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Biosensors and Bioelectronics (18734235)275
Emergence of recent pandemics/endemics e.g. COVID-19 and Dengue fever, demonstrated the necessity of development of strategies for swift adaptation of present biosensor for detection of the new emerging pathogens. However, development of a biosensor for a new target is time- and labor-consuming. In this study, we aimed to integrate the primer exchange reaction (PER), an isothermal technique that extends an initiator DNA with a user-defined single-stranded DNA tail, with bipolar electrochemistry. This integration led to the development of a universal biosensor, termed ViPER. We demonstrated the utility of the developed system to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic RNA as a model. The genomic RNA was reverse transcribed to a short cDNA and was tailed with a universal tag, consequently, the tagged cDNA was applied to an electrochemiluminescence integrated bipolar electrochemical biosensor (BPE-ECL). ECL signals were recorded using a digital camera and analyzed by ImageJ. The platform demonstrated a linear response over a wide dynamic range of 10−7-10−17 M for the target nucleic acid with a detection limit of 2.31 × 10−17 M for synthetic targets. The biosensor could also successfully discriminate between biological RNA samples from infected and non-infected individuals. This study introduces the potential of DNA-based visual biosensors for detecting single-stranded RNAs in low-equipped environments, and it holds promises for further development of an ultrasensitive method for various human RNA-based viral pathogens. Moreover, we can design a platform with a predetermined DNA probe sequence for a vast variety of different targets, simply by changing the PER input. © 2025 Elsevier B.V.
Scientific Reports (20452322)13(1)
In recent years, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as the cause of the coronavirus disease (COVID-19) global pandemic, and its variants, especially those with higher transmissibility and substantial immune evasion, have highlighted the imperative for developing novel therapeutics as sustainable solutions other than vaccination to combat coronaviruses (CoVs). Beside receptor recognition and virus entry, members of the SARS-CoV-2 replication/transcription complex are promising targets for designing antivirals. Here, the interacting residues that mediate protein–protein interactions (PPIs) of nsp10 with nsp16 and nsp14 were comprehensively analyzed, and the key residues’ interaction maps, interaction energies, structural networks, and dynamics were investigated. Nsp10 stimulates both nsp14’s exoribonuclease (ExoN) and nsp16’s 2′O-methyltransferase (2′O-MTase). Nsp14 ExoN is an RNA proofreading enzyme that supports replication fidelity. Nsp16 2′O-MTase is responsible for the completion of RNA capping to ensure efficient replication and translation and escape from the host cell’s innate immune system. The results of the PPIs analysis proposed crucial information with implications for designing SARS-CoV-2 antiviral drugs. Based on the predicted shared protein–protein interfaces of the nsp16-nsp10 and nsp14-nsp10 interactions, a set of dual-target peptide inhibitors was designed. The designed peptides were evaluated by molecular docking, peptide–protein interaction analysis, and free energy calculations, and then further optimized by in silico saturation mutagenesis. Based on the predicted evolutionary conservation of the interacted target residues among CoVs, the designed peptides have the potential to be developed as dual target pan-coronavirus inhibitors. © 2023, The Author(s).
Purpose and methods: The emergence of coronavirus disease 2019 (COVID-19) has once again affirmed the significant threat of respiratory infections to global public health and the utmost importance of prompt diagnosis in managing and mitigating any pandemic. The nucleic acid amplification test (NAAT) is the primary detection method for most pathogens. Loop‐mediated isothermal amplification (LAMP) is a rapid, simple, sensitive, and specific epitome of isothermal NAAT performed using a set of four to six primers. Primer design is a fundamental step in LAMP assays, with several complexities and experimental screening requirements. To address this challenge, an online database is presented here. Its workflow comprises three steps: literature aggregation, data curation, and database and website implementation. Results: LAMPPrimerBank (https://lampprimerbank.mathematik.uni-marburg.de) is a manually curated database dedicated to experimentally validated LAMP primers, their peculiarities of assays, and accompanying literature, with a primary emphasis on respiratory pathogens. LAMPPrimerBank, with its user-friendly web interface and an open application programming interface, enables the accelerated and facile exploration, comparison, and exportation of LAMP primer sequences and their respective information from the massively scattered literature. LAMPPrimerBank currently comprises LAMP primers for diagnosing viral, bacterial, and fungal respiratory pathogens. Additionally, to address the challenge of false-positive results generated by nonspecific amplifications, LAMPPrimerBank computationally predicted and visualized the sizes of LAMP products for recorded primer sets in the database. Conclusion: LAMPPrimerBank, as a pioneering database in the rapidly expanding field of isothermal NAAT, endeavors to confront the two challenges of the LAMP: primer design and discrimination of false-positive results. © 2023, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.
