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DNA/RNA Nanoparticles Structures for siRNA Delivery Applications
Published in Loutfy H. Madkour, Nanoparticle-Based Drug Delivery in Cancer Treatment, 2022
Hong et al. demonstrated RCA-based enzymatic amplification of DNA nanostructures for the delivery of siRNAs (Figure 3.7). A self-assembled Y shaped DNA structure (159 nt) was used as the closed circular template for RCA to generate long amplified DNA products. Site-specific cleavage can be achieved by containing PstI endonuclease-specific sequences in the open loop of the Y-DNA junction. Intermolecular and intramolecular self-assembly of an elongated ss DNA product can form hybridization of palindromic PstI sites without the addition of helper DNA strands. The occurrence of the PstI palindromes in two otherwise unrelated DNA species is consistent with the hypothesis that they are related to mobile DNA sequences that either propagate or were once capable of propagating within mitochondria. The Neurospora PstI palindromes are found flanking genetic elements (e.g. the small and large rRNA genes, protein genes, and tRNA genes) as well as within the large rRNA gene new opposite end of the 2.3-kilobase intervening sequence. The presence of PstI palindromic sequences in two otherwise unrelated DNA species, Neurospora mtDNA and the Mauriceville plasmid, could be accounted for by common ancestry, by a common mechanism for intramolecular repetition of DNA sequences, or by some type of recombination event (e.g. transposition). Further insight into the function of the PstI elements may come when the Mauriceville plasmid has been more fully characterized with respect to genetic function and location of genetic elements such as the DNA replication origin and promoters. After treatment with PstI enzymes, the long ss DNA product is site-specifically cleaved to generate individual DNA fragments, which can later self-assemble to form Y-DNA nanostructures. The overhang sequences on each arm have been designed to form stable hybridization with FA-conjugated siRNAs. In this study, 1 pmol DNA template was amplified to approximately 1068 pmol elongated ss DNA, and 213 pmol of Y-DNA structure was produced. This approach is widely useful as a simple platform for the large-scale synthesis of various DNA nanostructures for therapeutic applications [104].
Heterologous expression of azurin from Pseudomonas aeruginosa in food-grade Lactococcus lactis
Published in Preparative Biochemistry and Biotechnology, 2019
For amplification of azurin gene, forward and reverse primers were designed according to the sequence of P. aeruginosa azurin (GenBank Accession No. M30389.1) by considering the recognition sites of restriction enzymes (F primer containing PstI digestion site: 5′-GTCTGCAGATGCTACGTAAACTCG-3′; R primer containing KpnI digestion site: 5′-TAGGTACCTCACTTCAGGGTCAGG-3′. The restriction enzyme sites were underlined). After genomic DNA of P. aeruginosa was isolated by PureLink™ Genomic DNA Mini Kit (Invitrogen, Waltham, MA), azurin gene was amplified by PCR using the genomic DNA and the primers. The PCR conditions were 4 min at 94 °C; 35 cycles of 1 min at 94 °C, 1 min at 54 °C, 1 min at 72 °C; 10 min at 72 °C. The PCR products were identified by 1% agarose gel electrophoresis. The azurin fragment was recovered using MEGAquick-spin™ Total Fragment DNA Purification Kit (Intron, Seongnam-si, Korea).
Engineered Pseudomonas putida for biosynthesis of catechol from lignin-derived model compounds and biomass hydrolysate
Published in Preparative Biochemistry & Biotechnology, 2022
T4 DNA ligase, restriction enzymes (EcoRI, HindIII, kpnI, BamHI, PstI), shrimp alkaline phosphatase were purchased from New England Biolabs USA. PrimeSTAR Max DNA polymerase (2×) high fidelity PCR master-mix was purchased from Clontech (DSS TaKaRa Bio India Pvt Ltd.). Dream Taq Green PCR Master Mix (2×) was procured from ThermoFisher Scientific (India). NucleoSpin® Gel and PCR Clean-up kit was purchased from Macherey-Nagel (MN, India). Miniprep kit was purchased from GeneAll USA. Primers were procured from Eurofins Genomics, India.