The base pairs are the contributors to the sequence-dependent recognition of nucleic acids, genetic information storage, and high fidelity of DNA polymerase replication. However, the wobble base pairing, where T pairs with G instead of A, reduces specific base-pairing recognition and compromises the high fidelity of the enzymatic polymerization. Via the selenium atomic probing at the 2-position of thymidine, we have investigated the wobble discrimination by manipulating the steric and electronic effects at the 2-exo position, providing a unique chemical strategy to enhance the base pair specificity. We report here the first synthesis of the novel 2-Se-thymidine ((Se)T) derivative, its phosphoramidite, and the Se-DNAs. Our biophysical and structural studies of the 2-Se-T DNAs reveal that the bulky 2-Se atom with a weak hydrogen-bonding ability can largely increase mismatch discriminations (including T/G wobble and T/C mismatched base pairs) while maintaining the (Se)T/A virtually identical to the native T/A base pair. The 2-Se atom bulkiness and the electronic effect are probably the main factors responsible for the discrimination against the formation of the wobble (Se)T/G base pair. Our investigations provide a potential novel tool to investigate the specific recognition of base pairs, which is the basis of high fidelity during replication, transcription, and translation. Furthermore, this Se-atom-specific substitution and probing are useful for X-ray crystal structure and function studies of nucleic acids.
The selective expression of Escherichia coli purine nucleoside phosphorylase ( PNP ) in solid tumors has been successfully used to activate two purine nucleoside analogs [ 9 -( 2 -deoxy -b -D -ribofuranosyl ) -6 -methylpurine ( MeP -dR ) and 9 -b -D -arabinofuranosyl -2 -fluoroadenine ( F -araA ) ] resulting in lasting tumor regressions and cures. E. coli PNP also cleaves 2 -fluoro -2 0 -deoxyadenosine ( F -dAdo ) to 2 -F -adenine, which is the toxic purine analog liberated from F -araA that has high bystander activity and is active against nonproliferating tumor cells. As F -dAdo is 3000 times better than F -araA as a substrate for E. coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E. coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents. Like MeP -dR and F -araA -5 0 -monophosphate ( FaraAMP, a prodrug of F -araA ), treatment of mice bearing D54 tumors that express E. coli PNP with F -dAdo resulted in excellent antitumor activity. Although F -dAdo was as active as MeP -dR and better than F -araAMP, it was not dramatically better than either compound because of its short plasma half -life and the limited activation of F -adenine to toxic metabolites. Regardless, these results indicated that F -dAdo was also an excellent prodrug for use with gene vectors that deliver E. coli PNP to tumor cells.
Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i) minimal interference with the exponential cell growth, (ii) minimal reduction in cellular host RNA levels, and (iii) reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.Despite the availability of infectious cDNA clones of the hepatitis C virus (HCV), efficient in vitro replication has not been observed (3). After transfection of subgenomic HCV RNA replicons that also express the neomycin phosphotransferase gene selection marker, HCV replication has been reported previously in the human hepatoma cell line Huh-7 (2, 21). Such HCV replicon-harboring cell lines could be cultivated for more than a year without signs of cytopathogenicity (26). High levels of HCV RNAs can be maintained in cells passaged under continuous selection with G418. In addition, high-level replication was reflected in the observed adaptations of the HCV replicon to the host cell (20).A tight coupling of the amount of intracellular HCV RNA and cell growth was observed. High levels of viral RNA were found in exponentially growing cells, but this was followed by a sharp decline in RNA levels when cells reached a confluent state (26,29). This suggests that cellular factors required for RNA replication and/or translation vary in abundance and become limited in resting cells. Several proteins have been suggested, but none of them has as yet been positively identified to be directly responsible (9,15,16,19,25,30). However, there are no reports in which the availability of the cellular nucleoside triphosphate pools were linked to the loss of steadystate level replication of HCV replicon RNA in confluent cells. It is well-known that confluent cells mainly depend on salvage nucleoside biosynthetic pathways, resulting in lowered concentrations of endogenous nucleoside pools. As most antimetabolite agents of the de novo nucleoside biosynthesis are known to chemically deplete nucleoside pools and induce a concentration-dependent cytostasis, evaluating these compounds against HCV replicon can shed light on the relationship between nucleoside pools and replicon dynamics. However, before doing so, a definition of specificity of antiviral ...
Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.
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