Aptamers are a class of single-stranded (ss) nucleic acid molecules generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that involves iterations of time-consuming and tedious selection, amplification, and enrichment steps. To compensate for the drawbacks of conventional SELEX, we have devised an in-silico methodology that facilitates a cost-effective and facile manner of aptamer selection. Here, we report the isolation of DNA aptamers against androgen receptors (ARs) using androgen response elements (ARE) that possess natural affinity toward AR. A virtual library of ARE sequences was prepared and subjected to a stringent selection criterion to generate a sequence pool having stable hairpin conformations and high GC content. The 3D-structures of the selected ss AREs were modeled and screened through rigid docking and molecular dynamic (MD) simulation to examine their potency as potential AR binders. The predicted sequences were further validated using direct enzyme-linked aptasorbent assay (ELASA), which includes the measurement of their binding affinity, specificity, and target discrimination properties under complex biological enviroments. A short, 15 nucleotides (nts), ssDNA aptamer, termed ARapt1 with the estimated Kd value of 5.5 ± 3 nm, was chosen as the most prominent aptamer against AR based on the coherence of both the in-silico and in-vitro evaluation results. The high target-binding affinity and selectivity of ARapt1 signify its potential use as a versatile tool in diagnostic applications relevant to prostate cancer and related diseases.
Viruses with small circular ssDNA genomes encoding a replication initiator protein can infect a wide range of eukaryotic organisms ranging from mammals to fungi. The genomes of two such viruses, a cyclovirus (CyCV-SL) and gemycircularvirus (GemyCV-SL) were detected by deep sequencing of the cerebrospinal fluids of Sri Lankan patients with unexplained encephalitis. One and three out of 201 CSF samples (1.5%) from unexplained encephalitis patients tested by PCR were CyCV-SL and GemyCV-SL DNA positive respectively. Nucleotide similarity searches of pre-existing metagenomics datasets revealed closely related genomes in feces from unexplained cases of diarrhea from Nicaragua and Brazil and in untreated sewage from Nepal. Whether the tropism of the cyclovirus and gemycircularvirus reported here include humans or other cellular sources in or on the human body remains to be determined.
A small plasmid designated pAR141 was isolated from Lactococcus lactis subsp. lactis M14 and its complete 1,594 base pair nucleotide sequence was determined. Analysis of the sequence indicated that this plasmid does not carry any industrially important determinants besides the elements involved in plasmid replication and control. The transcriptional repressor CopG and replication initiation protein RepB appeared as a single operon. A small countertranscribed RNA (ctRNA) coding region was found between the copG and repB genes. The double strand origin (dso) and single strand origin (sso) of rolling circle replicating (RCR) plasmids were also identified in pAR141, suggesting that this plasmid replicates by rolling circle (RC) mode. This observation was supported by S1 nuclease and Southern hybridization analyses.
A toxicity electrochemical DNA biosensor has been constructed for the detection of carcinogens using 24 base guanine DNA rich single stranded DNA, and methylene blue (MB) as the electroactive indicator. This amine terminated ssDNA was immobilized onto silica nanospheres and deposited on gold nanoparticle modified carbon-paste screen printed electrodes (SPEs). The modified SPE was initially exposed to a carcinogen, followed by immersion in methylene blue for an optimized duration. The biosensor response was measured using differential pulse voltammetry. The performance of the biosensor was identified on several anti-cancer compounds. The toxicity DNA biosensor demonstrated a linear response range to the cadmium chloride from 0.0005 ppm to 0.01 ppm (R2 = 0.928) with a limit of detection at 0.0004 ppm. The biosensor also exhibited its versatility to screen the carcinogenicity of potential anti-cancer compounds.
DNA polymerase β is a 39kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31kDa domain responsible for the polymerase activity and an 8kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S44/55 and S55 phosphorylation were less dramatic than S44 and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is likely the major contributor to structural fluctuations that lead to loss of enzymatic activity.
In this paper, we propose a metal-polydopamine (MPDA) framework with a specific molecular probe which appears to be the most promising approach to a strong fluorescence quencher. The MPDA framework quenching ability toward various organic fluorophore such as aminoethylcoumarin acetate, 6-carboxyfluorescein (FAM), carboxyteramethylrhodamine, and Cy5 are used to establish a fluorescent biosensor that can selectively recognize Hg2+ and Ag+ ions. The fluorescent quenching efficiency was sufficient to achieve more than 96%. The MPDA framework also exhibits different affinities with ssDNA and dsDNA. In addition, the FAM-labeled ssDNA was adsorbed onto the MPDA framework, based on their interaction with the complex formed between MPDA frameworks/ssDNA taken as a sensing platform. By taking advantage of this sensor, highly sensitive and selective determination of Hg2+ and Ag+ ions is achieved through exonuclease III signal amplification activity. The detection limits of Hg2+ and Ag+ achieved to be 1.3 and 34 pM, respectively, were compared to co-existing metal ions and graphene oxide-based sensors. Furthermore, the potential applications of this study establish the highly sensitive fluorescence detection targets in environmental and biological fields.