Iranian Journal of Science (27318095)(2)
The pandemic of severe acute respiratory syndrome 2 (SARS-CoV-2) revealed the necessity of diagnosis of the infected people to prevent the prevalence infection cycle. Many commercial pathogen diagnosis methods are based on the detection of genomic materials. Isothermal amplification methods such as loop-mediated-isothermal amplification (LAMP) are the method of choice in these cases. Reverse transcription steps are efficiently coupled to LAMP for the detection of pathogens with genomic RNAs such as SARS-CoV-2. Many detection systems for LAMP include fluorescent readout systems. Although such systems result in desirable limits of detection, the need for special instrumentation is the main dispute of such systems to become real point of care assays. In contrast, colorimetric detection methods would reduce costs and improve the applicability of the system. In this study one-step reverse transcription-LAMP reaction was established that enables visual detection of the SARS-CoV-2 genome. Nasopharyngeal RNA samples were first validated by reverse transcription quantitative polymerase chain reaction and then subjected to RT-LAMP. To lower the cost associated with the readout system equipment, malachite green (MG) was used. The color change of MG to blue allowed visual detection of the virus. Firstly, experiments were set up as two-step RT-LAMP reaction to identify the best primer sets. In addition, MG concentration was optimized with the significant colorimetric signal for the positive samples. Next, a one-step colorimetric method was developed for the detection of SARS-CoV-2 based on MG color shift in 2 h. © 2023, The Author(s), under exclusive licence to Shiraz University.
Nucleic Acids Research (03051048)(W1)
Nucleic acid cleaving DNAzymes are versatile and robust catalysts that outcompete ribozymes and protein enzymes in terms of chemical stability, affordability and ease to synthesize. In spite of their attractiveness, the choice of which DNAzyme should be used to cleave a given substrate is far from obvious, and requires expert knowledge as well as in-depth literature scrutiny. DNAzymeBuilder enables fast and automatic assembly of DNAzymes for the first time, superseding the manual design of DNAzymes. DNAzymeBuilder relies on an internal database with information on RNA and DNA cleaving DNAzymes, including the reaction conditions under which they best operate, their kinetic parameters, the type of cleavage reaction that is catalyzed, the specific sequence that is recognized by the DNAzyme, the cleavage site within this sequence, and special design features that might be necessary for optimal activity of the DNAzyme. Based on this information and the input sequence provided by the user, DNAzymeBuilder provides a list of DNAzymes to carry out the cleavage reaction and detailed information for each of them, including the expected yield, reaction products and optimal reaction conditions. DNAzymeBuilder is a resource to help researchers introduce DNAzymes in their day-to-day research, and is publicly available at https://iimcb.genesilico.pl/DNAzymeBuilder. © 2022 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research.
Sensors and Actuators B: Chemical (09254005)
Primer exchange reaction (PER) is an emergent method for non-templated synthesis of single stranded DNA molecules. PER has been shown to be effective in cell imaging systems and for detection of macromolecules. A particular application of PER is to detect a specific target nucleic acid. To this endeavor, two coupled DNA hairpins, a detector and an amplifier, play in accordance to extend a target nucleic acid with a concatemer DNA sequence. Here we introduced unified-amplifier based primer exchange reaction (UniAmPER) that beneficially extends the target by a unified-amplifier. The unified-amplifier operates as both detector and amplifier hairpins. The extension resulted in synthesis of concatemer G-rich sequences. The G-rich sequences were expected to form G-quadruplex (GQ) structures. Presence of the GQ structures were investigated by peroxidase activity of GQs in presence of hemin, H2°2 and 3,3′,5,5′-Tetramethylbenzidine (TMB) as well as by fluorescence signal generation upon intercalation of thioflavin T (ThT). The presented unified-amplifier in this study facilitates application of PER systems for development of colorimetric or fluorogenic biosensors. As a proof of principle, the method has been applied for detection of reversely transcribed cDNAs from clinical SARS-CoV-2 samples. © 2022
