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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. ...
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 . Elevated temperature (>65°C) and high pH also cause RNA degradation under physicochemical conditions    . 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 . ...
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. ...
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.
Investigation of the occurrence in hen oviduct of highly acidic nucleotides other than the known sulfated sugar nucleotide, UDP-N-acetylgalactosamine-4-sulfate, revealed the presence of four other sugar nucleotides, each bearing 1 or 2 sulfate residues. They are: (a) UDP-N-acetylgalactosamine-4,6-disulfate, the same compound that was previously synthesized from UDP-N-acetylgalactosamine-4-sulfate and 3'-phosphoadenosine-5'-phosphosulfate in the presence of a sulfotransferase preparation from hen oviduct; (b) UDP-N-acetylgalactosamine-4-sulfate-6-phosphate, i.e. a UDP-N-acetylgalactosamine derivative bearing a sulfate and a phosphate residue at position 4 and position 6, respectively, on the N-acetylgalactosamine moiety; (c) UDP-N-acetylglucosamine-6-phosphogalactose-sulfate, i.e a derivative of the known disaccharide-carrying nucleotide, UDP-N-acetylglucosamine-6-phosphogalactose, bearing a sulfate residue on the galactose moiety; and (d) UDP-galactose-sulfate, i.e. a UDP-galactose derivative bearing also a sulfate residue on the galactose moiety.
The diversity of sulfated sugar nucleotides suggests the existence of a common plan of sugar-nucleotide sulfation, with which the tissue makes striking changes in the biological activities or metabolic fates conferred on the sugar nucleotides.
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.
An intracellular, inorganic pyrophosphatase from Escherichia coli K-12 3000(λ) has been purified 500-fold to a state of apparent homogeneity. The enzyme catalyzes hydrolysis of inorganic pyrophosphate, tripolyphosphate, and tetrapolyphosphate with relative velocities of 1.000, 0.016, and 0.007, respectively. No activity whatsoever was found with a variety of other phosphate esters.
There was an absolute requirement for divalent cation in amounts suggesting stoichiometric combination with the substrate. At pH 9.1, Mg²⁺ and Mn²⁺ were maximally effective; at pH 7.5, Zn²⁺ and Co²⁺ were best.
Although no exchange reaction (³²Pi ⇄ PPi) could be demonstrated, a net reversal of the reaction was achieved by coupling through thymidine diphosphate glucose pyrophosphorylase and phosphoglucomutase to oxidation of glucose 6-phosphate to 6-phosphogluconic acid. The stoichiometry of the reverse reaction indicated utilization of 10% of the ³²Pi (trapped as thymidine triphosphate).
The enzyme was exceptionally stable. In the presence of 0.01 m Mg²⁺, it withstood a temperature of 80° for 10 min.
L’utilisation de siRNA est une nouvelle approche thérapeutique très prometteuse. Néanmoins leur transfection à visée thérapeutique est un réel défi. Les obstacles à franchir pour élaborer des agents de transfection sûrs et fiables sont nombreux. Afin de les contourner nous nous sommes attachés à la construction d’un système dynamique qui, à l’image des virus, est constitué de briques moléculaires, s’emboitant et interagissant avec des acides nucléiques selon des interactions supramoléculaires. Ainsi, nous avons élaboré des polymères supramoléculaires polycationiques à base de monomères de cyclodextrines pontées, fonctionnalisées par un groupement adamantyle. Ce type de conjugué pallie un problème manifeste dans la littérature concernant les assemblages de β-CD souvent insolubles ou bien auto-inclus. L’ajout éventuel d’une autre fonction cationique pour améliorer l’interaction avec les siRNA a aussi été réalisé. Ainsi, la capacité à s’auto-assembler de quatre composés a été étudiée par RMN-1H, RMN-ROESY, ITC, RMN-DOSY, et SANS. Par ailleurs, ces composés ont montré une certaine capacité à complexer et à protéger les siRNA. L’un de ces composés a de plus montré une bonne aptitude à transfecter des siRNA in vitro, sans induire de toxicité. Les assemblages CD-siRNA ont finalement été observés par cryo-TEM et ont montré la formation de fibres, organisées de manière hiérarchique et hautement coopérative. Nous avons ainsi créé des assemblages supramoléculaires uniques à base d’acides nucléiques, rappelant la structure, la taille et la fonction d’un virus.
