title: The double helix is dehydrated: evidence from the hydrolysis of acridinium ester-labeled probes.
abstract: A highly chemiluminescent reporter molecule, acridinium ester (AE), was tethered to single-stranded oligonucleotide probes and hybridized to complementary as well as mismatched target sequences. When tethered to single-stranded 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 double helix to strongly inhibit AE hydrolysis. To establish the molecular basis for these remarkable hydrolysis properties of AE-labeled probes, the binding and hydrolysis mechanisms of AE-labeled probes were examined. When tethered to single- or double-stranded nucleic acids, hydrolysis of AE was found to proceed by generalized base catalysis in which a base abstracts a proton from water and the resulting hydroxide ion then hydrolyzes AE. Analysis of 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 within 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-fold 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 five base pairs on either side of the mismatch.
authors.affiliation: Gen-Probe Incorporated, San Diego, California 92121, USA.
authors.forename: M
authors.initials: M
authors.lastname: Becker
authors.forename: V
authors.initials: V
authors.lastname: Lerum
authors.forename: S
authors.initials: S
authors.lastname: Dickson
authors.forename: N C
authors.initials: NC
authors.lastname: Nelson
authors.forename: E
authors.initials: E
authors.lastname: Matsuda
mesh_terms: D000166:Acridines / Q000737:chemistry / Q000378:metabolism*; D020137:Base Pair Mismatch; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism*; D004952:Esters; D006868:Hydrolysis; D006878:Hydroxides; D009690:Nucleic Acid Conformation; D014867:Water / Q000737:chemistry*
chemical_list: D000166:Acridines; D018736:DNA Adducts; D015342:DNA Probes; D004952:Esters; D006878:Hydroxides; C051802:triplex DNA; D014867:Water; D004247:DNA; C031356:hydroxide ion