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.
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.
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.
Nanoparticles have been investigated as flagging tests for the sensitive DNA recognition that can be utilized as a part of field applications to defeat restrictions. Gold nanoparticles (AuNPs) have been widely utilized due to its optical property and capacity to get functionalized with a mixed bag of biomolecules. This study exhibits the utilization of AuNPs functionalized with single-stranded oligonucleotide (AuNP-oligo test) for fast the identification of Human Papillomavirus (HPV). This test is displayed on interdigitated electrode sensor and supported by colorimetric assay. DNA conjugated AuNP has optical property that can be controlled for the applications in diagnostics. With its identification abilities, this methodology incorporates minimal effort, strong reagents and basic identification of HPV.
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.
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 study demonstrates the development of a liquid-based gate-control silicon nanowire biosensor for detection of specific single-stranded DNA (ssDNA) molecules. The sensor was fabricated using conventional photolithography coupled with an inductively coupled plasma dry etching process. Prior to the application of DNA to the device, its linear response to pH was confirmed by serial dilution from pH 2 to pH 14. Then, the sensor surface was silanized and directly aminated with (3-aminopropyl) triethoxysilane to create a molecular binding chemistry for biofunctionalization. The resulting Si‒O‒Si‒ components were functionalized with receptor ssDNA, which interacted with the targeted ssDNA to create a field across the silicon nanowire and increase the current. The sensor shows selectivity for the target ssDNA in a linear range from target ssDNA concentrations of 100 pM to 25 nM. With its excellent detection capabilities, this sensor platform is promising for detection of specific biomarkers and other targeted proteins.