Journal of Applied Microbiology (13652672)130(2)pp. 493-503
Aims: Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus. Methods and Results: To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA1) and fluorescent dimeric affibody (fA2) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA1 and fA2 had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that ‘fA2’ affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the ‘fA1’ affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results. Conclusions: The results show that ‘fA1’ affiprobe and ‘fA2’ affiprobe are two new efficient luminescent affiprobes for detecting S. aureus. Significance and Impact of the Study: We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits. © 2020 The Society for Applied Microbiology
Biochemical and Biophysical Research Communications (0006291X)524(2)pp. 405-410
Deoxyribozymes or DNAzyme are identified as catalytic DNA sequences which catalyze different chemical reactions. Ligating deoxyribozymes catalyze the formation of branched and linear products. Due to the lack of efficient read-out systems, there is no report on in vivo application of ligating deoxyribozymes. To expand the biological application of branched-RNA forming deoxyribozymes, we performed our study in order to suggest a practical toolkit for measurement of in vivo real-time activity of ligating deoxyribozymes. Further in vitro studies were designed to analyze the effects of the location of branch site on reverse transcriptase (RT) interference. With this toolkit even the activity of RT was measured precisely. Our results indicate that the activity of RT enzyme significantly affected by a 17 nt branched adaptor synthesized by 10DM24 ligating deoxyribozyme. The RT stalls at or near the RNA branch point during both initiation and elongation phases. The DNA synthesis is decreased 4.3 and 2.7 fold during initiation and elongation phases respectively. In conclusion, we introduce a general and practical toolkit called “DMLR” which is based on Real-time PCR method. The use of DMLR precisely determines RT behavior when encountered with any backbone modification with the ability of stopping the enzyme activity. © 2020 Elsevier Inc.
Analytical Methods (17599660)(6)
Single stranded nucleic acids, for e.g., exosomal microRNAs, have been utilized widely for the analysis of the pathological status of individuals in recent years. Template enhanced hybridization process (TeHyP) is a promising strategy for the detection and quantification of such nucleic acid biomarkers. In the TeHyP strategy, two separate DNA strands only assemble for their performance when the target template is present. A TeHyP strategy may be combined with a split G-quadruplex peroxidase mimic to be a colorimetric assay of single stranded nucleic acids. In this study, a special case of such TeHyP was designed and investigated for its detection performance for a microRNA that was reported as a biomarker for triple negative breast cancer. The performance of the colorimetric assay was analyzed comprehensively in the presence of biological media, for e.g., blood, urine, plasma, serum, and saliva. The impact of biological media on the peroxidase mimic activity of the TeHyP system in the presence of the target or its mutants has been quantitatively investigated here. © The Royal Society of Chemistry 2020.
Journal of Nanostructures (22517871)(3)
DNA-based nano-biosensors are emerging scope in the field of biosensors. Many synthetic single stranded functional DNAs have been applied for development of such sensors, recently. Immobilization of DNA oligonucleotides on the surface of gold nanoparticles is a key step in the development of most colorimetric nano-biosensors. The bound DNA is usually thiolated and forms Au-S covalent bond to the surface of gold nanoparticles. To this endeavor, the DNA must get reduced prior to immobilization. There are a variety of approaches for reduction of thiolated DNAs that mostly employ Dithiothreitol (DTT). DTT-based DNA reduction is not always complete and the sulfhydryl DNA (DSH) is not the sole product of the reaction. The results of the reduction of a thiolated DNA with DTT is analyzed in this paper with high performance liquid chromatography (HPLC) in order to find an optimal condition for DTT based reduction. Finally, the optimal condition is compared to TCEPbased reduction for the efficiency of DNA immobilization on the surface of gold nanoparticles. © 2019, University of Kashan.
