Locked nucleic acid: high-affinity targeting of complementary RNA for RNomics.

Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation. This conformational restriction is translated into unprecedented hybridization affinity towards complementary single-stranded RNA molecules. That makes fully modified LNAs, LNA/DNA mixmers, or LNA/RNA mixmers uniquely suited for mimicking RNA structures and for RNA targeting in vitro or in vivo. The focus of this chapter is on LNA antisense, LNA-modified DNAzymes (LNAzymes), LNA-modified small interfering (si)RNA (siLNA), LNA-enhanced expression profiling by real-time RT-PCR and detection and analysis of microRNAs by LNA-modified probes.

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.

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.

Convenient syntheses of 7-hydroxy-1-(hydroxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo

Synthesis of the diastereoisomeric LNA (locked nucleic acid) nucleosides 1-(2-O,4-C-methylene-alpha-L-ribofuranosyl)thymine (6) and 1-(2-O,4-C-methylene-alpha-L-xylofuranosyl)thymine (13) are reported via convenient reaction cascades from di-O-p-toluenesulfonyl and tri-O-methanesulfonyl nucleoside derivatives 3, 7, and 10.

The solution structure of a DNA duplex containing a single 1-(2-O,3-C-ethylene-beta-D-arabinofuranosyl)thymidine nucleoside.

The structure of a DNA duplex containing one 1-(2-O,3-C-ethylene-beta-D-arabinofuranosyl)-thymidine nucleoside (T5) modification was investigated by use of two-dimensional 1H NMR spectroscopy at 750 MHz. The structure of the d(CCGCT5AGCG):d(CGCTAGCGG) duplex (CT5AG) containing one of this 2'-O,3'-C-linked bicycloarabino conformational restricted modification has been determined. We obtained inter-proton distance bounds from NOESY spectra by including a complete relaxation matrix analysis. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. We also analyzed the fine structure of the cross peaks in a selective DQF-COSY spectra to determine the sugar conformations of the nucleotides. Forty final structures were generated for CT5AG from A-form and B-form dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.92A. The structures were observed to be markedly irregular compared to canonical B-DNA, especially in terms of large variations in propeller twist and buckle. Also, lack of stacking of two bases near the modification site is observed. The sugar conformations of all the unmodified nucleotides are close to pure C2'-endo conformation. The structural feature of CT5AG was discussed in relation to the thermal stability and resistance towards exonucleolytic degradation.

The solution structure of a locked nucleic acid (LNA) hybridized to DNA.

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.

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. 10-16 mer 2'-O-methyl oligoribonucleotides give sequence- and dose-dependent inhibition of Tat-dependent transcription of an HIV DNA template in HeLa cell nuclear extract. Inhibition is maintained for the substituted 12-mer analogues but is poorer for PNA and is not correlated with TAR binding strength.

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.

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. The phosphoramidite derivative of 15 was successfully incorporated into oligodeoxynucleotides using standard methods, and thermal denaturation studies showed moderate decreases in duplex stabilities of -2.1 and -1.5 degrees C per modification toward complementary DNA and RNA, respectively.

Synthesis of diastereomeric mixtures of 5'-C-hydroxymethylthymidine and introduction of a novel class of C-hydroxymethyl functionalised oligodeoxynucleotides.

Novel 5'-C-hydroxymethyl substituted derivatives of thymidine have been prepared by dihydroxylation of the 5'C-methylene nucleoside 1. Osmium tetraoxide catalysed dihydroxylation of 1 afforded a 3:2 epimeric mixture of diols 2, whereas asymmetric dihydroxylation using AD-mix-alpha and AD-mix-beta resulted in mixtures 3 and 4 of the two epimeric diols, both enriched with the same diastereomer. Nucleosides 2 were transformed into phosphoramidites 8 which were used for solid phase synthesis of oligodeoxynucleotides (ODNs) containing 5'-C-(hydroxymethyl) functionalised thymidine monomers. This novel class of C-hydroxymethyl modified ODN-analogues exhibited promising affinity towards both complementary DNA and RNA as well as resistance towards 3'-exonucleolytic degradation.

The adenine derivative of alpha-L-LNA (alpha-L-ribo configured locked nucleic acid): synthesis and high-affinity hybridization towards DNA, RNA, LNA and alpha-L-LNA complementary sequences.

