Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides

Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.

Molecular docking investigation of the amantadine binding to the enzymes upregulated or downregulated in Parkinson’s disease

<p class="ADMETabstracttext">Parkinson’s disease (PD) is a progressive neurodegenerative disease. Levodopa in combination with amantadine has a demonstrated efficacy in motility impairment. An extensive investigation of some enzymes described to be upregulated or downregulated in PD was made – adenylate kinase (AK), adenine phosphoribosyltransferase (APRT), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), nucleoside-diphosphate kinase 3 (NDK3), purine nucleoside phosphorylase 1 (PNP1), and ecto-5’-nucleotidase (NT5E). Also, creatine kinase (CK) was included in the study because it is one of the main enzymes involved in the regulation of the nucleotide ratio in energy metabolism. To date, there is no proven link between amantadine treatment of PD and these enzymes. Because there are many AKs isoforms modified in PD, the AK was the first investigated. The molecular docking experiments allow the analysis of the selective binding of amantadine – unionized (with –NH₂ group) and ionized form (with –NH₃⁺ group) – to the AKs’ isoforms implicated in PD. Using available X-ray 3D structures of human AKs in closed-conformation, it was demonstrated that there are notable differences between the interactions of the two forms of amantadine for the zebrafish AK1 (5XZ2), human AK2 (2C9Y), human AK5 (2BWJ), and AK from B.stearothermophilus. The cytosolic human AK1 and human AK2 mostly interact with ionized amantadine by AMP binding residues. The human AK5 interaction with ionized amantadine does not involve the residues from the catalytic site. Among other enzymes tested in the present study, APRT revealed the best results in respect of binding amantadine ionized form. The results offer a new perspective for further investigation of the connections between amantadine treatment of PD and some enzymes involved in purine metabolism.</p>

Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum

ABSTRACT We investigated the effect of antisense RNA (asRNA) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [AADC] and coenzyme A-transferase [CoAT]) of Clostridium acetobutylicum strain ATCC 824. First, we generated three strains, C. acetobutylicum ATCC 824 (pADC38AS), 824(pADC68AS), and 824(pADC100AS), which contain plasmids that produce asRNAs of various lengths against the AADC (adc) transcript. Western analysis showed that all three strains exhibit low levels of AADC compared to the plasmid control [ATCC 824(pSOS95del)]. By using computational algorithms, the three different asRNAs directed toward AADC, along with previously reported clostridial asRNAs, were examined for structural features (free nucleotides and components). When the normalized metrics of these structural features were plotted against percent downregulation, only the component/nucleotide ratio correlated well with in vivo asRNA effectiveness. Despite the significant downregulation of AADC in these strains, there were no concomitant effects on acetone formation. These findings suggest that AADC does not limit acetone formation and, thus, we targeted next the CoAT. Using the component/nucleotide ratio as a selection parameter, we developed three strains [ATCC 824 (pCTFA2AS), 824(pCTFB1AS), and 824(pCOAT11AS)] which express asRNAs to downregulate either or both of the CoAT subunits. Compared to the plasmid control strain, these strains produced substantially low levels of acetone and butanol and Western blot analyses showed significantly low levels of both CoAT subunits. These results show that CoAT is the rate-limiting enzyme in acetone formation and strengthen the hypothesis that the component/nucleotide ratio is a predictive indicator of asRNA effectiveness.

THE NATURE AND PROCESSING OF RIBOSOMAL RIBONUCLEIC ACID IN A DINOFLAGELLATE

Certain features of the dinoflagellate nucleus suggest that it represents a primitive form of eukaryotic nucleus. For this reason, it was of interest to characterize dinoflagellate ribosomal RNA (rRNA) and its mode of synthesis to determine if it also deviated from typical eukaryotic patterns. Gyrodinium cohnii was chosen for this examination. Gyrodinium ribosomal RNA species are 16 and 25s as judged by their sedimentation velocities in isokinetic sucrose gradients. These values are typical of higher plants. In addition, the RNA cosedimented precisely with rRNA from the ciliate Tetrahymena. Nucleotide ratio analyses revealed a GMP + CMP content of 46% for both species of rRNA. The kinetics of incorporation of a radioactive precursor into ribosomal RNA have also been studied, and it seems likely that the maturation of rRNA starts with the synthesis of a 38s molecule. This serves as precursor to the 16s species, and, after a 27s intermediate, the 25s ribosomal component. The process is similar to that in other eukaryotes. The structure of the nucleolus has also been examined, and is seen to be typically eukaryotic.

