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The structure of ribonucleic acid. I. Cyclic nucleotides produced by ribonuclease and by alkaline hydrolysis

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... It is plausible that aldehyde-alkali extraction might report the pool of free nucleotides i.e. soluble and not bound fraction. Second, degradation of nucleotides that are instable in alkaline conditions, such as cyclic nucleotides (Markham and Smith 1952), (p)ppGpp (Cashel and Kalbacher 1970). Third, incomplete lysis which gives a systematic error, overrepresentation of the content of larger cells, and obtained results are therefore also a function of cell size distribution (Dennis, Ehrenberg et al. 2004). ...
... RNA degradation depends on how prone the molecule is to in-line hydrolytic cleavage and attack by nucleases, oxidizers and chemical modifiers in the RNA's environment (10)(11)(12)(13). Amongst these degradation processes, in-line hydrolytic cleavage is a universal mechanism intrinsic to RNA. ...
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RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term ‘superfolder’ mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.
... For example, early genetic maps of tomato and maize contained 45 and 58 loci that were associated with various morpho logical traits (Butler, 1952;Emerson, 1932). Methods were developed that advanced un derstanding of DNA and RNA molecules, especially electrophoresis (Markham & Smith, 1952) and restriction enzymes (Linn & Arber, 1968), and they were quickly adopted by geneticists and breeders to develop genetic maps based on DNA markers and identify loci (Botstein, White, Skolnick, & Davis, 1980;Lander & Botstein, 1986). The use of DNA-based markers led to the "Mendelization" of quantita tive traits in which DNA markers were statistically associated with a phenotype to esti mate the number of loci and the effect of those loci in the expression of the trait under study (Lander & Botstein, 1989;Paterson et al., 1988). ...
... RNA is susceptible to the universal presence of ribonucleases (RNases) in both human cells, lab reagents and consumables [53]. Elevated temperature (>65°C) and high pH also cause RNA degradation under physicochemical conditions [54] [55] [56] [57]. As a drastic demonstration, without adequate RNase control, free RNA is nonamplifiable after 15 s of incubation in biological samples, such as plasma or serum [53]. ...
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Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To counter COVID-19 spreading, an infrastructure to provide rapid and thorough molecular diagnostics and serology testing is the cornerstone of outbreak and pandemic management. We hereby review the clinical insights with regard to using molecular tests and immunoassays in the context of COVID-19 management life cycle: the preventive phase, the preparedness phase, the response phase and the recovery phase. The spatial and temporal distribution of viral RNA, antigens and antibodies during human infection is summarized to provide a biological foundation for accurate detection of the disease. We shared the lessons learned and the obstacles encountered during real world high-volume screening programs. Clinical needs are discussed to identify existing technology gaps in these tests. Leverage technologies, such as engineered polymerases, isothermal amplification, and direct amplification from complex matrices may improve the productivity of current infrastructure, while emerging technologies like CRISPR diagnostics, visual end point detection, and PCR free methods for nucleic acid sensing may lead to at-home tests. The lessons learned, and innovations spurred from the COVID-19 pandemic could upgrade our global public health infrastructure to better combat potential outbreaks in the future.
... RNA degradation depends on how prone the molecule is to in-line hydrolytic cleavage and attack by nucleases, oxidizers, and chemical modifiers in the RNA's environment (10)(11)(12)(13). Amongst these degradation processes, in-line hydrolytic cleavage is a universal mechanism intrinsic to RNA. ...
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RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery, and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Motivated by the need for stabilized COVID-19 mRNA vaccines, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall rate of hydrolysis. To characterize the stabilization achievable through structure design, we compare optimization of AUP by conventional mRNA design methods to results from the LinearDesign algorithm, a new Monte Carlo tree search algorithm called RiboTree, and crowdsourcing through the OpenVaccine challenge on the Eterna platform. Tests were carried out on mRNAs encoding nanoluciferase, green fluorescent protein, and COVID-19 mRNA vaccine candidates encoding SARS-CoV-2 epitopes, spike receptor binding domain, and full-length spike protein. We find that Eterna and RiboTree significantly lower AUP while maintaining a large diversity of sequence and structure features that correlate with translation, biophysical size, and immunogenicity. Our results suggest that increases in in vitro mRNA half-life by at least two-fold are immediately achievable and that further stability improvements may be enabled with thorough experimental characterization of RNA hydrolysis.
