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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To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic acid) three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated with respect to duplex stabilities. The abasic LNA monomer (X(L), Figure 1) with the rigid carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the exceptional hybridization properties of LNA primarily originate from improved intrastrand nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives, obtained by cleavage of the C1'-O4' bond of an LNA monomer or complete removal of the O4'-furanose oxygen atom (Z(L) and dZ(L), respectively, Figure 1), were compared to their acyclic DNA counterpart (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives Z(L) and dZ(L) destabilize duplex formation even more than the flexible seco-DNA monomer Z.

Starting from lithium o-lithiobenzyl alkoxides and dichlorophosphines, a series of P-substituted 3H-2-1 -benzoxaphospholes has been prepared and converted into five-co-ordinate phosphoranes by addition of 4,5-dimethylo-benzoquinone or 4,4'-dimethylbenzil, or condensation with 4,5-dimethylpyrocatechol or pinacol. The variabletemperature n.m.r. spectra of these phosphoranes are discussed. In these systems phenylamino-and dimethylamino-groups have similar apicophilicities and are less apicophilic than hydrogen by some 4-5 kcal mol-l.

Neural cell adhesion molecule; Northern blotting; Oligonucleotide A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized the five NCAM mRNAs in rat brain.