The double helix is dehydrated: Evidence from the hydrolysis of acridiniumester-labeled probes

A highly chemiluminescent reporter molecule, acridinium ester (AE), was tet hered to single-stranded oligonucleotide probes and hybridized to complemen tary as well as mismatched target sequences. When tethered to single-strand ed probes, AE was readily hydrolyzed by water or hydroxide ion. In contrast , when hybridized to a complementary target, hydrolysis of the AE probe was markedly inhibited. Mismatches near AE eliminated the ability of the doubl e helix to strongly inhibit AE hydrolysis. To establish the molecular basis for these remarkable hydrolysis properties of AE-labeled probes, the bindi ng and hydrolysis mechanisms of AE-labeled probes were examined. When tethe red to single- or double-stranded nucleic acids, hydrolysis of AE was found to proceed by generalized base catalysis in which a base abstracts a proto n from water and the resulting hydroxide ion then hydrolyzes AE, Analysis o f the hydrolysis rates of AE bound to DNA revealed that AE binds the minor groove of DNA and that its hydrolysis is inhibited by low water activity wi thin the minor groove of the helix. Depending upon the sequence of the DNA, the water activity of the minor groove was estimated to be at least 2-4-fo ld lower than bulk solution. Hydrolysis measurements of AE tethered to RNA as well as RNA/DNA hybrids argued that the grooves of these double helices are also dehydrated relative to bulk solution. Remarkably, mismatched bases , regardless of their structure or sequence context, enhanced hydrolysis of AE by inducing hydration of the double helix that spread approximately fiv e base pairs on either side of the mismatch.