Quantitative analysis of shape-specific interactions of Rev response element with a positively charged Rev peptide by...

Human immunodeficiency virus type 1 (HIV-1) Rev protein is known to regulate the expression of proteins via binding to an RNA site termed the HIV Rev response element (RRE) presumably with a defined shape, mediated mainly by electrostatic interactions. We have developed a quantitative method based on CE-LIF detection for a systematic evaluation of interactions between a truncated RRE (tRRE) RNA and an HIV-1 Rev peptide. Employing a fluorescently labeled HIV-1 Rev protein fragment (RevF) as a probe, buffers were evaluated for the separation and detection as well as for the RNA shape-specific formation of the complex. Selection of an optimal buffer condition allowed us to perform quantitation of the tRRE-RevF complex formation and determine its dissociation constant. In addition, competitive inhibitions of the RNA-peptide interaction by some aminoglycosides were evaluated quantitatively by monitoring the complex peak, resulting in determination of IC(50) values. This sensitive and reliable CE-LIF-based method would be of interest in developing various screening systems for RNA interference in drug discovery.