Introduction: One of the commonly used techniques for mutation screening is High Resolution Melting (HRM) analysis. HRM is a post PCR method that relies on the detection of the fluorescent signals acquired due to the release of DNA intercalated dyes upon the melting of dsDNA to ssDNA. The method is simple, inexpensive and does not require post PCR-handling, making it suitable for high throughput screening. Methods: This study aimed to develop and validate HRM technique for the screening of two disease-associated single nucleotide polymorphisms (SNPs) namely BDNF rs6265 and DAT1 rs40184 using a total of 30 gDNA samples. The obtained results were confirmed and validated by sequencing. Results: HRM analysis showed that the predicted genotypes of BDNF rs6265 and DAT1 rs40184 among all the gDNA samples were in 100% concordance with the sequencing results, making it an accurate and sensitive method for the detection of SNPs. Conclusions: The application of HRM can accurately determine the genotype of BDNF rs6265 and DAT1 rs40184 SNPs, making it a promising tool for rapid and high-throughput screening of targeted SNPs in a large population study.
A gene sensor for rapid detection of the Human Papillomavirus 16 (HPV 16) which is associated with the appearance of cervical cancer was developed. The assay is based on voltammetric determination of HPV 16 DNA by using interdigitated electrodes modified with titanium dioxide nanoparticles. Titanium dioxide nanoparticles (NPs) were used to modify a semiconductor-based interdigitated electrode (IDE). The surface of the NPs was then functionalized with a commercial 24-mer oligomer DNA probe for HPV 16 that was modified at the 5' end with a carboxyl group. If the probe interacts with the HPV 16 ssDNA, the current, best measured at a working voltage of 1.0 V, increases. The gene sensor has has a ∼ 0.1 fM limit of detection which is comparable to other sensors. The dielectric voltammetry analysis was carried out from 0 V to 1 V. The electrochemical sensitivity of the IDE is 2.5 × 10-5 μA·μM-1·cm-2. Graphical abstract Schematic of an interdigitated electrode (IDE) modified with titanium dioxide nanoparticles for voltammetric determination of HPV 16 DNA by using an appropriate DNA probe.
Aptamers are a group of synthetic single-stranded nucleic acids. They are generated from a random library of single-stranded DNA or RNA by a technology named systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a repetitive process to select and identify suitable aptamers that show high affinity and specificity towards target cells. Great strides have been achieved in the design, construction, and use of aptamers up to this point. However, only a small number of aptamer-based applications have achieved widespread commercial and clinical acceptance. Additionally, finding more effective ways to acquire aptamers with high affinity remains a challenge. Therefore, it is crucial to thoroughly examine the existing dearth and advancement in aptamer-related technologies. This review focuses on aptamers that are generated by SELEX to detect pathogenic microorganisms and mammalian cells, as well as in cell-internalizing SELEX for diagnostic and therapeutic purposes. The development of novel aptamer-based biosensors using optical and electrical methods for microbial detection is reported. The applications and limitations of aptamers are also discussed.
The emergence of coronavirus disease 2019 (COVID-19) motivates continuous efforts to develop robust and accurate diagnostic tests to detect severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Detection of viral nucleic acids provides the highest sensitivity and selectivity for diagnosing early and asymptomatic infection because the human immune system may not be active at this stage. Therefore, this work aims to develop a label-free electrochemical DNA biosensor for SARS-CoV-2 detection using a printed circuit board-based gold substrate (PCBGE). The developed sensor used the nucleocapsid phosphoprotein (N) gene as a biomarker. The DNA sensor-based PCBGE was fabricated by self-assembling a thiolated single-stranded DNA (ssDNA) probe onto an Au surface, which performed as the working electrode (WE). The Au surface was then treated with 6-mercapto-1-hexanol (MCH) before detecting the target N gene to produce a well-oriented arrangement of the immobilized ssDNA chains. The successful fabrication of the biosensor was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). The DNA biosensor performances were evaluated using a synthetic SARS-CoV-2 genome and 20 clinical RNA samples from healthy and infected individuals through EIS. The developed DNA biosensor can detect as low as 1 copy per μL of the N gene within 5 minutes with a LOD of 0.50 μM. Interestingly, the proposed DNA sensor could distinguish the expression of SARS-CoV-2 RNA in a patient diagnosed with COVID-19 without any amplification technique. We believe that the proposed DNA sensor platform is a promising point-of-care (POC) device for COVID-19 viral infection since it offers a rapid detection time with a simple design and workflow detection system, as well as an affordable diagnostic assay.