Oxidative phosphorylation, adenine nucleotides, and phosphofructokinase in the livers of rats receiving thrombogenic diets

Diets of varying thrombogenic potencies were fed to rats for a period of 6 weeks. The thrombogenic potency of these diets was determined by the extent of the hepatic infarcts (consequent upon thrombosis in the hepatic vein) which occurred within 24 h after the injection of S. typhosa endotoxin. Those groups of rats receiving the low thrombogenic diets were more suitable as controls for the animals on the high thrombogenic diets than were normal rats fed standard laboratory chow. The thrombogenic potency of the diets was inversely related to the ratio of [AMP][ATP]/[ADP]2 found in the liver. A decrease in this ratio should favor platelet aggregation, and the results are thus in accord with the hypotheses implicating adenine nucleotides in thrombogenesis. The thrombogenic potency of the diets was directly related to the activity of liver phosphofructokinase. This increase in activity is probably linked to the disturbed adenine nucleotide metabolism. Oxidative phosphorylation mechanisms were investigated as possible causes for the changes in adenine nucleotides, but no important alteration in these reactions could be demonstrated. The exact mechanism by which the thrombogenic diets alter the adenine nucleotide ratio and the phosphofructokinase activity remains to be elucidated.Des rats ont été nourris pendant 6 semaines avec des régimes de forces thrombogènes variées. Cette capacité thrombogène est évaluée d'après la sévérité des infarcti hépatiques (provoqués par un thrombus dans la veine hépatique) que l'on trouve en dedans de 24 h après l'injection d'endotoxine de S. typhosa. Les groupes de rats sur régimes thrombogènes à force faible constituent de meilleurs groupes témoins que ne le sont des animaux, recevant un régime usuel. La force thrombogène de ces régimes est en relation inverse avec le rapport [AMP][ATP]/[ADP]2 que l'on trouve dans le foie. Un abaissement de ce rapport devrait favoriser l'agrégation des plaquettes sanguines; les résultats obtenus supportent les hypothèses selon lesquelles les nucléotides d'adénine sont impliqués dans la thrombogénèse. L'activité de la phosphofructokinase est augmentée en relation directe avec la force thrombogène des régimes. Cette augmentation est probablement liée au métabolisme anormal des nucleotides d'adénine. Aucun changement n'a pu être décelé dans les mécanismes de phosphorylation oxydative, qui pourraient provoquer des altérations dans le métabolisme des nucléotides d'adénine. Le mécanisme exact par lequel les régimes thrombogènes agissent au niveau des nucléotides d'adénine et de la phosphofructokinase est inexpliqué.

THE PHYSIOLOGY OF HOST-PARASITE RELATIONS: XVII. ACID-SOLUBLE NUCLEOTIDES IN RUST-INFECTED AND SENESCING WHEAT LEAVES

Acid-soluble nucleotides in senescing wheat leaves tended to decrease with time in a manner similar to the decline in RNA.2 Little change was apparent in the free nucleotide: RNA nucleotide ratio. Treatment with kinetin reduced, but did not prevent the loss of soluble nucleotides. The greatest losses occurred in the nucleoside diphosphates. Rust infection increased each of the soluble nucleotides even more rapidly than it increased RNA content. Rust uredospores, themselves, appeared to possess a large soluble nucleotide pool. Uridine and adenosine derivatives were the only nucleotides consistently found. Uredospores and rust-infected leaves differed from uninfected leaves in possessing a large amount of a UDP-N-acetylglucosamine complex. The results are discussed in relation to previous measurements of RNA in senescing and rust-infected leaves.