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A sulfotransferase system that catalyzed the sulfation of UDP-N-acetylgalactosamine in the presence of 3'-phosphoadenosine-5'-phosphosulfate was obtained in a particulate form from the isthmus of hen oviduct. The product of sulfation was identified as UDP-N-acetylgalactosamine-4-sulfate, the same compound that accumulates in the isthmus region during egg formation. Several attempts were made to solubilize the enzyme system following some of the procedures which were successful in solubilizing sulfotransferases for mucopolysaccharide sulfation. No indication was obtained, however, that the enzyme system which produces UDP-N-acetylgalactosamine-4-sulfate could be solubilized. Results from examination for substrate specificity and from kinetic studies indicated that the synthesis of UDP-N-acetylgalactosamine-4-sulfate is the principal function of this enzyme system. This was also supported by the finding that the enzyme system tends to be much lower in content in the adjacent magnum region (synonym: albumin-secreting region) where UDP-N-acetylgalactosamine-4-sulfate does not accumulate in more than trace amounts.
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Two distinct d-amino acid dehydrogenases, each showing absolute specificity for methylene blue or 2,6-dichloroindophenol, respectively, were isolated from Pseudomonas fluorescens (ATCC 11299B). The methylene blue-specific d-amino acid dehydrogenase was detectable only in extracts from d-tryptophan-grown cells and was purified about 40-fold. The 2,6-dichloroindophenol-specific d-amino acid dehydrogenase is constitutive, being present in all cell extracts irrespective of culture conditions, and it was purified about 65-fold. These enzymes appear to be flavoproteins in which the flavin adenine dinucleotide prosthetic group is tightly bound to the enzyme protein. The optimal pH of two enzymes was 7 to 8 and the Michaelis-Menten constant was 3 to 5 x 10⁻⁴m for each d-amino acid. Substrate specificity and thermal stability were observed to be somewhat different for the two enzymes. No inhibition of these enzymes was observed with metal chelating agents.
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The specificity of the enzyme purified from T2-infected Escherichia coli which catalyzes the phosphorylation of 5'-hydroxyl ends of ribonucleic acid and deoxyribonucleic acid has been examined. The enzyme also catalyzes the phosphorylation of 5'-hydroxyl groups of polynucleotides as well as 3'-mononucleotides. There is no detectable phosphorylation of nucleosides and adenosine 2'-phosphate is virtually inactive as a phosphate acceptor. The enzyme is also capable of using guanosine, cytidine, and uridine triphosphates as well as adenosine triphosphate as the phosphorylating agent. The enzyme catalyzes the quantitative phosphorylation of available 5'-hydroxyl groups occurring in a variety of different DNA preparations. When used in conjunction with alkaline phosphatase, the enzymatic phosphorylation of DNA measures the total amount of 5'-hydroxyl and 5'-phosphate ends present in DNA. The 5'-hydroxyl polynucleotide kinase was used to measure the amount of 5'-hydroxyl ends formed in DNA degraded by sonic oscillation and alkali and heat denaturation. In all of the cases, virtually no additional 5'-hydroxyl groups were formed. The 5'-hydroxyl kinase has also been detected in nuclei of rat liver and the activity partially characterized.
  • D M Brown
  • C A Dekker
  • A R Todd
Brown, D. M., Dekker, C. A. & Todd, A. R. (1952). J. chem. Soc. (in the Press).
  • P A Levene
  • S A Harris
Levene, P. A. & Harris, S. A. (1932). J. biol. Chem. 98, 9. Loring, H. S. & Luthy, N. G. (1951). J. Amer. chem. Soc. 73, 4215.
  • M G Sevag
  • D B Lackman
  • J Smolens
Sevag, M. G., Lackman, D. B. & Smolens, J. (1938). J. biol. Chem. 124, 425.
  • C E Carter
Carter, C. E. (1950). J. Amer. chem. Soc. 72, 1466.
  • D M Brown
  • D Magrath
  • A R Todd
Brown, D. M., Magrath, D. & Todd, A. R. (1952). J. chem. Soc. p. 2708.
  • W E Cohn
Cohn, W. E. (1951). J. cell. comp. Phy8iol. 38, Suppl. 1,21.
  • D M Brown
  • L J Haynes
  • A R Todd
Brown, D. M., Haynes, L. J. & Todd, A. R. (1950). J. chem. Soc. p. 408.
  • W E Cohn
Cohn, W. E. (1950). J. Amer. chem. Soc. 72, 2811.
  • D M Brown
  • A R Todd
Brown, D. M. & Todd, A. R. (1952 a). J. chem. Soc. p. 44.