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Published in Chad A. Mirkin, Spherical Nucleic Acids, 2020
Dwight S. Seferos, Andrew E. Prigodich, David A. Giljohann, Pinal C. Patel, Chad A. Mirkin
We prepared DNA-AuNPs and studied their nuclease stability using fluorescence spectroscopy and literature methods [16]. The DNA-AuNPs consist of a 13 ± 1 nm AuNP functionalized with a dense monolayer of oligonucleotides composed of a 20-base DNA sequence, a 10-base DNA linker, and propylthiol anchor. Each probe was allowed to hybridize with fluorescein-labeled DNA complements, and the enzyme deoxyribonuclease I (DNase I), a common endonuclease [20], was added to interrogate DNA-AuNP stability. Since DNase I is known to bind ssDNA (although with much lower affinity than dsDNA), we initially varied the nanoparticle surface coverage using duplex and single-stranded DNA. DNA-AuNPs were allowed to hybridize with different molar ratios of fluorophore-labeled complements (5, 10, 20, and 30 complements per DNA-AuNP). Hybridization was achieved by heating to 70°C and cooling for 12 h. After hybridization, the total dsDNA in each sample was adjusted to 50 nM. Next, the samples were treated with DNase I, and the rate of degradation was measured by a fluorescence-based assay. The results of these experiments reveal similar reaction rates in each sample. We conclude that dsDNA is the substrate of DNase I and the effect of ssDNA or dsDNA on the nanoparticle surface is similar. This is consistent with the ~500 times lower activity of DNase I for ssDNA [21].
Localized Surface Plasmon Resonance Spectroscopy with Applications to Chemistry
Published in Sarhan M. Musa, Nanoscale Spectroscopy with Applications, 2018
Another important application of the absorption-based colorimetric sensor is the detection of proteins. Many disease states are often associated with the presence of certain biomarker proteins or irregular protein concentrations. AuNPs have been successfully applied for colorimetric detection of proteins. A diverse range of functionalized AuNPs have been utilized for the detection of proteins [41–43]. For example, Mirkin et al. have developed a realtime colorimetric screening method for endonuclease activity by using DNA-mediated AuNP assemblies. Aggregates of gold nanoparticles interconnected by DNA duplexes are bluish-purple [44]. Cleavage of the duplexes by deoxyribonuclease I (DNase I) releases the nanoparticles, producing a bluish-purple-to-red color change (Figure 5.7). This method can be used to screen libraries of inhibitors of endonucleases in a high-throughput fashion by using either the naked eye or a simple colorimetric reader.
Methods and Protocols for In Vitro Animal Nanotoxicity Evaluation: A Detailed Review
Published in Vineet Kumar, Nandita Dasgupta, Shivendu Ranjan, Nanotoxicology, 2018
Venkatraman Manickam, Leema George, Amiti Tanny, Rajeeva Lochana, Ranjith Kumar Velusamy, M. Mathan Kumar, Bhavapriya Rajendran, Ramasamy Tamizhselvi
Comet assay is a single cell gel electrophoresis assay which is performed to check the breaks in the DNA strands in eukaryotic cells. In genotoxic evaluation of foreign materials, comet assay is commonly used, and with respect to nanomaterials, it has been extensively used to screen the genotoxicity from titanium dioxide nanoparticles, silver nanoparticles, and magnetic nanoparticles (Figure 12.15). During inflammatory and oxidative stress, as a balancing measure it is common to see cellular cytotoxicity from apoptotic death. DNA laddering assay is an agarose gel electrophoresis based visualization technique in which DNA fragments resulting from apoptosis are visualized. In apoptosis, caspase-activated DNase (CAD) cleaves DNA at inter-nucleosomal linker regions. Resulting DNA fragments of 180–185 bps and higher multiples of them are considered to be a key event during apoptosis. These fragmented DNA can be separated and visualized by using agarose gel electrophoresis, resulting in a characteristic “ladder” pattern.
