Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sugar moiety of nucleosides in solution is known to exist in a rapid dynamic equilibrium between extreme Northern and Southern conformations as defined in the pseudorotational cycle. In the present work, we describe how the bicyclo[3.1.0]hexane template fixes the ring pucker of 2'-deoxy-methanocarba-nucleosides 1-5 and 12 to values corresponding to either one of these two extreme conformations that are typical of nucleosides. The syntheses of the fixed Northern conformers 1-5 were performed by Mitsunobu coupling of the heterocyclic bases with the chiral carbocyclic alcohol 6 [(1R,2S,4R,5S)-1-[(benzyloxy)methyl]-2-(tert-butyloxy)-4-hydrox ybicyclo[3.1.0]hexane], while the synthesis of the Southern conformer, (S)-methanocarba-T (12), was reported earlier. Carbocyclic thymidine (carba-T, 13) was used as a reference, flexible carbocyclic nucleoside. Antiviral evaluation of these compounds revealed a very potent antiherpetic activity associated with the Northern thymidine analogue 2, which was more powerful than the reference standard acyclovir against both HSV-1 and HSV-2. (N)-Methanocarba-T (2) was further evaluated as a component of a short oligodeoxynucleotide (ODN) phosphorothioate (5'-CTTCATTTTTTCTTC-3') where all thymidines were replaced by 2. The expected thermodynamic stability resulting from the preorganization of the pseudosugar rings into a Northern conformation, typical of A-DNA, was evident by the increase in Tm of the corresponding DNA/RNA heteroduplex. However, the rigid A-tract ODN caused loss of RNase H recruitment. A detailed conformational analysis of (N)-methanocarba-T (2) and (S)-methanocarba-T (12), as representative examples of conformationally rigid pseudorotational antipodes, revealed that in addition to their different forms of ring pucker, (S)-methanocarba-T appears to be a rather stiff molecule with fewer low-energy conformational states available compared to (N)-methanocarba-T. The syn/anti-energy barrier for these nucleoside analogues is 5-6 kcal/mol higher than for common nucleosides.
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PMID:Nucleosides with a twist. Can fixed forms of sugar ring pucker influence biological activity in nucleosides and oligonucleotides? 880 62

Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH- RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH- RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl(2) in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH- RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH- RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH- RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.
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PMID:Purification of M-MLVH- RT on a 9-aminoethyladenine-(1,6-diamine-hexane)-triazine selected from a combinatorial library of dNTP-mimetic ligands. 2082 67