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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
Importantly, as mentioned above, RNA is much less stable than DNA and therefore presents some key methodological challenges. Most importantly, RNA degrading enzymes known as ribonuclease (RNase) can break down the RNA molecule into smaller fragments that are unusable for experiments. It is therefore recommended to always ensure good laboratory practice (i.e. always wearing clean gloves, a lab coat and cleaning all surfaces and equipment with RNase-inhibiting solution) and always use RNase-free plasticware and diethylpyrocarbonate (DEPC)-treated water when making relevant buffers and reagents.
Transport of Radiolabeled Enzymes
Published in Lelio G. Colombetti, Biological Transport of Radiotracers, 2020
Shindo et al.16 labeled muramidase with 13C and studied the product by means of NMR. Jaeck and Benz17 have synthesized ribonuclease radiolabeled with 14C on methi-onine-29; Noyer et al.18 have also labeled ribonuclease with 14C, but only on the “saccharide structure.’’ Since the entire ribonuclease molecule — of 124 amino acids — has been synthesized, it appears that it will be possible to synthesize other enzymes containing amino acids labeled with 14C.
“Biologically Active” RNA and the Immune Response*
Published in Edward P. Cohen, A. Arthur Gottlieb, Immune RNA, 2020
Cohen’s extracts, as well as Fishman’s, were impure and contained, in addition to RNA, both DNA and protein. However, the likely essential participation (if not elucidated) of RNA in the response was indicated by the extraordinary sensitivity of the active extracts to ribonuclease. Enzyme:substrate ratios as high as 1:700,000 inactivated the extracts (approximately 0.001 μg ribonuclease destroyed the biological activity of 700 μg of extract dissolved in saline in less than 10 min at 20°). Extracts dissolved in medium containing divalent cations were partially resistant to ribonuclease. In later studies Cohen found18 that divalent cations aid in stabilizing the secondary structure of the RNA (Figure 1); this is indicated by the heightened hyperchromicity of RNA dissolved in buffer containing calcium and magnesium relative to that observed for aliquots of the RNA solution dissolved in buffer without calcium and magnesium. This observation deserves special emphasis. Later sections of this book will present the capacity of RNA extracts injected into immunocompetent recipients, including humans, to affect the immune status of the recipient. Ribonuclease is present in blood and intercellular fluids and would be expected to quickly degrade such RNA. RNA dissolved in solutions containing salts in physiologic concentrations resisted degradation. Persistence of its biological activity was noted after incubation with ribonuclease if the reaction was performed in medium containing calcium and magnesium, but not if the reaction was performed in medium free of divalent cations.
Targeted Degradation of Structured RNAs via Ribonuclease-Targeting Chimeras (RiboTacs)
Published in Expert Opinion on Drug Discovery, 2023
Salma Haj-Yahia, Arijit Nandi, Raphael I. Benhamou
Recently a novel targeted degradation method was developed to induce RNA degradation. To mediate RNA decay, researchers exploit ribonucleases (RNases) that naturally regulate the RNA lifetime and recruit them to specific transcripts via a small molecule, or Ribonuclease Targeting Chimeras (RiboTaCs). These molecules play an important role in regulating gene expression and have been implicated in various diseases such as cancer and viral infections. This approach has been shown to be promising in various in-vitro and in-vivo models, but more research is needed to fully understand the potential and limitations of this technology. To date, RiboTaC has been shown to recruit only RNase L, which is a key player in the interferon-induced antiviral response. However, in order to enrich the RiboTaC toolbox, other Ribonucleases will need to be recruited. In this review, we will discuss the current state of the RiboTaC approach, including its mechanism of action, potential therapeutic applications, and future favorable prospects.
Emerging Drugs for the Treatment of Amyotrophic Lateral Sclerosis: A Focus on Recent Phase 2 Trials
Published in Expert Opinion on Emerging Drugs, 2020
Andrea Barp, Francesca Gerardi, Andrea Lizio, Valeria Ada Sansone, Christian Lunetta
ASOs are short (13–25 nucleotides), single-stranded nucleic acid, which selectively target and bind mRNA, altering its processing or translation. They can exert a gene targeting effect through different mechanisms, which include interaction with specific RNA binding proteins, RNA splicing or degradation of mRNA by the activation of ribonuclease H (RNase H) [181]. ASOs do not cross the blood–brain barrier, so they must be directly delivered into the cerebrospinal fluid (CSF), where they distribute throughout the brain and spinal cord [182]. In 2006, Smith and colleagues demonstrated that direct injections into the CSF via intra cerebroventricular injections of ASO against SOD1 reduced SOD1 mRNA through RNase H activity, in the brain and spinal cord of SOD1-rats, increasing their mean survival by 10 days [183]. This strategy was tested in patients with SOD1-linked ALS in a clinical trial after that they confirmed that intrathecal administration of the ASO in humans was well tolerated with no serious adverse events [184]. Following these preclinical results, a randomized, placebo-controlled phase 1 trial of intrathecally injected Tofersen (also called ISIS 333,611, BIIB067, and IONIS SOD1 Rx) was conducted in patients with SOD1 mutation, and there was a similar rate of adverse events between the two groups (88% in the placebo group vs. 83% in the treatment group) [184]. Thus, given the good safety profile, a randomized trial aimed at evaluating the clinical response was started and is now ongoing (NCT03070119) [185].
Advances in the discovery of microRNA-based anticancer therapeutics: latest tools and developments
Published in Expert Opinion on Drug Discovery, 2020
Kenneth K.W. To, Winnie Fong, Christy W.S. Tong, Mingxia Wu, Wei Yan, William C.S. Cho
This novel design made use of the conjugation of artificial ribonucleases or catalytic peptides to mediate the degradation of the targeted miRNAs [71,72]. A peptide nucleic acid (to mediate specific miRNA binding) has been conjugated with deithylenetriamine or a three-amino acid peptide HGG with Cu2+ co-factor (to mediate the oxidative or acid-base cleavage of miR-1323) for the treatment of neuroblastoma [73]. Another more recently developed conjugate system consisted of specific oligonucleotides and the catalytic peptide [(LRLR)G]2 that cleave the oncogenic miRNAs (miR-21 and miR-17) by trans-esterification reaction [71,72]. These new artificial ribonucleases were shown to effectively downregulate the targeted miRNAs, thereby restoring key tumor-suppressor protein expressions and inhibiting cancer proliferation without appreciable adverse effects.