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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].
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Published in Chad A. Mirkin, Spherical Nucleic Acids, 2020
Hui Li, Bohan Zhang, Xueguang Lu, Xuyu Tan, Fei Jia, Yue Xiao, Zehong Cheng, Yang Li, Dagoberto O. Silva, Henri S. Schrekker, Ke Zhang, Chad A. Mirkin
To determine if these conjugates exhibit properties characteristic of SNAs, we first examined their stability toward DNase I, an endonuclease that primarily cleaves dsDNA. Due to the dense oligonucleotide arrangement and the high local salt concentration associated with the nucleic acid corona, SNAs exhibit enhanced resistance to certain nucleases. A Förster resonance energy transfer (FRET)-based assay was utilized to analyze nuclease stability, which involves a fluorescein-modified strand hybridized to a dabcyl (quencher)-labeled complementary strand. In this assay, upon DNase I degradation, an increase of the fluorescence signal is observed due to the separation of the fluorophore−quencher pair, which can be used to measure degradation kinetics (Fig. 96.5A). Significantly, initial degradation half-lives of the POSS- and C60-based duplexes are ca. 1.4 and 2.7 times longer, respectively, than that of the free duplex of the same sequence (Fig. 96.5B). For other SNAs shown in the literature, the half-lives are typically between 2 and 5 times longer than the free duplex [11,12,15].
Gene expression in human umbilical vein endothelial cells exposed to fine particulate matter: RNA sequencing analysis
Published in International Journal of Environmental Health Research, 2022
Zhixiang Zhou, Mengnan Qin, Sara Khodahemmati, Wenke Li, Bingyu Niu, Jiangshuai Li, Yanghua Liu, Jingfeng Gao
We treated HUVEC cells with PM2.5 at a concentration of 100 μg/mL for 24 hours, and total RNA was extracted from control and PM2.5-treated cells using a MirVana kit (AM1560, Ambion, USA) according to the manufacturer’s instructions. The quality and concentration of RNA were measured using an Agilent RNA 6000 Nano Reagents kit (Agilent Technologies, USA) by an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) prior to further processing. Extracted total RNA was first treated with DNase I to degrade any possible DNA contamination. We isolated Poly-A mRNA using the oligo (dT) magnetic beads and fragmented it into small pieces of about 200 bp. Random hexamer primers were used to synthesize the first-strand cDNA by reverse transcription, and RNase H and DNA polymerase we were added to synthesize second-strand cDNA, followed by PCR amplification. After qualitative and quantitative analysis by Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System respectively, the cDNA libraries were subjected to RNA-sequencing via Illumina HiSeqTM 2000 (BGI, Shenzhen, China).