Biochimie (03009084)165pp. 161-169
Deoxyribozymes are synthetic and single stranded DNAs that are capable of catalysis of a variety of reactions, including cleavage of DNA substrates. Deoxyribozymes are usually characterized by analytical single-turnover kinetic assays, however, for many applications e.g. characterization of the reaction products, semi-preparative and preparative reactions are required. At such scales, there is a lack of comprehensive analysis and conditions that supports high amount of products in an appropriate time-scale are vaguely guessed by researchers. In this report, catalytic activity of an oxidizing DNA-cleaving deoxyribozyme, F-8(X), was comprehensively inspected in semi-preparative (10 μM substrate) scale. A 60 nucleotides long synthetic DNA sequence was selected as the target DNA for this study. The DNA sequence was originated from a single stranded DNA virus. Investigations revealed high yield in the presence of optimal concentration of oxidizing agents. The optimal conditions have been applied for scale-up of the reaction to preparative (40 μM substrate) and multi-turnover reactions to achieve highest amount of product in a cost-, time- and labor-effective manner. Such a comprehensive analysis of a deoxyribozyme's activity in semi-preparative scale provides a platform for expanded applications of DNA catalysts as a tool in chemical biology. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)
Pertanika Journal Of Science And Technology (01287680)(3)
Many colorimetric biosensors utilise citrate-capped gold nanoparticles (AuNPcit) in combination with functional macromolecules. Proper function of the utilised macromolecules is strongly dependent on the buffer systems. However, it is well known that solvents and buffers might cause aggregation of nanoparticles. A comprehensive and systematic investigation on the effect of buffer composition, concentration and pH on the aggregation of AuNPcit is reported in this study. Distinct aggregation behaviours were observed in acidic and basic pH. In acidic pH, the increase in pH, caused stabilisation of AuNPcit, while in basic pH, the stability was dependent on the ionisation degree of the applied buffer. Theoretical analyses revealed that ionic buffer species act as citrate competitors and control aggregation of AuNPcit. Understanding the fundamental principles of competition between citrate and buffer components allows scientists to choose orthogonal conditions for development of gold nanoparticle-based biosensors which guarantee stability of gold nanoparticles and proper folding of macromolecules simultaneously. © 2018 Universiti Putra Malaysia Press.
Analytical Biochemistry (00032697)
Lateral flow assays (LFAs) have promising potentials for point-of-care applications. Recently, many LFAs have been reported that are based on hybridization of oligonucleotide strands. Mostly, biotinylated capture DNAs are immobilized on the surface of a nitrocellulose membrane via streptavidin interactions. During the assay, stable colorful complexes get formed that are visible by naked eyes. Here, we present an inexpensive and unique design of LFA that applies unmodified oligonucleotides at capture lines. The presented LFA do not utilize streptavidin or any other affinity protein. We employ structural switch of molecular beacons (MB) in combination with base stacking hybridization (BSH) phenomenon. The unique design of the reported LFA provided high selectivity for target oligonucleotides. We validated potential applications of the system for detection of DNA mimics of two microRNAs in multiplex assays. © 2017 Elsevier Inc.
RSC Advances (20462069)7(86)pp. 54835-54843
The 10-23 deoxyribozyme is considered as sequence-specific "molecular scissors" for RNA molecules. Extensive investigations have been reported for this deoxyribozyme in vitro and in eukaryotic host cells. However, few investigations are reported in the literature on the activity of this deoxyribozyme inside bacterial cells. The available reports focus on the cleavage of target mRNAs that encode for proteins which are responsible for the viability of bacterial colonies. Hence, the growth of bacterial cells was blocked and the main readouts in such studies were colony counts or optical density at 600 nm. In the current study, blue-white screening was utilized as a novel readout for analysis of the activity of the 10-23 deoxyribozymes in viable bacterial cells. Two deoxyribozymes were designed to target the α-peptide fragment from β-galactosidase (lacZ) mRNA at two different positions, i.e. 5′ untranslated region and translated region. Control experiments were performed utilizing DNA oligos that lacked the catalytic core. The 3′-3 inverted thymidine modified deoxyribozymes were compared with unmodified ones to analyze the effect of such modification in prokaryotic cells. The activity of the designed deoxyribozymes caused a significant retardation in the formation of the blue-color in colonies with deoxyribozymes. Miller assay confirmed the blue-white screening results. This report showed a proof of concept for application of blue-white screening as a readout system for the activity of the 10-23 deoxyribozyme that is a model RNA-cleaving deoxyribozyme. The result of this report can promote future investigations on the activity of deoxyribozymes in prokaryotic cells. © 2017 The Royal Society of Chemistry.
Applied Biological Chemistry (24680834)(6)
In recent years, many nucleic acid-based lateral flow assays (NALFAs) have been developed for rapid and simple detection of various analytes including DNA sequences. In a NALFA, target molecules are applied within a small volume of a rehydrating buffer. The analyte flows laterally to reach the capture molecules at where it forms a colorimetric signal. Usually, in NALFAs, capture molecules are modified for maximized adsorption on the surface. In most cases, the modification is a biotin. The biotinylated capture DNA is held at capture line by interaction with streptavidin. However, there is a demand on methods that permit utilizing unmodified capture molecules and allow a cost-effective development process. Here, we report on a biotin- and streptavidin-free model NALFA. We also present a systematic investigation on the effect of various rehydrating buffers’ composition and concentration. In addition, the impacts of a protein blocker, detergents and chaotropic and kosmotropic agents on the intensity of the signal over background were analyzed. It has been demonstrated that simultaneous presence of sodium dodecyl sulfate and bovine serum albumin doubles the intensity of visible bands in the presented unmodified NALFA. Finally, this paper presents an optimized cost-effective model system that can be adapted for hybridization-based NALFAs. © 2017, The Korean Society for Applied Biological Chemistry.
