Expanding the design horizon of antisense oligonucleotides with alpha-L-LNA

Abstract

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.

Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals

“…Interestingly, the MOE 16mer 2a had greatly reduced potency, whereas the potency of the 16mer LNA 2b was improved relative to the 18mer LNA ASO 1b. These results are consistent with a previous study of LNA ASOs (37), in which the high affinity of LNA facilitated good target reduction with 16mer LNA ASO designs.…”

Evaluation of LNA-Modified DNAzymes Targeting a Single Nucleotide Polymorphism in the Large Subunit of RNA Polymerase II

“…The DNAzymes and LNAzymes were synthesized by Santaris Pharma A/S (Hørsholm, Denmark), as described previously (Frieden et al, 2003). In all ODNs, 5-methyl-C was used.…”

Locked Nucleic Acid: High-Affinity Targeting of Complementary RNA for RNomics

“…Also Elmén et al (2004) demonstrated that LNA/DNA mixmers activate RNase H when the mixmer has a DNA stretch of 6 nt. In accordance, Frieden et al (2003a) concluded that a DNA gap size between 7 and 10 nt is optimal for LNA/DNA/LNA antisense gapmers. The overall conclusion is that antisense LNA oligonucleotides can be designed to elicit RNase H activity while still containing LNA monomers for improved binding and target accessibility (Jepsen and Wengel 2004).…”