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.

Figure 1

Chemical structures of β-d-LNA and α-l-LNA.

Figure 2

Down-regulation of Luciferase expression by gapmers (1–12) with PS in the gap and PO in the flanks containing an increasing gap size from 7 to 10 nt directed against three different motifs (1, 2 and 3) at 2 nM. The melting temperatures (T_m) for the different gapmers are also included in the graph.

Figure 3

Down-regulation of Luciferase expression by fully thiolated oligonucleotides (all PS, 17–21) with reduced size and various gap sizes (12 and 14mers) at 2 nM. The 16mer (all PS, 13–16) is included as a reference. The melting temperatures (T_m) for the different gapmers are also included in the graph.

Figure 4

Down-regulation of Luciferase expression by oligonucleotides 14 and 15, and the corresponding mismatch-containing control oligonucleotides 35 and 36 directed against motif 3 at 2 nM.

Figure 5

Kinetic study of oligonucleotides containing α-l-LNA at the 3′-end (t₁₀T^αt and t₉T^α₂t) against a 3′-exonuclease (SVPD), in order to evaluate the nucleolytic stability. The graph also shows the corresponding controls (t₁₂, t_s12, t₁₀Tt and t₉T₂t). The assays were performed using 26 µg/ml oligonucleotide, 0.3 µg/ml enzyme at 37°C in a buffer of 50 mM Tris–HCl, 10 mM MgCl₂, pH 8. Aliquots of the enzymatic digestion were removed at the indicated times. In a separate assay, the enzyme was shown to maintain its activity under these conditions for at least 2 h (data not shown). The oligonucleotides were synthesized on deoxynucleoside-support, t. Upper case for LNA and lower case for DNA.

Figure 6

Kinetic study of different oligonucleotide gapmers (16mers) with an increasing deoxynucleotide gap against S1-endonuclease, in order to evaluate the nucleolytic stability. The graph also shows the stability of t_s16, T₁₆ and T^α₁₅T against S1-endonuclease. The assay was performed using 1.5 µM oligonucleotide and 16 U/ml enzyme at 37°C in a buffer of 30 mM NaOAc, 100 mM NaCl, 1 mM ZnSO₄, pH 4.6. Aliquots of the enzymatic digestion were removed at the indicated times. In a separate assay, the enzyme was shown to maintain its activity under these conditions for at least 2 h (data not shown). Upper case for LNA and lower case for DNA.

Figure 7

Down-regulation of Luciferase expression by different gapmers (9, 22–25) and chimeras (26–34) containing α-l-LNA and β-d-LNA at 2 and 50 nM. Note: oligonucleotides with and without FAM were tested and compared, and no significant difference was appreciated between the free and FAM-labeled ones.

Figure 8

Electrophoresis analysis of ³²P-labeled target RNA degradation products. The chimeras (27, 28, 30, 31 and gapmer 22) containing α-l-LNA and the labeled RNA were incubated with E.coli RNase H1 and aliquots were taken at 0, 10, 20 and 30 min, electrophoresed and reaction products visualized by autoradiography. A gapmer (9) containing β-d-LNA was included in the gel for reference. In the drawing, the line is DNA, the rectangle LNA and the gray shadow corresponds to α-l-LNA residues.