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Insufficient efficacy and͞or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a highaffinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA͞DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA͞DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA͞DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.A ntisense oligonucleotides designed according to straightforward base-pairing rules have been useful in functional genomics efforts, and there also has been recent clinical progress in developing antisense drugs (1-5). The key objective in the field, however, remains the identification of oligonucleotide analogs that provide the possibility to achieve high in vivo efficacy in the absence of significant toxicity (1-3).To date, all human antisense studies, as well as the vast majority of studies on other species, have relied on the use of phosphorothioate DNA analogs (where one nonbridging phosphate oxygen has been replaced). Although phosphorothioates are markedly more resistant to degrading enzymes than DNA, their DNA-binding capacity (relating to potency when used as antisense agents) is low, and they are well known to cause nonspecific protein binding, largely because of their polyanionic nature. The latter phenomenon contributes to a toxicity profile that limits many applications (6, 7). For example, when injected into the brain, phosphorothioates can cause severe tissue damage, especially with repeated or prolonged administration schedules (7,8). Such phosphorothioate-induced toxic reactions are thought to be reduced but not absent in second-generation antisense agents, like mixed backbone oligonucleotides (containing phosphorothioates in combination with oligodeoxyribonucleotides or oligoribonucleotides) (9).Interestingly, conformational restriction has been successfully applied in recent years to the design of high-affinity oligonucleotides. Several analogs containing bi-and tricyclic carbohydrate moieties have displayed enhanced duplex stability (10-20) and most notably so locked nucleic acids (LNA) (Fig. 1). LNA induces unprecedented increases in the thermal stability (melting temperature, T m ) of duplexes toward complementary DNA and RNA (⌬T m ͞LNA monomer ϭ ϩ 3 to ϩ 11°C compared with the corresponding DNA reference). By virtue of their bicyclic structure, the furanose ring of the LNA monomers is locked in a 3Ј-endo conformation, thus structurally mimicking the standard RNA monomers. Moreover, LNA͞LNA duplex formation has been shown to constitute the most stable...

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We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2'-O, 4'-C-methylene bridge. We have investigated a modified 9-mer single stranded oligonucleotide as well as three 9- and 10-mer modified oligonucleotides hybridized to unmodified DNA. The single-stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson-Crick base-pairing with all the nucleotides in anti-conformation. By use of selective DQF-COSY spectra we determined the ratio between the N-type (C3'-endo) and S-type (C2'-endo) sugar conformations of the nucleotides. In contrast to the corresponding single-stranded DNA (ssDNA), we found that the sugar conformations of the single-stranded LNA oligonucleotide (ssLNA) cannot be described by a major S-type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N-type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N-type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S-type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N-type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases.

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A novel efficient method for the synthesis of locked nucleic acid (LNA) monomers is described. The LNA 5',3'-diols containing thymine, 4-N-acetyl- and 4-N-benzoylcytosine, 6-N-benzoyladenine, and 2-N-isobutyrylguanine as nucleobases were prepared via convergent syntheses. The method is based on the use of the common sugar intermediate 1,2-di-O-acetyl-3-O-benzyl-4-C-methanesulfonoxymethyl-5-O-methanesulfonyl-D-erythro-pentofuranose (8) that easily can be prepared from D-glucose in multigram scale. Four different nucleobases were stereoselectively coupled to 8 using a modified Vorbrüggen procedure to give the corresponding 4'-C-branched nucleoside derivatives. Subsequent ring closing furnished the protected LNA nucleosides. The 5'-O-mesyl groups were efficiently displaced by nucleophilic substitution using sodium benzoate. Saponification of the 5'-benzoates followed by catalytic removal of the 3'-O-benzyl groups afforded the free LNA diols. The exocyclic amino groups of adenosine and cytidine were selectively acylated to give 4-N-acetyl- or 4-N-benzoyl-LNA-C and 6-N-benzoyl-LNA-A. The isobutyryl group of guanine was retained during the preparation of 2-N-isobutyryl-LNA-G. The LNA-T diol and base-protected LNA diols can be directly converted into LNA-phosphoramidites for automated chemical synthesis of LNA containing oligonucleotides.

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