Britta Mynster Dahl

The conformations of locked nucleic acids (LNA)

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. Copyright

Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes.

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.

LNA stereoisomers: xylo-LNA (β-D-xylo configured locked nucleic acid) and α-L-LNA (α-L-ribo configured locked nucleic acid)

Synthesis of xylo-LNA containing one 2′-O,4′-C-methylene-β-D-xylofuranosyl thymine nucleotide monomer and α-L-LNAs containing one or four 2′-O,4′-C-methylene-α-L-ribofuranosyl thymine nucleotide monomer(s) has been accomplished using phosphoramidite chemistry with pyridine hydrochloride as activator; oligothymidylate α-L-LNA displays strongly enhanced affinity towards complementary RNA.

Phosphoramidites derived from tertiary alcohols. Why do they sometimes couple with low efficiency

Abstract tert -Alkyl phosphoramidites are somewhat sterically hindered, but give phosphites in good yields with tetrazole catalysis when the coupling time with alcohols is prolonged. Low yields of phosphotriesters are caused by elimination of the tertiary alkyl group during the subsequent oxidation of the phosphite with iodine/water/pyridine, and can be avoided by the use of tert -butyl hydroperoxide as the oxidant.

Incorporation of α- and β-LNA (Locked nucleic acid) monomers in oligodeoxynucleotides with polarity reversals

Abstract The thymidine monomers of LNA with both α- and β-configuration are incorporated with polarity reversals (i.e., with 3′–3′ and 5′–5′ junctions) in oligodeoxynucleotides with β- and α-configuration, respectively. A 5′- O -phosphoramidite of the β-LNA monomer is synthesised. Large destabilisations of duplexes with both complementary DNA and RNA are observed for oligodeoxynucleotides containing the α-LNA monomer, whereas a duplex with complementary RNA of an α-oligodeoxynucleotide containing the β-LNA monomer is not destabilised.

xylo-Configured Oligonucleotides (XNA, Xylo Nucleic Acids): Synthesis and Hybridization Studies

Abstract We report synthesis and high-affinity hybridization of fully modified home-thymine 2′-deoxy and 2′-deoxy-2′-fluoro xylo nucleic acids.

Spirophosphoranes derived from 3H-2,1-benzoxaphospholes

Starting from lithium o-lithiobenzyl alkoxides and dichlorophosphines, a series of P-substituted 3H-2-1-benz-oxaphospholes has been prepared and converted into five-co-ordinate phosphoranes by addition of 4,5-dimethyl-o-benzoquinone or 4,4′-dimethylbenzil, or condensation with 4,5-dimethylpyrocatechol or pinacol. The variable-temperature 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–1.

Improved synthesis of oligonucleotides with an allylic backbone. Oligonucleotides containing acyclic, achiral nucleoside analogues: N-1 or N-9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases

An improved phosphoramidite method is described to prepare oligonucleotides modified with the acyclic, achiral monomers 1. Examination of dimers, prepared on solid support or in solution, showed that phosphortriester dimers containing the allylic unit 1 were unstable towards bases, whereas phosphordiester dimers were stable. Phosphordiester dimers were obtained by replacing cyanoethyl phosphoramidites 2 with phosphoramidites 3, which gave phosphordiesters directly upon oxidation. The phosphordiester dimers were found to be stable towards capping and oxidation, but were somewhat labile towards acids. By reducing the contact time to acids during detritylation it was possible to prepare oligonucleotides containing 4 or 8 modified A, G or T units. The modified oligonucleotides hybridized to complementary DNA and RNA, although with reduced affinity (DeltaT(m) per modification -1 to -5 degrees C).

Preparation and Properties of a New Type of Acyclic, Achiral Nucleoside Analogue

Abstract Preparation of the nucleoside analogues 1 and incorporation of 1, B = T, in deoxyribooligonucleotides by the phosphoramidite method is described. A two-step deprotection procedure was developed to reduce cleavage of the modified allylic unit. The binding properties of the modified oligonucleotides towards complementary DNA and RNA has been evaluated by Tm measurements showing a ΔTm of −2 to −6.5°C per modification. An oligonucleotide with two modifications at the 3′-end showed considerable resistance towards cleavage by a 3′-exonuclease. No antiviral activity against HIV-1 or HSV-1 was found for 1, B = G or T, or for any of the trihydroxy derivatives 5.