Oligonucleotides were melted in buffer containing 100 mM NaCl, 20 mM sodium cacodylate, 0.5 mM Na 2 EDTA, pH 7.0. The relatively low NaCl concentration kept melting temperatures in the reasonable range even when there were multiple LNA substitutions. Oligonucleotide single-strand concentrations were calculated from absorbencies above 80 C and single-strand extinction coefficients were approximated by a nearest-neighbor model (65, 66) . It was assumed that 2 0 -Omethyl RNA and RNA strands with identical sequences have identical extinction coefficients. Absorbancy versus temperature melting curves were measured at 260 nm with a heating rate of 1 C/min from 0 to 90 C on a Beckman DU 640 spectrophotometer with a water cooled thermoprogrammer. Melting curves were analyzed and thermodynamic parameters were calculated from a two-state model with the program MeltWin 3.5 (67) . For almost all sequences, the DH derived from T m À1 versus ln (C T /4) plots is within 15% of that derived from averaging the fits to individual melting curves, as expected if the two-state model is reasonable.