexes has two G-quadruplex structures and the other has only one G-quadruplex structure. In the absence of the inputs, all three G-quadruplex structures are active and produce a high-intensity signal, while in the other three states, including the presence of miR-218, the presence of miR-215, and the presence of both inputs, respectively, one, two, and zero G-quadruplex structures are active. Therefore, the designed system can identify two different biomarkers simultaneously with different signal ratios, which can easily distinguish the different states of the circuit. This strategy is very promising to identify diseases in which any combination of biomarkers leads to a particular disease.

Developing a colorimetric nucleic acid-responsive DNA hydrogel using DNA proximity circuit and catalytic hairpin assembly.

The development of powerful techniques for sensitive detection of nucleic acids has attracted much attention for fabricating accurate biosensors in various fields, such as genomics, clinical diagnostics, and forensic sciences. Up to now, different systems have been introduced, the majority of which are expensive, time-consuming, and relatively low selectivity/limit of detection. These limitations caught our attention to fabricate a nucleic acid responsive system by combining three layers of signal amplification strategy, namely a split proximity circuit (SPC), a catalytic hairpin assembly (CHA), and a DNA hydrogel. Herein, by SPC operation, two initiators and a target strand were assembled and activated the CHA reaction in the presence of three 5'-cytosine (C)-rich hairpins. Then, produced C-rich embedded three-way junction structures could form i-motif structures under acidic environment followed by a transition from sol to gel state. To acquire a quantitative and colorimetric measurement, gold nanoparticles (GNPs) were used that encapsulated and sediment by the gel formation. The resulting platform detected the target with a limit of detection of 1 pM and considerable selectivity.

Multiplex monitoring of Alzheimer associated miRNAs based on the modular logic circuit operation and doping of catalytic hairpin assembly.

In recent years, DNA logic gates have been extensively applied in developing multiplex processing platforms to provide an accurate decision on the diagnosis of multi-factor diseases. In this work, we presented a new cascaded logical operator by combining different modules for computational monitoring of four miRNAs related to Alzheimer disease (has-miR-143-3p, has-miR-18b-5p, has-miR-424-5p, and has-miR-93-5p). Herein, three sequential logic gates were programed that upon entering the miRNA inputs, delivered the trigger strand of CHA (catalytic hairpin assembly) reaction through a cyclic amplification. Afterward, the product of the CHA reaction, three-way junction, could induce the gold nanoparticles aggregation. This phenomenon led to generate a blue color of the solution that enabled visualizing and quantitative measurement of the assay. The output signals were recorded through reading the absorbance intensity transition, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Taken together, the proposed assay by taking advantage of excellent generality, naked eye observation, the 4-plex detection, simplicity, enzyme-free nature, and two-steps process without any immobilization and washing has addressed the limitation of the previous systems. Moreover, the amplified monitoring of low-abundant of target miRNAs was accomplished with a limit of detection as low as 5 pM.

Colorimetric nanoplatform for visual determination of cancer cells via target-catalyzed hairpin assembly actuated aggregation of gold nanoparticles.

According to aptamer-mediated hairpin DNA cascade amplifier and gold nanoparticles aggregation, an optical platform for cancer cells determination has been proposed. High-affinity chimeric aptamers were used for cancer cell detection and also as an initiator for beginning hairpin assembly to construct three-way junction (3WJ) nanostructures. These three hairpins were modified at 3' ends with biotin. In the presence of target cell, chimeric aptamer binds to its ligand on cell surface and initiates 3WJ nanostructures formation. These 3WJ nanostructures interact with streptavidin-modified gold nanoparticles (AuNPs) via non-covalent biotin-streptavidin interactions and create a crossover lattice of nanoparticles. This event leads to AuNPs aggregation and red-shifting. The results were confirmed by gel electrophoresis and UV-visible spectrophotometry. The dynamic range of this assay is 25 to 107 cells with a detection limit of 10 cells which is respectively 9 and 4 times more significant than the sensitivity of AuNP-based approaches without amplification and enzyme-mediated signal amplification. Graphical abstract.

Colorimetric detection of miRNA-21 by DNAzyme-coupled branched DNA constructs.

