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RNA
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Concerning the 3′-terminal sequence, Sugiyama (1965) showed that alkaline hydrolysis of MS2 RNA releases a single nucleoside, namely adenosine. Thus, the genome was recognized as a linear, not a circular polynucleotide. Labeling of the 3′-end with tritiated borohydride allowed isolation of an oligonucleotide containing the labeled 3′ after hydrolysis with RNase T1. The viral RNA was uniformly labeled with 32P and terminally labeled with 3H, and only a single 3H-labeled peak of 8 to 10 residues was obtained after enzymatic digestion. The last step consisted of the actual nucleotide sequence determination that was achieved by partial hydrolysis with venom exonuclease, separation of the partial products, and determination of the nucleotide composition GUUACCACCCAOH -3′ (De Wachter and Fiers 1967). Weith and Gilham (1967) independently came to the same sequence for the 3′-end of the f2 RNA.
Analysis of Small RNA Species: Phylogenetic Trends
Published in S. K. Dutta, DNA Systematics, 2019
Mirko Beljanski, Liliane Le Goff
Plasmid containing mini-cells synthesize the RNA of about 4S to a considerable extent.87 Multiple drug-resistant plasmid NR1 codes for about 10 small RNAs ranging from 60 to 120 nucleotides. They have been characterized by RNase T1 fingerprinting. Some hybridize with RN1 DNA digested with restrition endonuclease, indicating that they originate in “the resistance transfer factor region of the plasmid genome.” Several of these small RNAs are associated with DNA fragments that contain origins of replication.86 One stable species of these RNAs migrates at the position of host tRNA. This component contains about 75 nucleotides carrying terminal-CCA residues at 3′. No modified bases have been detected. Thus, this small RNA is not tRNA. Since it has a-CCA residue, one should determine if it is capable of binding amino acids. Two 5S rRNA have been identified. They have no similarity with E. coli 5S rRNA.86 Two RNA species contain 60 to 65 nucleotides possessing pUUAAGp at 5′, which indicates that they were not primary transcripts; they possibly resulted through nuclease action on larger RNAs (absence of 5′-PPP). It has been suggested that they might serve as primers for plasmid DNA replication. Curiously enough, a small RNA of about 60 nucleotides has been found solely in the nucleus of plasmid-infected cells. Using a constructed plasmid DNA carrying the 32 nucleotide intervening sequence, it was possible to demonstrate that this small RNA is complementary to an intervening sequence.88 Sixty nucleotide RNA does not possess a 5′-terminal cap. In contrast to the nucleus, the cytoplasm of uninfected cells contains several small RNA species: 185, 160, 87, 77, and 73 nucleotides. The most abundant is an RNA with 185 nucleotides. These RNAs would seem to act in the translational process. However, direct proof is still unavailable.
Tear Film miRNAs and Their Association With Human Dry Eye Disease
Published in Current Eye Research, 2022
Andrew D. Pucker, William Ngo, Cameron K. Postnikoff, Henry Fortinberry, Jason J. Nichols
The supernatant was then treated with RNase and DNase to remove any RNA or DNA that may be associated with the outside of the EVs.2,36 The single RNase and DNase treatment was performed by subjecting samples to (1 mL final volume with enzymes) 100 µL of DNA digest buffer (Qiagen, RNase-Free DNase Set; product number: 1023460), 100 µL of DNase1 (Roche, 10 mg/mL; product number: 112849320001), and 180 µL of RNase (Invitrogen RNase cocktail, 50 U/mL RNase A, 20 000 U/mL RNase T1; product number: AM2286). Samples were then incubated at 37 °C for 30 min to allow the enzymes to act while at the same time keeping the general conditions across samples constant; the samples were then stored on ice for 15 min to significantly reduce further RNase/DNase enzymatic activity during the process of EV isolation.
Non-invasive targeted iontophoretic delivery of cetuximab to skin
Published in Expert Opinion on Drug Delivery, 2020
Maria Lapteva, Marwa A. Sallam, Alexandre Goyon, Davy Guillarme, Jean-Luc Veuthey, Yogeshvar N. Kalia
As mentioned above, it was considered that the electrotransport of macromolecules would have to depend mainly on the electroosmotic solvent flow [15], but that the ‘convective solvent flow driven transport was insufficient to overcome the diffusional resistance of the stratum corneum’ [17]. However, the successful transdermal iontophoretic delivery of small proteins (cytochrome C (12.4 kDa), RNase A (13.6 kDa), RNase T1 (11.1 kDa), and hbFGF (17.4 kDa) has been demonstrated as being due to electromigration [17] – even the delivery of hbFGF was still attributable up to 75% to EM (at a current density of 0.5 mA/cm2) and electroosmosis had only a secondary role. In contrast, the cutaneous delivery of CTX, which has an MW ~9-fold greater than hbFGF, does depend on electroosmosis. Furthermore, the results demonstrate that convective solvent flow enables the electrotransport of CTX across an intact stratum corneum thereby overcoming its diffusional resistance. However, the skin remains a formidable barrier to transport since no transdermal permeation was observed within the timeframe of the experiment. The absence of negative charges in the deeper skin layers was suggested in studies comparing iontophoretic and passive transport of acetaminophen across heat separated dermis – thus EO would no longer operate in these regions [25]. Further screening investigations with proteins of various sizes and physicochemical properties will be needed to determine (i) the molecular weight cutoff for proteins for epidermal/dermal iontophoretic delivery in 8 h, (ii) the maximum molecular weight possible for transdermal iontophoretic delivery in 8 h, and (iii) the protein molecular weight threshold at which electroosmosis supersedes electromigration as the dominant electrotransport mechanism.