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From Computational RNA Structure Prediction to the Design of Biologically Active RNA-Based Nanostructures
Published in Peixuan Guo, Kirill A. Afonin, RNA Nanotechnology and Therapeutics, 2022
W.K. Kasprzak, Bruce A. Shapiro
From the structural point of view RNA is a polymer chain containing a combination of four nucleotides with a directionality corresponding to the natural order of RNA chain synthesis (transcription), from the 5′ to 3′ end. The bases that give nucleotides their names, usually referred to by the single letters A, G, C, and U, are Adenine, Guanine, Cytosine, and Uracil. A primary structure of RNA is defined by a sequence of nucleotides. Conceptually single-stranded, RNA chains are sufficiently flexible to fold onto themselves and base pair Gs with Cs and As with Us in the canonical, Watson-Crick scenario. An additional, so-called “wobble” base pair G-U is also frequent. In general, all kinds of non-canonical interactions are found in natural RNAs. The pattern of paired and unpaired nucleotides defines the secondary structure of a sequence. Pairing interactions stabilize the folded RNA. Unpaired regions, out of which emanate two or more helices (or branches), are called loops, and they tend to destabilize the secondary structure.
Recombinant DNA technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
For example, a poly-A tail may be added to the 3′-end of the RNA to make it analogous to eukaryotic mRNA (oligo-T is now used as primer). This reaction is catalyzed by the enzyme poly-A polymerase. The appropriate oligonucleotide primer (oligo-T for eukaryotic mRNA) is annealed with the mRNA. These primers will base pair to the 3′-end of mRNA. Reverse transcriptase extends the 3′-end of the primer using the mRNA molecule as a template. This produces an RNA—DNA hybrid molecule, the DNA strand being the cDNA. The RNA strand is digested by either RNase H or alkaline hydrolysis. This frees the single-stranded cDNA. Curiously, the 3′-end of this cDNA serves as its own primer and provides the free 3′-OH required for the synthesis of its complementary strand. Therefore, a primer is not required for this step. The complementary strand of the cDNA single strand is synthesized either by the reverse transcriptase itself or by E. coli DNA polymerase. This generates a hairpin loop in the cDNA. The hairpin loop is cleaved by a single strand specific nuclease to yield a regular DNA duplex.
Use of Recombinant DNA Technology for Engineering Mammalian Cells to Produce Proteins
Published in Anthony S. Lubiniecki, Large-Scale Mammalian Cell Culture Technology, 2018
The 3′ end of eukaryotic mRNA is formed by polyandenylation, which involves cleavage of the precursor mRNA at a specific site and then polymerization of about 200 adenylate residues, poly (A), to the newly generated 3′ end (151). Removal of the polyadenylation site decreases expression up to 10-fold (152). Two sequences important for polyadenylation have been identified. The first is a highly conserved hexanucleotide AAUAAA, present 11–30 nucleotides upstream of most polyadenylation sites, which forms the recognition sequence for the cleavage and polyadenylation reaction. Deletions or point mutations in this sequence prevent the appearance of properly polyadenylated mRNA in vivo (153–156). There is also a requirement for a sequence downstream of the poly (A) site for efficient cleavage and polyadenylation (157, 158). A loose consensus sequence for this potential second element was identified as either a U-rich or a G + U-rich tract (159). However, removal of this sequence in some instances has no effect on the efficiency of polyadenylation (160) but may influence the position of 3′ processing (161). This sequence appears to be required for the formation of a precleavage complex (162).
Diabetic retinopathy progression associated with haplotypes of two VEGFA SNPs rs2010963 and rs699947
Published in Egyptian Journal of Basic and Applied Sciences, 2023
Haider Ali Alnaji, Rabab Omran, Aizhar H. Hasan, Mohammed Qasim Al Nuwaini
Genotyping. Genomic DNA was extracted from whole blood in EDTA tubes using G-spin™ Total DNA Extraction Mini Kit (iNtRON Biotechnology, Korea) and stored at −20°C in the biotechnology laboratory of Babylon University until the genotyping. The genotyping of the SNPs in the study were tetra primers ARMS-PCR technique. In brief, the outer primers of rs2010963 and rs699947 SNPs generate the confirmative amplicon for the 5’ UTR and promoter region where the SNPs resides. The inner reverse primers were designed for the mutant allele by mismatching the 3’ end of the primer. Also, the third nucleotide of the 3’ end was mismatched to increase the specificity. The PCR steps were as follows: 40 cycles of denaturation at 95°C for 35 s and annealing at 65°C decreased gradually every cycle to reach the lowest annealing at 58°C for 40 s and 72°C for extension and final extension for 40 s and 7 min respectively. The PCR cycles are preceded by initial denaturation at 95°C for 2 min for activation. Followed by agarose gel electrophoresis (2% and 75 V) for 1 hour. The SNPgen® tool was used to design rs2010963 primers, whereas rs699947 primers were obtained from an Elfaki et al. study [16], as shown in Table 1.
Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications
Published in Preparative Biochemistry & Biotechnology, 2023
Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain, Sumit K. Singh
The selection of terminator sequence also plays an important role in regulating recombinant protein synthesis. In most organisms, TAA, TAG, and TGA are utilized as stop codons, but in E. coli, TAA is more preferentially used than the other two codons.[61] Efficient termination of transcription may lead to lower cellular energy expenditure reducing the host cell’s metabolic load.[41] There is one more additional advantage of the transcription terminator as it forms a secondary structure at the 3′ end of mRNA, which helps improve mRNA stability and thereby enhances recombinant protein production.[62] Occasionally, the translocation of the protein to the periplasm is required for proper protein folding and formation of disulfide bonds. Signal peptides are added before target gene to direct the movement of the protein toward the periplasm. Some plasmids such as p5 series of pMAL vector and pET-22 vector contain the signal peptide at the 5′ region of the MCS. Addition of a signal peptide upstream of the target gene can improve the recombinant protein expression.[63]
Re-Analysis of Non-Small Cell Lung Cancer and Drug Resistance Microarray Datasets with Machine Learning
Published in Cybernetics and Systems, 2023
Çiğdem Erol, Tchare Adnaane Bawa, Yalçın Özkan
All genes obtained as a result of the analyzes and their distribution according to frequencies are shared in the findings section (Tables 2 and 3). It is thought that genes with high frequency in the same data set should also be considered as potential candidates. As a result; ELOVL7, HMGA2, SAT1, RRM1, IER3, SLC7A11, and U2AF1 genes were found in at least 2 different datasets. Pathways for 7 genes obtained as a result of our research and their links are given in parentheses; ELOVL7 (Synthesis of very long-chain fatty acyl-CoAs), HMGA2 (Formation of Senescence-Associated Heterochromatin Foci), SAT1 (Interconversion of polyamines, Arginine and Proline metabolism), RRM1 (Glutathione metabolism, Pyrimidine metabolism, Purine metabolism, Mitochondrial DNA Depletion Syndrome-3), IER3 (PI5P, PP2A, and IER3 Regulate PI3K/AKT Signaling, Gastrin_CCK2R_240212), SLC7A11 (Amino acid transport across the plasma membrane, Basigin interactions, Transport of inorganic cations/anions and amino acids/oligopeptides), U2AF1 (Transport of Mature mRNA derived from an Intron-Containing Transcript, pre-mRNA splicing, RNA Polymerase II Transcription Termination, mRNA 3′-end processing).