A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology

A new method to measure the aminoacylation of tRNA based upon the use of th e scintillation proximity assay (SPA) technology has been developed. The as say detects incorporation of radiolabeled amino acids into cognate tRNA, ca talyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic condit ions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregate s, while the radiolabeled amino acid substrate remains in solution, resulti ng in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by meas urement of steady-state kinetic constants and inhibitor binding constants f or a range of aaRS enzymes in comparison with data from standard, trichloro acetic acid-precipitation-based assays. In all cases, the data were quantit atively comparable, Although the radioisotopic counting efficiency of the S PA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stabilit y) of the SPA assays was at least equivalent to the separation-based method s. The assay was also shown to work well in miniaturized 384-well microtite r plate formats, resulting in considerable reagent savings. In summary, a n ew method to characterize aaRS activity is described that is faster and mor e amenable to high-throughput screening than traditional methods. (C) 2000 Academic Press.