LNA guanine and 2,6-diaminopurine (D) phosphoramidites have been synthesized as building blocks for antisense oligonucleotides (ON). The effects of incorporating LNA D into ON were investigated. As expected, LNA D containing ON showed increased affinity towards complementary DNA (Delta Tm +1.6 to +3.0 degrees C) and RNA (Delta Tm +2.6 to +4.6 degrees C) ON. To evaluate if LNA D containing ON have an enhanced mismatch sensitivity compared to their complementary LNA A containing ON thermal denaturation experiments towards singly mismatched DNA and RNA ON were undertaken. Replacing one LNA A residue with LNA D, in fully LNA modified ON, resulted in higher mismatch sensitivity towards DNA ON (Delta Delta Tm -4 to >-17 degrees C). The same trend was observed towards singly mismatched RNA ON (Delta Delta Tm D-a = -8.7 degrees C and D-g = -4.5 degrees C) however, the effect was less clearcut and LNA A showed a better mismatch sensitivity than LNA D towards cytosine (Delta Tm +5.5 degrees C).