The effects of LNA substitutions are approximately additive when LNA nucleotides are spaced by at least one 2 0 -O-methyl nucleotide Table 2 contains thermodynamic parameters measured for duplexes having more than one LNA substitution and Table 3 compares the stabilities at 37 C with those predicted from four simple models. The first model, labeled 'additivity', predicts the DG 37 for duplex formation in the 5 0 ACUACCA/ 3 0 UGAUGGU series by adding the free energy increments measured for single LNA substitutions in the same context to the DG 37 for duplex formation in the absence of LNA nucleotides. The second model predicts the DG 37 (kcal/mol) for duplex formation with the following equation as deduced from fitting the data in Tables 1 and 2 Here, DG 37 (2 0 -O-MeRNA/RNA) is the free energy change at 37 C for duplex formation in the absence of any LNA nucleotides, n 5 0 tL is the number of 5 0 terminal LNAs, n iAL/UL and n iGL/CL are the number of internal LNAs in AU and GC pairs, respectively, n 3 0 tU and n 3 0 tAL/CL/GL are the number of Here, T m À1 is the inverse melting temperature in kelvin, R is the gas constant, 1.987 cal K À1 mol À1 , C T is the total oligonucleotide strand concentration, and both strands have the same concentration. Table 1 . Thermodynamic parameters of duplex formation between RNA and 2 0 -O-methyl oligoribonucleotides with and without a single LNA substitution a Oligonucleotides RNA Average of curve fits 3 0 terminal LNAs that are U or not U, respectively. Both methods that use experimental data for DG 37 (2 0 -O-MeRNA/RNA) provide reasonable predictions that are within 1 kcal/mol of the measured value (Table 3) . Two other methods that use nearest-neighbor models to approximate DG 37 (2 0 -O-MeRNA/RNA) provide somewhat less accurate, but still reasonable predictions as described below. The duplex with the worst prediction, 5 0 G M U L U M C L G M G L /3 0 CAAGCC has a 5 0 G M U L /3 0 CA nearest neighbor, consistent with this motif being unusually unstable by $1.2 kcal/mol. Thus, it is likely that the DG 37 of Equation 1 should be made less favorable by 1.2 kcal/mol for every internal 5 0 G M U L /3 0 CA nearest neighbor in a duplex. Evidently, the effects of multiple LNA substitutions are approximately additive when the LNAs are spaced by at least 1 nt.