GFP fusion promotes the soluble and active expression of a pea actin isoform (PEAc1) in Escherichia coli
Published in Preparative Biochemistry & Biotechnology, 2023
Shaobin Zhang, Yiqing Wang, Xin Jiang, Zhanyong Wang
Inhibiting DNase I activity is a basic characteristic of actins.[43] DNase I hydrolyzes DNA, increasing the absorption of the system at 260 nm. The inhibition is due to the spatial barrier to the interaction of Glu13 on DNase I with the substrate DNA.[44] It has been shown that amino acid residues 168–226 on actin play an important role in the interaction with DNase I and have little to do with the N and C-terminal amino acid residues in monomeric actin located on the surface of the molecule away from the DNase I binding region.[45] His-PEAc1-GFP inhibited DNase I activity more strongly than either PEAc1 or His-PEAc1 (Figure 10). All (or most) of PEAc1 and His-PEAc1 did not fold to form the correct spatial structure, could not interact with DNase I (or had weak binding to DNase I), and had little effect on their hydrolytic DNA activity. In contrast, His-tag and GFP fused in His-PEAc1-GFP did not affect the binding of PEAc1 and DNase I. Instead, GFP promoted the correct folding of PEAc1. The binding of PEAc1 with the correct spatial structure to DNase I inhibited the activity of DNase I in hydrolyzing DNA. As expected, His-GFP did not exhibit any DNase I inhibition activity. Therefore, the GFP fusion tag did not obviously influence the interaction between PEAc1 and DNase I, and it promoted the soluble expression of PEAc1.
Isolation of endophytic bacteria from the medicinal, forestal and ornamental tree Handroanthus impetiginosus
Published in Environmental Technology, 2022
Mauro Enrique Yarte, María Inés Gismondi, Berta Elizabet Llorente, Ezequiel Enrique Larraburu
Strain identification was based on 16S rRNA gene sequences. Individual colonies of isolated strains were boiled in 50 μl bidestilled water a 95°C for 15 min [21] and clarified by centrifugation. An aliquot (1 μl) of the extracted DNA was used as a source of DNA template in a PCR reaction containing primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 518R (5′-ATTACCGCGGCTGCTGG-3′), which amplify approximately 500 bp of the V1 and V3 variable regions of the 16S rRNA gene [22,23]. Each PCR reaction was performed using 1 U GoTaq DNA polymerase (Promega), 0.2 mM each dNTPs, 0.5 μM each primer, 1.5 mM MgCl2 and PCR Buffer 1X in a final volume of 20 μl. Amplifications were carried out in a thermocycler (GeneAmp PCR System 9700, Applied Biosystems) with an initial denaturation step at 94°C for 2 min; 30 cycles at 94°C for 30 s, 60°C for 30 s (hybridization) and 72°C for 40 s (elongation); and one final elongation step at 72°C for 10 min. As a positive control, we used the DNA from Azospirillum brasilense Cd (ATCC 29710). As a negative control, we replaced the DNA template with sterile DNase-free water. The PCR products were run on a 1% (w/v) agarose gel and visualized by ethidium bromide staining under UV light. A 500 bp fragment was excised, purified using the ADN Puriprep GP-Kit (INBIO Highway, Tandil, Argentina) and quantified using NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). The samples were sequenced at Unidad de Genómica, Instituto de Agrobiotecnología y Biología Molecular, INTA-CONICET, Castelar, Argentina.
Pequi enriched diets protect Drosophila melanogaster against paraquat-induced locomotor deficits and oxidative stress
Published in Journal of Toxicology and Environmental Health, Part A, 2019
Sandra Mara Duavy, Assis Ecker, Gerson Torres Salazar, Julia Loreto, José Galberto Martins Da Costa, Nilda Vargas Barbosa
The total RNA was treated with DNase I (Invitrogen), and complementary DNA (cDNA) was synthesized with M-MLV reverse transcriptase (RT) enzyme and random primers using the manufacturer’s protocol (Invitrogen). The quantitative real-time polymerase chain reaction (qRT-PCR) was performed with 20 µl PCR mixture containing 1µL RT product (cDNA) as the template, 1x PCR buffer, 25mM dNTPs, 0.2 mM of each primer of interest, 1.5–5 mM MgCl2, 0.1x SYBR Green I (molecular probes), and 1U Taq DNA polymerase (Invitrogen). The thermal cycle was carried out using a StepOne Plus real-time PCR system (Applied Biosystems, NY) according to the following protocol: activation of the Taq DNA polymerase at 95°C for 5 min, followed by 40 cycles of 15 sec at 95°C, 15 sec at 60°C, and 25 sec at 72°C. The fluorescent signal produced from the amplification was acquired at the end of the polymerization step at 72°C. Threshold and baselines were manually determined using the StepOne Software v2.3 (Applied Biosystems, NY) and the CT (cycle threshold) value for each sample was calculated and recorded using 2−ΔΔCT (Livak and Schmittgen 2001). For each well, analyzed in triplicate, the ΔCT was obtained by subtracting the GPDH CT value from the CT value of the gene of interest. Levels of gene expression in all groups are presented as a ratio of the standard control group value. The experiment was repeated three times with independently isolated RNA samples.