Free energy parameters for predicting stabilities of 2 0 -O-methyl RNA/RNA and 2 0 -O-methyl RNA-LNA/RNA duplexes with the Individual Nearest-Neighbor Hydrogen Bonding (INN-HB) model (64) were obtained by multiple linear regression with the program Analyse-it v.1.71 (Analyse-It Software, Ltd, Leeds, England; www.analyse-it. com) which expands Microsoft Excel. Analyse-It was also used to obtain parameters for enhancement of stabilities of 2 0 -O-methyl RNA/RNA duplexes by substitution of LNA nucleotides internally and/or at the 3 0 end when the LNAs are separated by at least one 2 0 -O-methyl nucleotide. Results from T m À1 versus ln (C T /4) plots were used as the data for the calculations. 3 show typical data from optical melting curves, and Table 1 lists the thermodynamic parameters for the helix to coil transition with either no or one LNA nucleotide in the primarily 2 0 -O-methyl strand of a hybrid with a Watson-Crick complementary RNA strand.