Chemistry - A European Journal (09476539)(11)
Catalytic DNAs, also known as deoxyribozymes, are of practical value for the synthesis of structurally or topologically complex RNAs, but little is known about the molecular details of DNA catalysis. We have investigated a deoxyribozyme that catalyzes the formation of a specific intramolecular 2′,5′-phosphodiester bond to produce lariat RNA, which is an important biological intermediate in eukaryotic mRNA splicing. The results of combinatorial mutation interference analysis (CoMA) allowed us to shrink the catalytic core to 70% of its original length and revealed that the essential part of the deoxyribozyme sequence contained more than 50% guanosines. Nucleotide analogue interference mapping (dNAIM) and dimethyl sulfate interference (DMSi) experiments provided atomic details of individual guanosine functional groups. Additional spectroscopic experiments and structural probing data identified conformational changes upon metal-ion binding and catalysis. Overall, this comprehensive analysis of the DNA-catalyzed reaction has provided specific insights into the synthesis of 2′,5′-branched RNA, and suggested the general features of deoxyribozymes that catalyze nucleic acid ligation reactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Journal of the American Chemical Society (00027863)(22)
A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb3+ as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2′-OH groups of internal adenosines in in vitro transcribed RNA. The DNA-catalyzed 2′,5′-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ∼160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA. © 2014 American Chemical Society.
Journal of the American Chemical Society (15205126)(34)
Most deoxyribozymes (DNA catalysts) require metal ions as cofactors for catalytic activity, with Mg2+, Mn2+, and Zn2+ being the most represented activators. Trivalent transition-metal ions have been less frequently considered. Rare earth ions offer attractive properties for studying metal ion binding by biochemical and spectroscopic methods. Here we report the effect of lanthanide cofactors, in particular terbium (Tb 3+), for DNA-catalyzed synthesis of 2′,5′-branched RNA. We found up to 104-fold increased ligation rates for the 9F7 deoxribozyme using 100 μM Tb3+ and 7 mM Mg2+, compared to performing the reaction with 7 mM Mg2+ alone. Combinatorial mutation interference analysis (CoMA) was used to identify nucleotides in the catalytic region of 9F7 that are essential for ligation activity with different metal ion combinations. A minimized version of the DNA enzyme sustained high levels of Tb3+-assisted activity. Sensitized luminescence of Tb 3+ bound to DNA in combination with DMS probing and DNase I footprinting results supported the CoMA data. The accelerating effect of Tb 3+ was confirmed for related RNA-ligating deoxyribozymes, pointing toward favorable activation of internal 2′-OH nucleophiles. The results of this study offer fundamental insights into nucleotide requirements for DNA-catalyzed RNA ligation and will be beneficial for practical applications that utilize 2′,5′-branched RNA. © 2013 American Chemical Society.
Angewandte Chemie - International Edition (15213773)(45)
Complete analysis! Combinatorial mutation interference analysis (CoMA) is a highly efficient method to identify catalytically essential nucleotides in deoxyribozymes by the simultaneous assessment of all possible mutations in the active site of the catalyst. The application of CoMA for two different deoxyribozymes revealed indispensable guanosine nucleotides for DNA-catalyzed RNA ligation. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Practical application of enzymatic nucleic acids has received more attention in recent years. Understanding the mechanism of catalysis and availability of information on potentials and limitations of these enzymes expands their application scope. A general approach for characterization of functional macromolecules including enzymatic nucleic acids is to perturb a specific set of condition and to follow the perturbation effect by biophysical and biochemical methods. This chapter reviews several perturbation strategies for functional nucleic acids, including deletion, mutation, and modifications of backbone and nucleobases, and consequent kinetic analysis, spectroscopic investigations, and probing assays. In addition to single point mutation and modifications, different combinatorial approaches for perturbation interference analysis provide reliable high amounts of data in a time-effective manner. The chapter compares various combinatorial perturbation interference analysis methods, that is, combinatorial mutation interference analysis (CoMA), nucleotide analogue interference mapping for RNA and DNA (NAIM and dNAIM), chemical and enzymatic combinatorial nucleoside deletion scanning (NDS), and dimethyl sulfate interference (DMSi). © 2017, Springer International Publishing AG.