Synthesis of a 9-mer alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) containing three 9-(2-O,4-C-methylene-alpha-L-ribofuranosyl)adenine nucleotide monomer(s) has been accomplished. The work involved synthesis of the bicyclic adenine nucleoside via a condensation reaction between L-threo-pentofuranose derivative 1 and 6-N-benzoyladenine followed by C2'-epimerization. Hybridization studies demonstrated very strong duplex formation with 9-mer complementary DNA, RNA, LNA and alpha-L-LNA target sequences.

Incorporation of (R)- and (S)-3',4'-seco-thymidine into oligodeoxynucleotides: hybridization properties and enzymatic stability.

Novel flexible oligodeoxynucleotide analogues containing (R)- and (S)-3',4'-seco-thymidine were synthesized on an automated DNA-synthesizer using the phosphoramidite approach. Oligodeoxynucleotide analogues (17-mers) having one or three modifications in the middle or one or two modifications in the ends were evaluated with respect to hybridization properties and enzymatic stability. 3'-End-modified oligomers were stable towards 3'-exonuclease degradation and displayed acceptable hybridization properties.

Oligonucleotides containing 4'-C-aminomethyl-2'-modified thymidines show increased binding affinity towards DNA and RNA.

Oligonucleotides containing 4'-C-aminomethyl-2'-O-methyl or 4'-C-aminomethyl-2'-deoxy-2'-fluoro modified thymidines have been synthesized. Compared with the corresponding oligodeoxynucleotide reference these novel oligonucleotide analogues display increased binding affinity towards complementary single stranded DNA as well as RNA. The possible effect of the positively charged 4'-C-aminomethyl group has been investigated.

Synthesis of a novel bicyclic nucleoside restricted to an S-type conformation and initial evaluation of its hybridization properties when incorporated into oligodeoxynucleotides.

The phosphoramidite (1S,3R,4S)-3-(2-cyanoethoxy(diisopropylamino)phosphinoxymethyl)-5-N-(4-monomethoxytrityl)-1-(uracil-1-yl)-5-aza-2-oxabicyclo[2.2.1]heptane 18 of a novel bicyclic nucleoside structure was synthesized from the known 1-(3'-deoxy-beta-D-psicofuranosyl)uracil 3. Conformational analysis of its structure verified its expected S-type furanose conformation, and the secondary amino group in the 4'-position allowed for incorporation into oligonucleotides using 5' --> 3' directed oligonucleotide synthesis as previously described for phosphoramidates. Thermal denaturation studies showed rather large decreases in duplex stabilities of -4.3 and -2.7 degrees C per modification toward complementary DNA and RNA, respectively.

Stereocontrolled synthesis of LNA dinucleoside phosphorothioate by the oxathiaphospholane approach.

The application of the oxathiaphospholane approach for the stereocontrolled synthesis of LNA dinucleoside phosphorothioate is described. The reaction of ring opening condensation proceeds in CH3CN solution in high yield and with over 96% stereoselectivity. One of diastereomers of LNA dinucleoside phosphorothioate (presumably R(P)) was found to be readily digested by svPDE.

Synthesis and properties of alpha- and beta-oligodeoxynucleotides containing alpha- and beta-1-(2-O-methyl-D-arabino-furanosyl)thymine.

Synthesis of the alpha- and beta-anomer of 2'-OMe-araT (alpha- and beta-1-(2-O-methyl-D-arabinofuranosyl)thymine) and their incorporation into oligodeoxynucleotide (ODN) analogues is described. Condensation of the key arabinofuranose derivative with silylated thymine afforded the alpha-anomer and the beta-anomer which were converted into the respective phosphoramidite building blocks. Automated synthesis of beta-ODNs containing beta-2'-OMe-araT (by use of standard beta-amidites and phosphoramidite building block 9b) and alpha-ODNs containing alpha-2'-OMe-araT (by use of alpha-T-amidite and phosphoramidite building block 9a) allowed evaluation of their properties. With regard to 3'-exonucleolytic degradation, 3'-end incorporation of either beta- or alpha-2'-OMe-araT resulted in considerable stabilization compared to unmodified beta-ODNs. Thermal stabilities of duplexes formed between modified ODNs and both unmodified DNA and RNA were evaluated and compared to unmodified controls. In all experiments stable duplexes were formed, but whereas beta-ODNs containing beta-2'-OMe-araT showed moderately lowered thermal stabilities towards both DNA and RNA, alpha-ODNs containing alpha-2'-OMe-araT exhibited significantly increased melting points (compared to beta-ODN controls) when complexed with RNA. These results illustrate the potential of using arabino-configurated nucleosides as modified monomers in biologically active ODN-analogues, either as, e.g., 2'-O-alkylated or 2'-O-functionalized derivatives.