In the early part of the 20th Century, the nature of nucleic acid and what its role was within the cell were a bit of a mystery. DNA itself was first isolated as far back as 1869 by the Swiss chemist Johann Friedrich Miescher. He separated nuclei from the cytoplasm of cells and then isolated an acidic substance from these nuclei that he called nuclein1. Chemical tests by Miescher showed that nuclein contained large amounts of phosphorus and no sulphur, characteristics that differentiated it from proteins1. The first step in determining the structure of nucleic acid (either DNA or RNA) would be to identify its precise composition. RNA was considered a more approachable target for composition analysis because the simple treatment of RNA with hydroxide rapidly and completely hydrolyses the molecule to its individual component nucleotides. DNA, on the other hand, is resistant to such treatment.
Die DNA von fünf Mikroorganismen, Proteus mirabilis 273, dessen UV ‐sensibler Mutante P. mirabilis PG 672, einer stabilen zellwandlosen L‐Form von P. mirabilis VI sowie die der Actinomyceten Streptomyces chrysomallus und Str. griseus, wurde mit Hilfe der Dünnschicht‐ und Säulenchromatographie auf ihren Gehalt an seltenen Basen untersucht. In beiden Streptomyces‐Stämmen konnte nur 5‐MC nachgewiesen werden. Die zellwandlose L‐Form von P. mirabilis VI und der Stamm P. mirabilis 273 enthalten neben 5‐MC noch 6‐MAP, der Stamm P. mirabilis PG 672 zwar 6‐MAP aber nur sehr wenig 5‐MC.
A method is described for the isolation and purification of bound coumarin from seeds of Melilotus alba. Synthetic coumarinic acid β-glucoside has been prepared and its properties compared with those of bound coumarin. The results of these experiments confirm that bound coumarin and coumarinic acid β-glucoside are identical.
Les modifications post-transcriptionnelles sont fondamentales dans différents aspects pour assurer les rôles biologiques des acides ribonucléiques (ARN) dans le processus de traduction. La caractérisation chimique des modifications est par conséquent essentielle afin de permettre la compréhension des intérêts de ces modifications. Les travaux, présentés dans ce manuscrit de thèse, décrivent la mise en œuvre d’une nouvelle approche basée sur le couplage de l’électrophorèse capillaire à la spectrométrie de masse de haute résolution (CE-MS) au service de l’étude des modifications d’ARN. Ici, une stratégie sur deux niveaux a été développée. Le premier niveau d’analyse a permis de déterminer la nature des modifications présentes sur un ARN. Le second niveau, s’inspirant d’une stratégie « bottom-up », a permis de localiser les modifications sur les structures primaires des ARN. La méthodologie développée a permis de caractériser, sans ambiguïté, un grand nombre de modifications sur différents types d’ARN et notamment des ARN de transfert, ouvrant la voie à la CE-MS comme un nouvel outil d’analyse pour la caractérisation de modifications d’ARN.
Exonuclease VII from Escherichia coli is specific for single-stranded DNA, degrading denatured DNA, single-stranded regions extending from the ends of duplex DNA, or displaced single-stranded regions. Hydrolysis is initiated at both 3′ and 5′ termini. Exonuclease VII can also excise thymine dimers from duplex DNA following treatment of the DNA with the Micrococcus luteus endonuclease specific for thymine dimers. The enzyme has no detectable activity on RNA or DNA-RNA hybrid molecules. The limit products of exonuclease VII action are oligonucleotides bearing 5′-phosphoryl and 3′-hydroxyl termini predominantly in the range of tetramers to dodecamers; no mononucleotide products have been observed. The enzyme acts in a processive fashion, initially releasing large, acid-insoluble oligonucleotides which can be degraded further to produce a limit product of acid-soluble oligonucleotides. The possible role of exonuclease VII in recombination and in the repair of ultra-violet damage to DNA is discussed.
RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.
Copolymers containing AMP and dAMP residues have been synthesized with polynucleotide phosphorylase from Micrococcus luteus. The rate of copolymerization depends upon the input ratio of ADP to dADP. It also depends, in a nonlinear way, upon enzyme concentration. Copolymerization proceeds most readily when Mn²⁺ replaces Mg²⁺ and preparation of polymers in Mn²⁺ therefore permits incorporation of a higher proportion of dAMP residues into copolymer. In the presence of either cation, the ratio of AMP to dAMP residues in copolymer is always higher than the ratio of ADP to dADP in the substrate mixture. The size of the copolymer poly (rAn,dAm) is quite large and identical to that of the homopolymer, polyA. Polymers synthesized in Mn²⁺ are more heterogenous in size than polymers synthesized in Mg²⁺.