Fluctuation of nucleic acid expression and ultrasensitive and specific detection of these variations in expression is a crucial subject in molecular medicine and clinical theranostics. A novel DNAzyme-coupled branched hybridization chain reaction (b-HCR) assay is reported for efficient signal-amplified detection of miRNA in this study. This assay was composed of a translator (T) hybridized with miR-21 to initiate the first HCR by hairpin 1 (H1) and hairpin 2 (H2). The primary HCR provided a backbone chain for numerous branches budding through hairpin 3 (H3) and hairpin 4 (H4) assembles. In the presence of hemin, the G-rich domains embedded in H1 and H4 produce an active G-quadruplex DNAzyme upon exposure to a target that could catalyze the oxidation of colorless substrate to colored product. The present approach has the potential to be used for quantitative detection of miR-21 with a sensitivity and a dynamic range of 1 pM and 1 pM to 1 nM, respectively.

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.

DNA hydrogel-empowered biosensing.

DNA hydrogels as special members in the DNA nanotechnology have provided crucial prerequisites to create innovative gels owing to their sufficient stability, biocompatibility, biodegradability, and tunable multifunctionality. These properties have tailored DNA hydrogels for various applications in drug delivery, tissue engineering, sensors, and cancer therapy. Recently, DNA-based materials have attracted substantial consideration for the exploration of smart hydrogels, in which their properties can change in response to chemical or physical stimuli. In other words, these gels can undergo switchable gel-to-sol or sol-to-gel transitions upon application of different triggers. Moreover, various functional motifs like i-motif structures, antisense DNAs, DNAzymes, and aptamers can be inserted into the polymer network to offer a molecular recognition capability to the complex. In this manuscript, a comprehensive discussion will be endowed with the recognition capability of different kinds of DNA hydrogels and the alternation in physicochemical behaviors upon target introducing. Finally, we offer a vision into the future landscape of DNA based hydrogels in sensing applications.

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.

Target-triggered three-way junction in conjugation with catalytic concatemers-functionalized nanocomposites provides a highly sensitive colorimetric method for miR-21 detection.

With the great advances in DNA nanotechnology, scientists have shown interest in developing dynamic nanostructures for theranostic applications, analyte sensing and cargo delivery. Here, we present a specific enzyme-free ultrasensitive platform based on a multilayer coupled signal amplification strategy to quantify miR-21 molecule. The biosensor was integrated based on three signal amplification gadgets, namely a translator-mediated catalytic hairpin assembly (CHA), a multilayer DNA concatemer on the surface of gold decorated magnetic nanoparticle (GMNP), and a DNAzyme-mediated catalytic signal amplification. MiR-21 mediates the release of a DNA translator from an immobilized duplex to engage in a CHA reaction using three hairpins, including a GMNP-conjugated hairpin 1 (H1), biotin-labeled hairpin 2 (H2) and a GMNP-conjugated hairpin 3 (H3) to form a three-way junction (3WJ). Meanwhile, a plenty of initiator strand 0 (S0) on GMNPs - each of which has been bifunctionalized with S0/H1 or S0/H3 - drive several multilayer peroxidase-mimicking DNAzyme concatemers in the presence of two accessory oligonucleotides; strand 1 (S1) and strand 2 (S2). Since a G-rich sequence was attached at the 5'-end of S1 strand, in the presence of hemin cofactor, an active G-quadruplex DNAzyme with peroxidase activity was formed. The concatemers on the surface of GMNPs can convert a colorless substrate to a green product. The biosensor can detect as low as 1 aM of miR-21 and provide an excellent capability to discriminate single-base mismatches. The required time for the formulation of the assay reagents is about three days and the reaction time for the detection of miR-21 takes place in less than four hours.

Aptamer-integrated DNA nanoassembly: A simple and sensitive DNA framework to detect cancer cells.

