A high throughput method to investigate oligodeoxyribonucleotide hybridization kinetics and thermodynamics.

Abstract

We describe a high throughput microtiter-based assay to measure binding of oligodeoxyribonucleotides to nucleic acid targets. The assay utilizes oligodeoxyribonucleotide probes labeled with a highly chemiluminescent acridinium ester (AE). Reaction of AE with sodium sulfite renders it non-chemiluminescent. When an AE-labeled probe hybridizes to a target nucleic acid AE is protected from reaction with sodium sulfite and thus remains chemiluminescent. In contrast, unhybridized probe readily reacts with sodium sulfite and is rendered non-chemiluminescent. Hybridization of an AE-labeled probe to a target nucleic acid can therefore be detected without physical separation of unhybridized probe by treatment of the hybridization reaction with sodium sulfite and measurement of the remaining chemiluminescence. Using this method we measured hybridization rate constants and thermodynamic affinities of oligodeoxyribonucleotide probes binding to simple synthetic targets as well as large complex biological targets. The kinetic and thermodynamic parameters were measured with a high degree of accuracy and were in excellent agreement with values measured by other established techniques.