Oligonucleotide analogues containing 4'-C-(hydroxymethyl)uridine: synthesis, evaluation and mass spectrometric analysis.

2',3'-Di-O-tert-butyldimethylsilyl-4'-C-(hydroxymethyl)uridine was synthesized and converted into the phosphoramidite building blocks 9 and 13. Novel oligodeoxynucleotide analogues containing 4'-C-hydroxymethyl linked phosphodiester internucleoside linkages and 3'-hydroxyl linked phosphodiester internucleotide linkages were synthesized on an automated DNA-synthesizer. The latter modification introduced an additional 4'-C-hydroxymethyl functionality. Oligodeoxynucleotides with one or two modifications in the middle or in the ends of 17-mers, 15-mers and 14-mers have been evaluated with respect to hybridization properties and enzymatic stability. Compared to unmodified oligomers, 3'-end-modified oligodeoxynucleotides were stabilized towards 3'-exonucleolytic degradation, but showed moderately to strongly lowered hybridization properties towards complementary DNA. However, more promising results were obtained in melting experiments with complementary RNA where only small decreases in melting temperature were detected. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to identify products from syntheses of the modified oligodeoxynucleotide analogues.

Synthesis and evaluation of oligodeoxynucleotides containing acyclic nucleosides: introduction of three novel analogues and a summary.

Novel flexible oligodeoxynucleotides containing (S)-1-(2,3-dihydroxypropyl)thymine or 2',3'-seco-thymidine nucleoside analogues were synthesized on an automated DNA-synthesizer. Oligodeoxynucleotides with one, two or three acyclic nucleosides incorporated in the middle or in the ends of 17-mers have been evaluated. 3'-End-modified oligomers were significantly stabilized towards 3'-exonucleolytic degradation compared to unmodified analogues and showed acceptable hybridization properties as measured by UV experiments. For oligodeoxynucleotide analogues containing the three novel acyclic monomers in the middle, a more pronounced reduction in duplex stability was observed. All oligodeoxynucleotides containing acyclic nucleoside analogues made so far are evaluated with respect to stability towards 3'-exonucleolytic degradation and hybridization properties.

Stereoselective synthesis, chemistry and antiviral evaluation of 1-(2,3-dideoxy-3-C-hydroxymethyl-beta-D-threo-pentofuranosyl)thymine derivatives.

A series of novel 3'-C-branched 2',3'-dideoxynucleosides have been synthesized and evaluated as anti-HIV agents. Hydroboration of 2',3'-dideoxy-3'-C-methylene nucleoside proceeded regio- and stereoselectively to give 1-(2,3-dideoxy-3-C-hydroxy methyl-5-O-trityl-beta-D-threo-pentofuranosyl)thymine (5) after oxidation. 3'-C-Chloromethyl and 3'-C-iodomethyl 5'-O-protected 2',3'-dideoxynucleosides 9 and 12 were obtained from 5 by reaction with carbon tetrachloride/triphenylphosphine and methyltriphenoxyphosphonium iodide, respectively. Arbuzov reaction of 12 with triethyl phosphite afforded 3'-C-(diethyl-phosphono)methyl 5'-O-protected 2',3'-dideoxynucleoside 14. Compounds 5, 9, 12 and 14 were detritylated to give 1-(3-C-chloromethyl-2,3-dideoxy-beta-D-threo-pentofuranosyl)thymine (10), 1-(2,3-dideoxy-3-C-hydroxymethyl-beta-D-threo-pentofuranosyl)-thymine (11), 1-(2,3-dideoxy-3-C-iodomethyl-beta-D-threo-pentofuranosyl)thymine (13) and 1-(2,3-dideoxy-3-C-(O,O'-diethylphosphono)methyl-beta-D-threo- pentofuranosyl)thymine (15), respectively. These nucleoside analogues were evaluated for antiviral activity against human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1) in vitro. Compound 5 demonstrated selective antiviral activity against HIV-1 but not HSV-1.