Biomedical subjects
Publications and source records attributed to Troels Koch.
The first reported synthesis of 2'-amino-LNA purine nucleosides via a transnucleosidation is accomplished enabling the preparation of oligonucleotides incorporating 2'-amino-LNA with all four natural bases.
Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform.
Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.
Allele-specific inhibition (ASI) is a new strategy to treat cancer through a vulnerability created by the loss of large segments of chromosomal material by loss of heterozygosity (LOH). Using antisense approaches, it is possible to target single nucleotide polymorphisms (SNP) in the remaining allele of an essential gene in the tumor, thus killing the tumor while the heterozygous patient survives at the expense of the other nontargeted allele lost by the tumor. In this study, the feasibility of using locked nucleic acid (LNA)-modified DNAzymes (LNAzymes) of the 10-23 motif as allele-specific drugs was investigated. We demonstrate that incorporation of LNA into 10-23 motif DNAzymes increases their efficacy in mRNA degradation and that, in a cell-free system, the 10-23 motif LNAzyme can adequately discriminate and recognize an SNP in the large subunit of RNA polymerase II (POLR2A), an essential gene frequently involved in LOH in cancer cells. However, the LNAzymes, optimized under in vitro conditions, are not always efficient in cleaving their RNA target in cell culture, and the efficiency of RNA cleavage in cell culture is cell type dependent. The cleavage rate of the LNAzyme is also much slower than RNase H-recruiting DNA phosphorothioate antisense oligonucleotides. Moreover, compared with DNA phosphorothioates, the ability of the LNAzymes to differentially knock down two POLR2A alleles in cultured cancer cells is limited.
Methods and protocols for automated synthesis and purification of locked nucleic acid (LNA), a class of oligonucleotides obeying the Watson-Crick base-pairing rules but displaying unprecedented binding affinities toward complementary deoxyribonucleic acid (cDNA) and ribonucleic acid (RNA), is described. LNA and LNA-DNA chimeras containing phosphordiester or phosphorothioate linkages, or a mixture thereof, can be assembled by standard DNA synthesizers using 2-cyanoethyl DNA phosphoramidites and 2-cyanoethyl LNA phosphoramidites. Compared to the standard protocols used for DNA synthesis, slightly longer coupling time and oxidation time are needed for efficient oligomerization of LNA phosphoramidites. When the LNA has been assembled, it is removed from the solid support as the 5'-end-O-dimethoxytrityl (DMT) protected LNA oligomer by treatment with concentrated aqueous ammonia that also removes the phosphate and nucleobase protecting groups. The crude DMT protected LNA product can be purified using, for example, reversed-phase chromatography.
LNA guanine and 2,6-diaminopurine (D) phosphoramidites have been synthesized as building blocks for antisense oligonucleotides (ON). The effects of incorporating LNA D into ON were investigated. As expected, LNA D containing ON showed increased affinity towards complementary DNA (Delta Tm +1.6 to +3.0 degrees C) and RNA (Delta Tm +2.6 to +4.6 degrees C) ON. To evaluate if LNA D containing ON have an enhanced mismatch sensitivity compared to their complementary LNA A containing ON thermal denaturation experiments towards singly mismatched DNA and RNA ON were undertaken. Replacing one LNA A residue with LNA D, in fully LNA modified ON, resulted in higher mismatch sensitivity towards DNA ON (Delta Delta Tm -4 to >-17 degrees C). The same trend was observed towards singly mismatched RNA ON (Delta Delta Tm D-a = -8.7 degrees C and D-g = -4.5 degrees C) however, the effect was less clearcut and LNA A showed a better mismatch sensitivity than LNA D towards cytosine (Delta Tm +5.5 degrees C).
Oligonucleotides containing Locked Nucleic Acids (LNA) to various extents and at various positions were evaluated for antisense activity, RNase H recruitment, nuclease stability and thermal affinity. In this work, two different diastereoisomers of LNA were studied: the beta-D-LNA and the alpha-L-LNA (abbreviated as beta-D-LNA and alpha-L-LNA). Our findings show that the best antisense activity with 16mer gapmers containing beta-D-LNA (oligonucleotides containing consecutive segments of LNA and DNA with a central DNA stretch flanked by two LNA segments, LNA-DNA-LNA) is found with gap sizes between 7 and 10 nt. The optimal gap size is motif-dependent, and requires the right balance between gap size and affinity. Compared to beta-D-LNA, alpha-L-LNA shows superior stability against a 3'-exonuclease. The design possibilities of alpha-L-LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of alpha-L-LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA, alpha-L-LNA and beta-D-LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with alpha-L-LNA still recruit RNase H and show good levels of antisense activity, while the same design with beta-D-LNA results in a drop in antisense potency. Our findings indicate that alpha-L-LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.
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. After substitution of the triflate with azide a basic reduction affords the desired 2'-amino-LNA structure, i.e., compound 18. This new synthesis strategy towards 2'-amino-LNA improves the overall yield significantly and converges the syntheses of 2'-oxy-LNA and LNA analogues.
The stabilizing properties of LNA and alpha-L-LNA oligonucleotides against endo- and 3'-exonucleases have been evaluated.
2'-Amino-LNA phosphoramidite (10) was synthesised by means of a new strategy, which is convergent with the synthesis of 2'-oxy-LNA up until a late stage intermediate (1).
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