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 β-D-LNA and α-L-LNA). Our findings show that the best antisense activity with 16mer gapmers containing β-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 β-D-LNA, α-L-LNA shows superior stability against a 3′-exonuclease. The design possibilities of α-L-LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of α-L-LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA α-L-LNA and β-D-LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with α-L-LNA still recruit RNase H and show good levels of antisense activity, while the same design with β-D-LNA results in a drop in antisense potency. Our findings indicate that α-L-LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.