Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes

Abstract

Nearest-neighbor thermodynamic parameters of the 'universal pairing base' deoxyinosine were determined for the pairs I.C, I.A, I.T, I.G and I.I adjacent to G.C and A.T pairs. Ultraviolet absorbance melting curves were measured and non-linear regression performed on 84 oligonucleotide duplexes with 9 or 12 bp lengths. These data were combined with data for 13 inosine containing duplexes from the literature. Multiple linear regression was used to solve for the 32 nearest-neighbor unknowns. The parameters predict the T(m) for all sequences within 1.2 degrees C on average. The general trend in decreasing stability is I.C > I.A > I.T approximately I. G > I.I. The stability trend for the base pair 5' of the I.X pair is G.C > C.G > A.T > T.A. The stability trend for the base pair 3' of I.X is the same. These trends indicate a complex interplay between H-bonding, nearest-neighbor stacking, and mismatch geometry. A survey of 14 tandem inosine pairs and 8 tandem self-complementary inosine pairs is also provided. These results may be used in the design of degenerate PCR primers and for degenerate microarray probes.

Figure 1

The proposed inosine-Watson–Crick configurations (–7).

Figure 2

Comparison of experimental versus predicted free energies of all two-state duplexes from Table 1.