The giant freshwater prawn holds a significant position as a valuable crustacean species cultivated in the aquaculture industry, particularly well-known and demanded among the Southeast Asian countries. Aquaculture production of this species has been impacted by Macrobrachium rosenbergii nodavirus (MrNV) infection, which particularly affects the larvae and post-larvae stages of the prawn. The infection has been recorded to cause mortality rates of up to 100% among the affected prawns. A simple, fast, and easy to deploy on-site detection or diagnostic method is crucial for early detection of MrNV to control the disease outbreak. In the present study, novel single-stranded DNA aptamers targeting the MrNV capsid protein were identified using the systematic evolution of ligands by exponential enrichment (SELEX) approach. The aptamer was then conjugated with the citrate-capped gold nanoparticles (AuNPs), and the sensitivity of this AuNP-based aptasensor for the detection of MrNV capsid protein was evaluated. Findings revealed that the aptamer candidate, APT-MrNV-CP-1 was enriched throughout the SELEX cycle 4, 9, and 12 with the sequence percentage of 1.76%, 9.09%, and 12.42%, respectively. The conjugation of APT-MrNV-CP-1 with citrate-capped AuNPs exhibited the highest sensitivity in detecting the MrNV capsid protein, where the presence of 62.5 nM of the viral capsid protein led to a significant agglomeration of the AuNPs. This study demonstrated the practicality of an AuNP-based aptasensor for disease diagnosis, particularly for detecting MrNV infection in giant freshwater prawns.
Nanoparticle-mediated bio-sensing promoted the development of novel sensors in the front of medical diagnosis. In the present study, we have generated and examined the potential of titanium dioxide (TiO2) crystalline nanoparticles with aluminium interdigitated electrode biosensor to specifically detect single-stranded E.coli O157:H7 DNA. The performance of this novel DNA biosensor was measured the electrical current response using a picoammeter. The sensor surface was chemically functionalized with (3-aminopropyl) triethoxysilane (APTES) to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO2 nanoparticles while maintaining the sensing system's physical characteristics. The complement of the target DNA of E. coli O157:H7 to the carboxylate-probe DNA could be translated into electrical signals and confirmed by the increased conductivity in the current-to-voltage curves. The specificity experiments indicate that the biosensor can discriminate between the complementary sequences from the base-mismatched and the non-complementary sequences. After duplex formation, the complementary target sequence can be quantified over a wide range with a detection limit of 1.0 x 10(-13)M. With target DNA from the lysed E. coli O157:H7, we could attain similar sensitivity. Stability of DNA immobilized surface was calculated with the relative standard deviation (4.6%), displayed the retaining with 99% of its original response current until 6 months. This high-performance interdigitated DNA biosensor with high sensitivity, stability and non-fouling on a novel sensing platform is suitable for a wide range of biomolecular interactive analyses.
Multi-wall carbon nanotubes (MWCNTs) were modified to design a new DNA biosensor. Functionalized MWCNTs were equipped with gold nanoparticles (GNPs) (~15nm) (GNP-MWCNTCOOH) to construct DNA biosensors based on carbon-paste screen-printed (SPE) electrodes. GNP attachment onto functionalized MWCNTs was carried out by microwave irradiation and was confirmed by spectroscopic studies and surface analysis. DNA biosensors based on differential pulse voltammetry (DPV) were constructed by immobilizing thiolated single-stranded DNA probes onto GNP-MWCNTCOOH. Ruthenium (III) chloride hexaammoniate [Ru(NH3)6,2Cl(-)] (RuHex) was used as hybridization redox indicator. RuHex and MWCNT interaction was low in compared to other organic redox hybridization indicators. The linear response range for DNA determination was 1×10(-21) to 1×10(-9)M with a lower detection limit of 1.55×10(-21)M. Thus, the attachment of GNPs onto functionalized MWCNTs yielded sensitive DNA biosensor with low detection limit and stability more than 30days. Constructed electrode was used to determine gender of arowana fish.
The seeds of Acalypha wilkesiana have been used empirically by traditional healers in Southwest Nigeria together with other plants as a powder mixture to treat patients with breast tumours and inflammation.
