Functionalized LNA (locked nucleic acid): high-affinity hybridization of oligonucleotides containing N-acylated and N-alkylated 2'-amino-LNA monomers.

A molecular dynamics (MD) simulation, synthesis and very efficient hybridization for a series of N-acylated and N-alkylated derivatives of 2'-amino-LNA are reported.

Synthesis of 2'-amino-LNA: a new strategy.

In this paper we present revised and significantly improved synthetic routes to 2'-amino-LNA (locked nucleic acid). The optimal route is convergent with the synthesis of LNA monomers ("2'-oxy-LNA") via a common intermediate obtained by a mild deacetylation for the liberation of the 2'-hydroxy group to give compound 23 without the concomitant ring closure that affords the 2'-oxy-LNA skeleton. After inversion of the stereochemistry at C2' and triflate formation at the 2'-hydroxy group a new common intermediate 16 is obtained which gives easy access to a range of other analogues exemplified by the introduction of a sulfur nucleophile leading to the 2'-thio-LNA structure.

NMR solution structure of dsDNA containing a bicyclic D-arabino-configured nucleotide fixed in an O4'-endo sugar conformation.

[3.2.0]bcANA is a D-arabino-configured bicyclic nucleotide with a 2'-O,3'-C-methylene bridge.

N-methylpiperazinocarbonyl-2',3'-BcNA and 4'-C-(N-methylpiperazino)methyl-DNA: introduction of basic functionalities facing the major groove and the minor groove of a DNA:DNA duplex.

Piperazino-functionalized 2',3'-BcNA and 4'-C-hydroxymethyl-DNA are appropriate molecular architectures for the introduction of basic functionalities facing the major groove and the minor groove of nucleic acid duplexes, respectively. 4'-C-(N-Methylpiperazino)methyl-DNA monomers induce significantly increased thermal stability of a DNA:DNA duplex.

The alpha-L-ribo-isomers of RNA and LNA (locked nucleic acid).

The phosphoramidite approach has been used for the automated synthesis of alpha-L-LNA, alpha-L-RNA, and oligomers composed of mixtures of alpha-L-LNA, alpha-L-RNA and DNA monomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. alpha-L-LNAs were shown to be significantly stabilized towards 3'-exonucleolytic degradation.

Xylo-Configured oligonucleotides (XNA, xylo nucleic acid): synthesis of conformationally restricted derivatives and hybridization towards DNA and RNA complements.

Xylo-Configured oligonucleotides (XNA) containing a novel conformationally restricted 2'-deoxy-2'-fluoro-beta-D-xylofuranosyl nucleotide monomer, a novel conformationally locked 2'-amino-2'-deoxy-2'-N,4'-C-methylene-beta-D-xylofuranosyl nucleotide monomer, and a known 2'-deoxy-beta-D-xylofuranosyl nucleotide monomer (XNA monomers) have been synthesized and their hybridization towards DNA and RNA complements studied. Thermal denaturation studies of nine-mer mixed-base sequences composed of a mixture of XNA monomers and DNA monomers revealed preferential hybridization towards RNA complements relative to DNA complements. For 14-mer homo-thymine XNAs containing thirteen XNA monomers, stable complexes towards single-stranded DNA and RNA were formed at pH 7.

A substituted triaza crown ether as a binding site in DNA conjugates.

Synthesis of an asymmetrically substituted triaza crown ether, its incorporation into the 3'-end and 5'-end of ninemer oligonucleotides, and the influence of various alkanediamine ligands on duplex thermostabilities are reported.

LNA: a versatile tool for therapeutics and genomics.

Locked nucleic acid (LNA) is a nucleic acid analogue that displays unprecedented hybridization affinity towards complementary DNA and RNA. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising molecule for the development of oligonucleotide-based therapeutics.

alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) recognition of DNA: an NMR spectroscopic study.

We have used NMR and CD spectroscopy to study and characterise two alpha-L-LNA:DNA duplexes, a nonamer that incorporates three alpha-L-LNA nucleotides and a decamer that incorporates four alpha-L-LNA nucleotides, in which alpha-L-LNA is alpha-L-ribo-configured locked nucleic acid. Both duplexes adopt right-handed helical conformations and form normal Watson-Crick base pairing with all nucleobases in the anti conformation. Deoxyribose conformations were determined from measurements of scalar coupling constants in the sugar rings, and for the decamer duplex, distance information was derived from 1H-1H NOE measurements.

Methylphosphonate LNA: a locked nucleic acid with a methylphosphonate linkage.

Synthesis of an oligonucleotide containing one methylphosphonate locked nucleic acid (LNA) thymine monomer using the phosphoramidite approach is described. The binding affinity of this 9-mer methylphosphonate LNA towards complementary DNA and RNA oligonucleotides was increased compared to the reference DNA, but decreased compared to the reference LNA. In the 9-mer sequence context studied, introduction of a single methylphosphonate LNA monomer, contrary to a single LNA monomer, efficiently inhibits 3'-exonucleolytic degradation.

LNAzymes: incorporation of LNA-type monomers into DNAzymes markedly increases RNA cleavage.

Incorporation of two alpha-L-LNA/LNA nucleotides into each of the two binding arms of a "10-23" DNAzyme has been accomplished and the RNA cleavage with these novel LNAzymes studied. In comparison with the unmodified DNAzyme, the LNAzymes show significantly improved cleavage of the phosphodiester backbone at the target nucleotide in a small RNA substrate (58n RNA) under single-turnover conditions. The LNAzymes show efficient multiple turnover.

