Triplex formation with 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) having C3'-endo conformation at physiological pH

Abstract

Antigenes, which are substances that inhibit gene expression by binding to double-stranded DNA (dsDNA) in a sequence-specific manner, are currently sought for the treatment of various gene-related diseases. As such antigenes, we developed new nuclease-resistant oligopyrimidine nucleotides that are partially modified with 2'-O,4'-C-ethylene nucleic acids (ENA), which are constrained in the C3'-endo conformation and can form a triplex with dsDNA at physiological pH. It was found that these oligonucleotides formed triplexes similarly to those partially modified with 2'-O,4'-C-methylene nucleic acids (2',4'-BNA or LNA), as determined by UV melting analyses, electromobility shift assays, CD spectral analyses and restriction enzyme inhibition assays. In our studies, oligonucleotides fully modified with ENA have delta torsion angle values that are marginally higher than those of 2',4'-BNA/LNA. ENA oligonucleotides present in 10-fold the amount of dsDNA were found to be favorable in forming triplexes. These results provide useful information for the future design of triplex-forming oligonucleotides fully modified with such nucleic acids constrained in the C3'-endo conformation considering that oligonucleotides fully modified with 2',4'-BNA/LNA do not form triplexes.

Figure 1

(A) Structures of 2′,4′-BNA/LNA and ENA and (B) a sequence of a triplex. The arrow indicates the cleavage site of MlnI, a restriction enzyme. The 5′ -end of a pyrimidine-rich strand of the dsDNA was labeled with fluorescein (FAM).

Figure 2

Electrophoretic mobility shift assay of triplex formation at pH 7.2. (A) dsDNA:TFO, 1:1; (B) dsDNA:TFO, 1:10. dsDNA of 5 µM and TFO of 5 or 50 µM were used. A solution of 50 mM Tris–HCl (pH 7.2 at 20°C) and 10 mM MgCl₂ was used as the running buffer. MlnI reaction buffer described in Materials and Methods was used as the reaction buffer.

Figure 3

CD spectra of triplexes. (A) dsDNA:TFO, 1:1; (B) dsDNA:TFO, 1:10. The buffer was 10 mM sodium cacodylate (pH 6.8), 200 mM NaCl and 20 mM magnesium chloride. Red, TFO only; purple, dsDNA only; green, complex between TFO and the dsDNA; blue, CD spectra of the complex between TFO and the dsDNA (green) minus that of the dsDNA (purple) and TFO (red).

Figure 4

Inhibition of MlnI cleavage due to triplex formation.

Figure 5

Stability of oligo(dT₉) and mixmers with ENA or 2′,4′-BNA/LNA residues in rat plasma. Closed circles, 5′-TTTTTTTTT-3′, where T is 2′-O,4′-C-ethylene thymidine; open diamonds, T is 2′-O,4′-C-methylene thymidine; open squares, natural DNA oligo(dT₉).

Scheme 1. Synthesis of 4-triazole thymidine derivative. Reagents and conditions: (i) [(iPr)₂N]₂P(OC₂H₄CN), N,N-diisopropylammonium tetrazolide; (ii) 1,2,4-triazole, POCl₃, triethylamine, CH₃CN.