Purified Lang virions (1.4 × 10 11 ) were treated with 25 µg/ ml of NE in 40 µL of VDB at 37°C for the times indicated. Reactions were terminated as described above. To verify σ3 removal, the proteins from 5.0 × 10 10 particles were separated on SDS-12% polyacrylamide gels and visualized with Coomassie Brilliant Blue staining. Viral infectivity for each time point was determined by plaque assay on L929 cell monolayers. The influence of locked nucleic acid residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA. Understanding the thermodynamics of nucleic acid duplexes is important for many reasons. For example, such knowledge facilitates design of ribozymes (1), antisense and RNAi oligonucleotides (2) (3) (4) (5) (6) (7) (8) (9) , diagnostic probes including those employed on microarrays (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) and structures useful for nanotechnology (24) (25) (26) (27) . Many modified residues have been developed for such applications. Examples include propynylated bases (28) (29) (30) , peptide nucleic acids (5, (31) (32) (33) , N3 0 -P5 0 phosphoramidates (34-38) and 2 0 -O-alkyl RNA (39) (40) (41) (42) (43) . A modification that is particularly stabilizing in DNA and RNA duplexes (44) (45) (46) (47) (48) (49) (50) (51) is a methyl bridge between the 2 0 oxygen and 4 0 carbon of ribose to form a 'locked nucleic acid' or LNA as shown in Figure 1 . McTigue et al. (48) have shown that the enhanced stability due to a single LNA residue in a DNA duplex can be predicted from a nearestneighbor model.