3',4'-trans-linked bicyclic nucleosides locked in S-type conformations.

A novel class of 3',4'-trans-linked bicyclic nucleosides with locked S-type furanose conformations is introduced by synthesis of two model derivatives; one was obtained by cyclic ether formation and the other by ring-closing metathesis methodology.

Universal hybridization using LNA (locked nucleic acid) containing a novel pyrene LNA nucleotide monomer.

A novel pyrene LNA nucleotide monomer is shown to mediate universal hybridization when incorporated into a DNA strand or a 2'-OMe-RNA/LNA chimeric strand. For the latter, high-affinity universal hybridization without compromising the base-pairing selectivity of bases neighbouring the universal pyrene LNA nucleotide monomer is documented.

Oligodeoxynucleotides containing amide-linked LNA-type dinucleotides: synthesis and high-affinity nucleic acid hybridization.

Synthesis of three different amide-linked LNA-type dinucleotides and their incorporation into 9-mer and 17-mer oligodeoxynucleotides is described; compared to the reference DNA-RNA duplex, incorporation of one of the three dimers (5'-DNA*LNA dimer) induced significantly increased duplex thermostabilities.

Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids.

Locked nucleic acids (LNAs) containing one or more 2'-O,4'-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. unprecedented helical thermostability when hybridized with cognate RNA and DNA.

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties.

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands.

Alpha-L-RNA (alpha-L-ribo configured RNA): synthesis and RNA-selective hybridization of alpha-L-RNA/alpha-L-LNA chimera.

Synthesis of the novel alpha-L-ribofuranosyl phosphoramidite derivative was accomplished via the alpha-L-ribofuranosyl thymine nucleoside. Amidite was used in automated syntheses of chimeric oligonucleotides composed of mixtures of the novel alpha-L-RNA nucleotide monomer ((alphaL)T, alpha-L-ribo configured RNA), and DNA, LNA (T(L), locked nucleic acid) or alpha-L-LNA ((alphaL)T(L), alpha-L-ribo configured locked nucleic acid) nucleotide monomers. For alpha-L-RNA/DNA and alpha-L-RNA/alpha-L-LNA chimeras, RNA-selective hybridization was obtained, for alpha-L-RNA/alpha-L-LNA chimera we found increased binding affinity compared to the corresponding DNA:RNA reference duplex.

Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2'-O-methyl/LNA oligoribonucleotides.

The HIV-1 trans-activation responsive element (TAR) RNA 59-residue stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibition of these interactions blocks full-length HIV transcription and hence replication. We have found that three types of 12-residue oligonucleotide analogues, namely, a 2'-O-methyl oligoribonucleotide (OMe), a chimeric oligonucleotide containing 7xOMe and 5x5-methyl C locked nucleic acid (LNA) residues, and a peptide nucleic acid (PNA), inhibit Tat-dependent in vitro transcription in HeLa cell nuclear extract equally efficiently (50% inhibition at 100-200 nM) and sequence specifically.

Oligonucleotide analogue interference with the HIV-1 Tat protein-TAR RNA interaction.

The HIV-1 Tat protein interaction with its RNA recognition sequence TAR is an important drug target and model system for the development of specific RNA-protein inhibitors. 2'-O-methyl oligoribonucleotides complementary to the TAR apical stem-loop effectively block Tat binding in vitro. Substitution by 5-propynylC or 5-methylC LNA monomeric units into a 12-mer 2'-O-methyl oligoribonucleotide leads to stronger inhibition, as does a 12-mer PNA.

LNA (locked nucleic acid) and the diastereoisomeric alpha-L-LNA: conformational tuning and high-affinity recognition of DNA/RNA targets.

The remarkable binding properties of LNA (Locked Nucleic Acid) and alpha-L-LNA (the alpha-L-ribo configured diastereoisomer of LNA) are summarized, and hybridization results for LNA/2'-O-Me-RNA chimera and LNAs with a "dangling" nucleotide are introduced. In addition, results from NMR investigations on the furanose conformations of the individual nucleotide monomers in different duplexes are presented. All these data are discussed with focus on the importance of conformational steering of unmodified nucleotides in partly modified LNA and alpha-L-LNA sequences in relation to the unprecedented binding properties of LNA and alpha-L-LNA.

Locked nucleic acids: a promising molecular family for gene-function analysis and antisense drug development.

Locked nucleic acids (LNAs) are a family of conformationally locked oligonucleotide analogs inducing unprecedented binding affinity towards DNA/RNA target sequences. Importantly, by virtue of the structural resemblance of LNAs to natural nucleic acid monomers, a combination of LNA chemistry with other oligonucleotide chemistries can be exploited to fine-tune the properties towards optimized antisense drug development and target validation technology. The first promising antisense results from experiments with LNA in living animals are described.

Novel bicyclic nucleoside analogue (1S,5S,6S)-6- hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo[3.2.1]octane: synthesis and incorporation into oligodeoxynucleotides.

The novel bicyclic nucleoside (1S,5S,6S)-6-hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo[3.2.1]octane [2'-deoxy-1'-C,4'-C-(2-oxapropano)uridine] (15), expected to be restricted into an O4'-endo furanose conformation, was synthesized from the known 1-(3'-deoxy-beta-D-psicofuranosyl)uracil 5.