THERMODYNAMIC STABILITY AND SOLUTION CONFORMATION OF TANDEM G-CENTER-DOT-A MISMATCHES IN RNA AND RNA-CENTER-DOT-DNA HYBRID DUPLEXES

G.A mismatches form a variety of hydrogen-bonded structures in DNA, most of which destabilise the duplex. Tandem G A mismatches in the context YGAR (Y = pyrimidine, R = purine), however, form base pairs using the amino group of the guanine residue [Li, Y., Zon, G. and Wilson, W D. (1991) Proc. Natl Acad. Sci. USA 88, 26-30], which permits extensive base-base stacking, leading to a slight stabilisation of the helix [Ebel, S., Lane, A. N. and Brown, T. (1992) Biochemistry 31, 12083-12086]. We have measured the thermodynamic stability of several RNA and RNA DNA hybrid duplexes containing tandem G A mismatches. The RNA duplexes are intrinsically much more stable than the corresponding DNA duplexes and the mutations are destabilising in all cases. NOE and coupling-constant data show that all of the sugars are in the C3'-endo range of conformations, and glycosidic torsion angles are in the range -160 degrees to -180 degrees in r(CCACGAGUGG) r(CCACGAGUGG). Both sequential NOE intensities and circular-dichroism measurements indicate that the global conformation of the mismatched RNA is A-like. The N1H group of the mismatched guanine residue is not involved in hydrogen bonding with the adenine residue, indicating the presence of the amino-pairing scheme. Determination of the structure using 'loose' NMR-derived constraints shows that the potential energies of the imino-paired and amino-paired forms are similar, but substantially higher than energy-minimised RNA. Using tighter constraints derived from more extensive analysis of one-dimensional and two-dimensional NOE data showed that the amino-paired structure agrees with the constraint data better than the imino-paired structure, and also accounts for unusual chemical shifts and the lack of hydrogen bonding of the guanine N1H group. Resulting molecular models show that the amino-paired mismatches are not as extensively stacked on the neighbouring part of the duplex as in the B-DNA analogues, largely accounting for the lower thermodynamic stability in the RNA duplexes.