Azole carboxamide ribonucleoside triphosphates: Synthesis, site specific incorporation into RNA, and incorporation studies with bovine viral diarrhea virus (BVDV) polymerase
The introduction of modified nucleotides into RNA can greatly aid our understanding of its biological function. They can provide insight into the effects of individual nucleotides on RNA structure and on their interactions with RNA binding proteins. Approaches to synthesize RNA containing modified nucleotides are inefficient and limited.We have developed a method where modifications can be introduced at specific positions in RNA including the 3’- and the 5’-ends. This method involves the use of a combination of different enzymes including Therminator DNA polymerase, T4 DNA ligase, DNase I endonuclease, and RNase H ribonuclease and nucleoside triphosphates as substrates. The entire sequence of enzymatic reactions is carried out in a single Eppendorf ® tube omitting the need for intermediate purification steps. The modified nucleoside triphosphates used in this study were also tested as substrates for bovine viral diarrhea virus (BVDV) polymerase, a surrogate model for hepatitis C (HCV). The only FDA approved combination therapy for HCV is ribavirin plus α-interferon. The nucleoside analog, ribavirin, in its phosphorylated form can be misincorporated by the viral RdRp causing transition mutations. Viruses exist as a mixture of genetically heterogonous but related viral particles that have undergone the maximum amount of mutations without compromising viability or infectivity. Further increase in mutation rates can lead to loss of genetic information and production of non-viable and non-infectious viruses. Based on this concept, we decided to test a number of azole carboxamide ribonucleoside triphosphates (ribavirin analogs) as substrates for BVDV RdRp. They differ from ribavirin and among themselves in the placement of heteroatoms within the 5- membered heterocyclic ring. Pre-steady state kinetic parameters of incorporation of ribavirin analogs by BVDV RdRp were determined. Our studies revealed that analogs 3 and 4 are incorporated across from a G base in the template with rates comparable to the correct ribonucleotide (within 10-fold) and used by the polymerase more efficiently than a mismatch nucleotide. They can either act as viral mutagens if the polymerase can extend beyond them or as chain terminators if the polymerase cannot extend beyond them. In either case, these results establish those analogs as potential antiviral drugs.
Bergstrom, Purdue University.
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