Thermodynamic stability and solution conformation of tandem G · A mismatches in RNA and RNA · DNA hybrid duplexes

Abstract

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. & 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. & 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° to ‐180° 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. Copyright © 1994, Wiley Blackwell. All rights reserved