A Non-Radioactive Microtitre Plate Reverse Transcriptase (RT) Assay, Based on Immobilized Template, for Screening of RT Activity Inhibitors and Evaluation of their Mode of Action

Abstract

A new sensitive colorimetric reverse transcriptase (RT) activity assay utilizing a 96-well microtitre plate format, with solid phase-conjugated polyadenylic acid (prA), was investigated for simple analyses of the RT inhibiting capacity and mode of action of various substances. Three different technical procedures using the assay were evaluated: (i) direct lC50 determinations with various substances, using four different combinations of primer and dNTP amounts; (ii) analyses of the capacity of the substances to interfere with the binding of RT to template or template-primer (BIC50); (iii) analyses of the capacity of the substances to destroy the template-primer in presence or absence of RT (TDC50). The assay was found to be useful for all three purposes using small amounts of recombinant RT. In the IC50 analyses, the test substances gave values similar to those reported for soluble RT assays, and the values varied in accordance with their known mode of action in relation to the combination of primer and dNTP amount used. Only one of the substances, prG, in addition to DNA and RNA gave true RT binding inhibition. The template destruction assay showed that chain terminating substances gave destruction at low inhibitor concentrations. Furthermore, this destruction was RT-dependent, in contrast to the destruction obtained with substances that can base-pair with the template or primer. For optimum information on mode of action of a given substance all three assay procedures should be used. The use of the assay in relation to the screening and analyses of new RT inhibitory substances and characterization of RT in primary isolates or plasma is discussed.