Abstract
A novel reverse transcriptase (RT) assay based on the combined use of macrobead-bound template and 125I-iododeoxyuridine-triphosphate (IUTP) was used to determine the IC50 values of various RT inhibitors. The results showed that this assay and the conventional assay gave similar IC50 values. The introduction of carrier bound template-primer, template, or primer also made it possible to design assays revealing the mechanism of action of various RT inhibitors. Unlabelled inhibitor substance could be incubated with carrier bound template-primer in the presence of excess enzyme, after which the inhibitor was removed and the residual template-primer function was analysed by RT assay. By this procedure it was found that chain elongation terminators like 2′,3′-dideoxy-TTP and 3′-azido-TTP destroyed the template-primer at low concentrations which corresponded to the amount of primer. In contrast, 20–200 times higher concentrations were needed for template-primer destruction when using substances continuously incorporated into the DNA, such as IUTP or TTP. Further, an inhibitor such as phosphonoformic acid (PFA) did not affect the template-primer at all. By excluding the excess RT in the first incubation, it was possible to determine whether or not the template-primer destruction of a given substance was enzyme dependent. Another feature of the macrobead bound template-primer, template, or primer useful for elucidation of the mechanism of action of RT inhibitors is that it can be used to study the interference between an inhibitor and the RTs binding to the template-primer, template, or primer. Briefly, the bead carrying the substrate is incubated with RT in the absence or presence of various inhibitor concentrations, followed by thorough wash. After this the bound RT activity is determined. Such analyses showed that, in contrast to different nucleic acids and oligonucleotides, the classic RT inhibitors either did not interfere or only interfered weakly with the binding of RT to the carrier bound template-primer, template, or primer. Due to the technical simplicity of this novel RT assay it is a far better tool to rapidly screen RT inhibitors than conventional procedures used to date. Further, the use of carrier bound template-primer, template, or primer offers a unique and simple technology for analysis of the mechanisms of action of different RT inhibitors and for analysis of the characteristics of different RT isozymes and mutated RT.