The development of powerful techniques to detect cancer cells at early stages plays a notable role in diagnosing and prognosing cancer patients and reducing mortality. This paper reports on a novel functional DNA nanoassembly capable of detecting cancer cells based on structural DNA nanotechnology. DNA nanoassemblies were constructed by the self-assembly of a DNA concatemer to a plenty of sticky-ended three-way junctions. While an aptamer moiety guided the nanoassembly to the target cancer cell, the peroxidase-mimicking DNAzymes embedded in the nanoassemblies were used as the sensing element to produce colorimetric signals. As proof-of-concept, as low as 175 cancer cells were detected by the assay, and color change was clearly distinguished by the naked eyes. The proposed system enjoys potential applications for point-of-care cancer diagnosis, with its excellent sensitivity and selectivity.

Protective Effect of Neuropeptide Apelin-13 on 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Dopaminergic Cells: Involvement of Its Antioxidant and Antiapoptotic Properties.

Parkinson's disease (PD) is a severe neurodegenerative disorder characterized by the loss of brain dopaminergic neurons. Beside pharmacologic and symptomatic treatment of PD the neuroprotective therapy has recently attracted more attention. Apelin, a novel neuropeptide, and its receptors have numerous reported roles in regulating brain functions. In addition, this peptide has potent neuroprotective effects in some neurodegenerative situations. In this study, the effects of apelin-13 were investigated in a cell model of PD. Human neuroblastoma SH-SY5Y cell damage was induced by 150 µM 6-hydroxydopamine (6-OHDA) and the cells viability was examined by MTT assay. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by fluorescence spectrophotometry method. Immunoblotting analysis was also employed to evaluate cytochrome c release and caspase-3 activity. Data showed that 6-OHDA could decrease cell viability and mitochondrial membrane potential and increase intracellular ROS, cytochrome c, and cleaved caspase-3 levels. Pretreatment of SH-SY5Y cells with apelin-13 (5 and 10 nM) significantly prevented the mentioned biochemical and molecular markers of 6-OHDA-induced neurotoxicity. Furthermore, the results showed that apelin receptor and PI3K signaling contributed to the observed protective effects of apelin. The results suggest that apelin-13 has protective effects against dopaminergic neural toxicity and its antioxidant and antiapoptotic properties are involved, at least in part, in such protection.

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.

Signal Amplification Technologies for the Detection of Nucleic Acids: from Cell-Free Analysis to Live-Cell Imaging.

Due to their unique properties, such as programmability, ligand-binding capability, and flexibility, nucleic acids can serve as analytes and/or recognition elements for biosensing. To improve the sensitivity of nucleic acid-based biosensing and hence the detection of a few copies of target molecule, different modern amplification methodologies, namely target-and-signal-based amplification strategies, have already been developed. These recent signal amplification technologies, which are capable of amplifying the signal intensity without changing the targets' copy number, have resulted in fast, reliable, and sensitive methods for nucleic acid detection. Working in cell-free settings, researchers have been able to optimize a variety of complex and quantitative methods suitable for deploying in live-cell conditions. In this study, a comprehensive review of the signal amplification technologies for the detection of nucleic acids is provided. We classify the signal amplification methodologies into enzymatic and non-enzymatic strategies with a primary focus on the methods that enable us to shift away from in vitro detecting to in vivo imaging. Finally, the future challenges and limitations of detection for cellular conditions are discussed.

Implementing a two-layer feed-forward catalytic DNA circuit for enzyme-free and colorimetric detection of nucleic acids.

In the present study, a highly sensitive and specific bio-sensing platform for enzyme-free and colorimetric detection of nucleic acids has been developed. The biosensor is composed of two DNA nanostructures and two fuel strands that construct the foundation of a feed-forward catalytic DNA circuit. Upon binding the target strand to a specific DNA nanostructure, the circuit is run in order that at the end a hemin-binding aptamer, with the ability to convert a colorless substrate into a colored substance is released. Based on this strategy, 4 pM of the target DNA can be easily detected in serum samples by naked eyes after only a two-hour incubation with the circuit; meanwhile, if the incubation time is extended to 3 h, the biosensor can detect 1 pM of the target DNA. Besides the elevated sensitivity, the circuit can truly discriminate a spurious target containing one nucleotide mismatch with high specificity. Overall, the enzyme-free catalytic DNA circuit can be used as a sensitive alternative method to enzyme-based biosensors for the specific and cost-effective detection of nucleic acids.

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.