Abstract
The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3′-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3′-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.
Highlights
According to the binding modes, anti-gene ONs are grouped as: (a) TFOs that bind to the polypurine strand in the major groove of double-strand DNA (dsDNA) by Hoogsteen (HG) or reverse HG hydrogen bonds between the bases forming a triplex structure[2, 3, 7,8,9]; (b) ONs that bind to one of the DNA strands by Watson-Crick (WC) hydrogen bonds leading to the displacement of the other strand
We found that locked nucleic acid (LNA) substituted ONs show conformation rearrangements, both in single and duplex strand states, which are beneficial for triplex formation, and the results were confirmed by binding experiments using electrophoretic mobility shift assay (EMSA)
In the absence of BQQ, 100% triplex formation was reached at 1:25 ratio of dsDNA:TFO (Fig. 2b), and in the presence of BQQ, ON2-5′DNA binding was completed at the lowest dsDNA:TFO ratio, demonstrating for the first time the ability of BQQ to intercalate and stabilize triplex structures formed by LNA-modified TFOs
Summary
According to the binding modes, anti-gene ONs are grouped as: (a) TFOs that bind to the polypurine strand in the major groove of dsDNA by Hoogsteen (HG) (parallel orientation) or reverse HG hydrogen bonds (antiparallel) between the bases forming a triplex structure[2, 3, 7,8,9]; (b) ONs that bind to one of the DNA strands by Watson-Crick (WC) hydrogen bonds leading to the displacement of the other strand In the latter case a double-strand invasion (DSI) complex is efficiently formed by oligomers containing locked nucleic acid (LNA)[10, 11] or peptide nucleic acid (PNA)[12,13,14,15,16,17]. A clear effect on TFO binding to dsDNA is observed when LNA substitution takes part at the 3′-end of the ON in contrast to the 5′-end
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