Signal amplification system for DNA hybridization assays based on in vitro expression of a DNA label encoding apoaequorin.

Abstract

The development of hybridization assays based on an apoaequorin-encoding DNA label is reported. The constructed label contains the T7 RNA polymerase promoter, the apoaequorin coding sequence and a downstream (dA/dT)(30). In the captured target configuration, biotinylated target DNA (233 bp) was captured on streptavidin-coated microtiter wells and hybridized to a poly(dT)-tailed detection probe. In the sandwich-type assay, the target DNA was hybridized simultaneously with an immobilized capture probe (through biotin/streptavidin) and a poly(dT)-tailed detection probe. In both configurations, the hybrids were reacted with poly(dA)-tailed apoaequorin DNA. The DNA label was subjected to in vitro transcription/translation to produce apoaequorin, which was converted to active aequorin in the reaction mixture. Generated aequorin was determined by its characteristic Ca(2+)-triggered bioluminescence. Each DNA label was estimated to produce 156 aequorin molecules. As low as 0.25 and 0.5 amol of target DNA were detected with the sandwich-type and captured target hybridization assays, respectively, with a linear range spanning four orders of magnitude. In comparison, captured target hybridization assays using photoprotein aequorin or firefly luciferase-encoding DNA labels were able to detect 25 and 20.5 amol of target DNA, respectively. The dramatic improvement in sensitivity observed with the proposed systems is attributed to amplification introduced by in vitro expression of apoaequorin DNA into multiple active aequorin molecules.