title: A non-radioactive assay system for screening for inhibitors of RNA-dependent reverse transcriptase activity; an analysis using aurintricarboxylic acid and plant flavonoids.
abstract: Ongoing and extensive screening for potentially selective and potent inhibitors of RNA-dependent DNA polymerases (reverse transcriptases) is important for the discovery of therapeutic agents active against retroviruses. Traditional systems for assaying the activity of reverse transcriptase enzymes have relied upon the use of radioactive nucleotides for monitoring complementary DNA (cDNA) synthesis. Moreover, such assays have also typically been programmed by the addition of synthetic template-primers. The recent trend, however, is towards the use of more natural nucleic acid template-primer-directed systems, which provides for a more realistic estimation of the activity of potential therapeutic anti-reverse transcriptase agents. A novel agarose gel-electrophoretic method originally developed to evaluate DNA polymerase activity during enzyme purification was adapted for screening purposes. The system was modified so that one can detect the synthesis of non-radioactively labeled cDNA synthesized in both DNA and RNA template-directed reverse transcription reactions. Such assays are programmed with either M13mp9 single-stranded DNA or rabbit globin mRNA as appropriate templates. Following incubation of reaction mixtures, agarose gels are used to determine the activity of the DNA-dependent DNA polymerase and RNA-dependent DNA polymerase activities of Maloney murine leukemia virus. This is done through a detection of the amount of double-stranded nucleic acid molecules (either DNA:DNA or RNA:DNA hybrids) generated by the enzyme in the presence and absence of various known enzyme inhibitors. The assay systems that have been developed will be useful in initial, general, rapid, inexpensive and safer screening programs for potential inhibitors of both the DNA- and RNA-dependent DNA-polymerase functions of reverse transcriptase enzymes. The system was tested and evaluated with potent inhibitors of reverse transcription, such as aurintricarboxylic acid and with several plant flavonoids known to be moderately potent inhibitors of reverse transcriptases. The details concerning the RNA-dependent DNA polymerizing system and some of our results are presented.
authors.affiliation: Department of Chemistry, Butler University, Indianapolis, Indiana 46208, USA.
authors.forename: G
authors.initials: G
authors.lastname: Spedding
mesh_terms: D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis; D004353:Drug Evaluation, Preclinical; D005419:Flavonoids / Q000494:pharmacology*; D005816:Genetic Complementation Test; D051379:Mice; D010944:Plants / Q000737:chemistry; D012313:RNA; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011817:Rabbits; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013698:Templates, Genetic
chemical_list: D005419:Flavonoids; D018894:Reverse Transcriptase Inhibitors; D001312:Aurintricarboxylic Acid; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase