Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

Abstract

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.

Figure 1.

LNA-targeting plasmids with six consecutive binding sites. (a) Target sequence in the plasmid with six binding sites (BS in bold) (separated by two bases in between), and schematic representation of the bisLNA-20 construct and its TFO- and Watson–Crick (WC) arm components. (b) Quantification of different bisLNA constructs using the GEFA assay. Concentrations used ranged from 0.05 to 12 µM and hybridizations were done in low salt phosphate buffer (pH 5.8) at 37°C for 16—20 h. (i) Comparison between constructs bisLNA-20 and TFO-LNA-20 co-incubated with WC-LNA-20. The two curves obtained correspond to the independent fluorescent signals coming from either TFO-LNA-20 (Cy3 signal detected) or WC-LNA-20 (Cy5 signal detected). The two different signals allow to quantify how much of each construct is bound to the plasmid independently of each other, despite of being co-incubated. (ii) Comparison between constructs with different LNA/DNA composition in the TFO- or WC arm. (iii) Comparison between bisLNA constructs having different linker compositions.

Figure 2.

Representative gels showing the PAGE-shift assay after RE digestions of LNA-hybridized plasmid pEGFPLuc/G6. Asterisks indicate the positions of the shifted DNA fragments. The highest shift occurs when the fragment’s target sites are saturated and the lowest shift occurs with the occupancy of only a single target site of the fragment. The letters ‘i–vi’ on the side of the gels represent the number of target sites occupied in each position (ranging from i = 1 to vi = 6). Below position ‘i’ and indicated by BSF (Binding Site containing Fragment) remains the unbound fraction of the DNA fragment.

Figure 3.

Binding and DSI of bisLNA-m30 and bisLNA-m44 to plasmid pDel-1. (a) Sequence of the binding site in the human MYC promoter region. In bold is highlighted the original 15-mer target-site sequence. Also represented are the schematic pictures of bisLNA-m30 and the WC-arm-extended bisLNA-m44. (b) Quantification of binding by GEFA (i), and the DSI determined by the S1 nuclease assay, after overnight hybridizations at 37°C (ii), and DSI after hybridizations for 72 h at 37°C (iii).

Figure 4.

DSI efficiency comparison between different modified bisLNA-m44-derivatives. (a) Comparison between bisLNA-m44, bisLNA-m44-3t, bisLNA-m44-7w and WC-LNA-m44, having different TFO or Watson–Crick arm lengths. Hybridizations performed overnight at 37°C. Applying two-way ANOVA with Bonferroni posttest significant differences are found: bisLNA-m44 versus WC-LNA-m44 at 1.35 µM and 4.05 µM (***P < 0.001) and 12 µM (*P < 0.05). (b) Comparison between bisLNAs containing different LNA content and positioning as described in Table 3. LNAs were hybridized overnight at 37°C. ***P < 0.001 found for m44e versus m44 and m44e versus m44a at 12 µM (two-way ANOVA with Bonferroni posttest).

Figure 5.

Agarose electrophoretic mobility shift assay showing the relative binding strength of different bisLNAs to binding site containing fragment (BSF) of pDel-1 after RE digestion-induced linearization of plasmid. Hybridization was performed overnight at 37°C.

Figure 6.

DSI efficiency for bisLNAs hybridized for 72 h at 37°C with pDel-1 and pDel-1-derived mutant plasmids. The mutant plasmids had either a single (mut_1) or double mutation (mut_2) on the target homopurine sequence. This mutation was either purine-to-purine (A→G) or purine-to-pyrimidine (A→C). (a) Hybridizations with bisLNA-m44. (b) Side by side comparison between hybridizations of pDel-1 and pDel-1 mut_1G (with an A→G mutation) with bisLNA-m44 and bisLNA-m44e.

Figure 7.

DSI efficiency for bisLNA-m(P2)37 hybridized overnight or for 72 h with pDel-1 (the cognate site being located at the P2 promoter region of the c-MYC gene).

Figure 8.

CAA base modification and analysis of bisLNA or LNA devoid of the TFO portion binding to pEGFPLuc-bisBSf using primer extension reaction and PAGE. (a) Analysis of the pyrimidine-rich strand (D-loop). Lanes 1 and 2: control plasmid, 3 and 4: plasmid hybridized with bisLNA-m44, and lanes 5 and 6: hybridized with WC-LNA-m44. Lanes 1, 3 and 5 are controls and lanes 2, 4 and 6 contain 2% CAA-treated plasmids. The DNA sequence complementary to the LNA binding-site (underlined) and flanking sequences are shown. CAA-modified nucleotides within the BS and in the flanking regions are indicated with ← or with *. (b) Analysis of the LNA-binding, purine-rich strand. Lanes 1 and 2: control plasmid, 3 and 4 (lane 4 appears as a split lane): plasmid hybridized with bisLNA-m44 and lanes 5 and 6: hybridized with WC-LNA-m44. Lanes 1, 3 and 5 are controls and lanes 2, 4 and 6 contains 2% CAA-treated plasmids. DNA sequence of the LNA-binding site (underlined) and flanking regions are shown. CAA-modified nucleotides are indicated. CAA base modifications were made on linearized plasmid.