A genomic DNA-based colorimetric assay is described for the detection of the early growth factor receptor (EGFR) mutation, which is the protruding reason for non-small cell lung cancer. A DNA sequence was designed and immobilized on unmodified gold nanoparticles (GNPs). The formation of the respective duplex indicates the presence of an EGFR mutation. It is accompanied by the aggregation of the GNPs in the presence of monovalent ions, and it indicates the presence of an EGFR mutation. This is accompanied by a color change from red (520 nm) to purple (620 nm). Aggregation was evidenced by transmission electron microscopy, scanning electron microscopy and atomic force microscopy. The limit of detection is 313 nM of the mutant target strand. A similar peak shift was observed for 2.5 μM concentrations of wild type target. No significant peak shift was observed with probe and non-complementary DNA. Graphical abstract Schematic representation of high-specific genomic DNA sequence on gold nanoparticle (GNP) aggregation with sodium chloride (NaCl). It illustrates the detection method for EGFR mutation on lung cancer detection. Red and purple colors of tubes represent dispersed and aggregated GNP, respectively.
Human papillomavirus (HPV) is one of the most common sexually transmitted disease, transmitted through intimate skin contact or mucosal membrane. The HPV virus consists of a double-stranded circular DNA and the role of HPV virus in cervical cancer has been studied extensively. Thus it is critical to develop rapid identification method for early detection of the virus. A portable biosensing device could give rapid and reliable results for the identification and quantitative determination of the virus. The fabrication of electrochemical biosensors is one of the current techniques utilized to achieve this aim. In such electrochemical biosensors, a single-strand DNA is immobilized onto an electrically conducting surface and the changes in electrical parameters due to the hybridization on the electrode surface are measured. This review covers the recent developments in electrochemical DNA biosensors for the detection of HPV virus. Due to the several advantages of electrochemical DNA biosensors, their applications have witnessed an increased interest and research focus nowadays.
Aptamers are single-stranded DNA or RNA oligonucleotides generated by SELEX that exhibit binding affinity and specificity against a wide variety of target molecules. Compared to RNA aptamers, DNA aptamers are much more stable and therefore are widely adopted in a number of applications especially in diagnostics. The tediousness and rigor associated with certain steps of the SELEX intensify the efforts to adopt in silico molecular docking approaches together with in vitro SELEX procedures in developing DNA aptamers. Inspired by these endeavors, we carry out an overview of the in silico molecular docking approaches in DNA aptamer generation, by detailing the stepwise procedures as well as shedding some light on the various softwares used. The in silico maturation strategy and the limitations of the in silico approaches are also underscored.
Aptamers are short oligonucleotides that possess high specificity and affinity against their target. Generated via Systematic Evolution of Ligands by Exponential Enrichment, (SELEX) in vitro, they were screened and enriched. This review covering the study utilizing bioinformatics tools to analyze primary sequence, secondary and tertiary structure prediction, as well as docking simulation for various aptamers and their ligand interaction. Literature was pooled from Web of Science (WoS) and Scopus databases until December 18, 2020 using specific search string related to DNA aptamers, in silico, structure prediction, and docking simulation. Out of 330 published articles, 38 articles were assessed in the analysis based on the predefined inclusion and exclusion criteria. It was found that Mfold and RNA Composer web server is the most popular tool in secondary and tertiary structure prediction of DNA aptamers, respectively. Meanwhile, in docking simulation, ZDOCK and AutoDock are preferred to analyze binding interaction in the aptamer-ligand complex. This review reports a brief framework of recent developments of in silico approaches that provide predictive structural information of ssDNA aptamer.
The discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotin-streptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplification step in SELEX are reviewed.
Two isolates of a novel babuvirus causing "bunchy top" symptoms were characterised, one from abacá (Musa textilis) from the Philippines and one from banana (Musa sp.) from Sarawak (Malaysia). The name abacá bunchy top virus (ABTV) is proposed. Both isolates have a genome of six circular DNA components, each ca. 1.0-1.1 kb, analogous to those of isolates of Banana bunchy top virus (BBTV). However, unlike BBTV, both ABTV isolates lack an internal ORF in DNA-R, and the ORF in DNA-U3 found in some BBTV isolates is also absent. In all phylogenetic analyses of nanovirid isolates, ABTV and BBTV fall in the same clade, but on separate branches. However, ABTV and BBTV isolates shared only 79-81% amino acid sequence identity for the putative coat protein and 54-76% overall nucleotide sequence identity across all components. Stem-loop and major common regions were present in ABTV, but there was less than 60% identity with the major common region of BBTV. ABTV and BBTV were also shown to be serologically distinct, with only two out of ten BBTV-specific monoclonal antibodies reacting with ABTV. The two ABTV isolates may represent distinct strains of the species as they are less closely related to each other than are isolates of the two geographic subgroups (Asian and South Pacific) of BBTV.