The “Discussion” includes a general discussion of the effect of Mn²⁺ on reactions catalyzed by M. luteus polynucleotide phosphorylase.
Polynucleotide phosphorylase of Micrococcus luteus catalyzes the addition of a single dAMP moiety from dADP onto an oligoribonucleotide. Further addition of either dAMP or AMP residues to the resulting (Ap)ndA is very difficult. The chain elongation reaction proceeds faster in the presence of Mn²⁺ than in the presence of Mg²⁺.
The enzyme also catalyzes an exchange between the β-phosphate of dADP and ³²Pi, but only in the presence of either an oligoribonucleotide bearing an unesterified C-3′-hydroxyl group or of ADP. Similar results are obtained with Form I (primer-independent) and Form T (primer-dependent) enzyme. It appears that ³²Pi-dADP exchange occurs by the addition of a dAMP residue to the end of a polyribonucleotide (or oligoribonucleotide) chain and the subsequent phosphorolysis of the internucleotide bond. Furthermore, no evidence for the formation of a dAMP-enzyme complex could be obtained. Incubation of the enzyme with dADP as the sole substrate results, rather, in the formation of a limited amount of (dA)n. This reaction also proceeds more readily in the presence of Mn²⁺ than in Mg²⁺.
The effect of 2'-cytidylic acid and 3'-cytidylic acid upon the binding of cupric ions by RNase has been studied by gel filtration, together with the effect of cupric ions upon binding of 2'- and 3'-cytidylic acids.
The results confirm that binding of 2'-CMP by RNase weakens its affinity for cupric ions; reciprocally, binding of cupric ion diminishes the affinity of RNase for 2'-CMP. The negative interactions between cupric ion and 2'-CMP upon binding to RNase have been shown to lead to distortion of the gel filtration ligand trough. At pH 5.5 these interactions are tentatively interpreted in terms of competition between 2'-CMP and Cu(II) for the strongest Cu(II)-binding site on RNase together with an acetate-dependent increase in the Cu(II) affinity of one of the weaker RNase sites in the presence of bound 2'-CMP.
Binding of 3'-CMP to RNase increases the affinity of RNase for 2 cupric ions; similarly binding of cupric ions to RNase increases its affinity for 3'-CMP. Analysis of the binding pattern of Cu(II) to the 3'-CMP-RNase complex indicates co-operative interactions between two Cu(II)-binding sites on the 3'-CMP-RNase complex; at least one of these sites differs from any on free RNase and could involve the phosphate group of 3'-CMP. In further contrast to free RNase, the Cu(II)-binding sites at pH 5.5 on the 3'-CMP-RNase complex have been shown to have a diminished affinity for the cupric ion-monoacetate complex relative to free cupric ion. This suggests that, on the average, Cu(II) is coordinated with more ligands on the 3'-CMP-RNase complex than on free RNase or, alternatively, that binding of Cu(II) to the complex occurs in a more sterically limited environment than on free RNase.
Isolated rat liver mitochondria are capable of incorporating labeled deoxynucleoside triphosphates into DNA. The reaction is linear from 1 to 2 hours, is sensitive to high concentrations of actinomycin D, and requires oxidative phosphorylation and Mg²⁺. Deoxynucleoside triphosphates serve as better precursors than monophosphates, and a partial dependency on all four triphosphates is demonstrated. That the labeled product is DNA is shown by its complete lability to DNase, its complete resistance to RNase and alkali, its proper buoyant density in CsCl, and its increase and then decrease in buoyant density upon being subjected to denaturing and then reannealing conditions. That the labeled DNA is mitochondrial in origin is shown by the occurrence of the incorporation process in the virtual absence of bacteria, the absence of nuclei or nuclear membranes in the mitochondrial preparation, and the failure of added DNase to inhibit significantly the incorporation process. Moreover, the buoyant density of the denatured and reannealed labeled product is identical with that of similarly treated authentic mitochondrial DNA in contrast to nuclear DNA. That the incorporation process leads to “genuine” mitochondrial DNA, i.e. DNA of ordered structure, is suggested by the labeling being internal rather than solely at the 3′-OH end and by the results of nearest neighbor frequency analysis. Controls on the latter procedure show that added dGTP is rapidly broken down in mitochondria, which accounts for the particularly low incorporation of dGTP. Isolation of closed circular DNA from the total DNA labeled by the isolated mitochondria indicates that label is associated with this form of DNA.
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.
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.