The Trapping of a Spontaneously “Flipped-Out” Base from Double Helical Nucleic Acids by Host−Guest Complexation with β-Cyclodextrin:  The Intrinsic Base-Flipping Rate Constant for DNA and RNA

Beta-cyclodextrin, which forms stable host-guest complexes with purine bases, induces the melting of RNA and DNA duplexes below their normal melting temperatures. Alpha-cyclodextrin, which does not form stable complexes, has no effect on either RNA or DNA. Gamma-cyclodextrin, which forms weaker complexes, has no effect on RNA and a smaller effect than beta-cyclodextrin on DNA. The rate of melting is kinetically first-order in duplex and, above about 20 mM beta-cyclodextrin, is independent of the beta-cyclodextrin concentration with a first-order rate constant, common to both RNA and DNA, of (3.5 +/- 0.5) x 10(-3) s(-1) at 61 degrees C (DNA) and at 50 degrees C (RNA). This is taken to be the rate constant for spontaneous "flipping out" of a base from within the duplex structure of the nucleic acids, the exposed base being rapidly trapped by beta-cyclodextrin. Like beta-cyclodextrin, nucleic acid methyltransferases bind the target base for methylation in a site that requires it to have flipped out of its normal position in the duplex. The spontaneous flip-out rate constant of around 10(-3) s(-1) is near the value of k(cat) for the methyltransferases (ca. 10(-3) to 10(-1) s(-1)). In principle, the enzymes, therefore, need effect little or no catalysis of the flipping-out reaction. Nevertheless, the flip-out rate in enzyme/DNA complexes is much faster. This observation suggests that the in vivo circumstances may differ from in vitro models or that factors other than a simple drive toward higher catalytic power have been influential in the evolution of these enzymes.