Cleavage of pyrene-stabilized RNA bulge loops by trans-(±)-cyclohexane-1,2-diamine

Abstract

Chemical agents that cleave HIV genome can be potentially used for anti-HIV therapy. In this report, the cleavage of the upper stem-loop region of HIV-1 TAR RNA was studied in a variety of buffers containing organic catalysts. trans-(±)-Cyclohexane-1,2-diamine was found to cleave the RNA with the highest activity (31%, 37°C, 18 h). Cleavage of the RNA in trans-(±)-cyclohexane-1,2-diamine buffer was also studied when the RNA was hybridized with complementary DNAs. A pyrene-modified C3 spacer was incorporated to the DNA strand to facilitate the formation of a RNA bulge loop in the RNA/DNA duplex. In contrast, unmodified DNAs cannot efficiently generate RNA bulge loops, regardless of the DNA sequences. The results showed that the pyrene-stablized RNA bulge loops were efficiently and site-specifically cleaved by trans-(±)-cyclohexane-1,2-diamine.

Figure 1

Oligonucleotide sequences and structure. (a) Oligonucleotide sequences and major cleavage sites. Arrow indicates the major cleavage sites when the RNA or RNA/DNA duplex was treated with trans-(±)-cyclohexane-1,2-diamine. The cleavage of 1/3, 1/4, 1/5, and 1/6 were not tested due to incomplete hybridization. (b) Chemical modifications of C3-spacer.

Figure 2

Cleavage of RNA 1 in buffers containing 20 mM NaCl, 1 mM Tris-EDTA (pH 8.0), and different organic catalysts (0.5 M, pH 8.0). Lane 1, no catalyst. Lane 2, trans-(±)-cyclohexane-1,2-diamine. Lane 3, glycine, Lane 4, propane-1,3-diamine. Lane 5, ethylenediamine. Lane 6, imidazole. Overall cleavage: Lane 1, 0%. Lane 2, 31%. Lane 3, 0%. Lane 4, 5%. Lane 5, 24%. Lane 6, < 1%.

Scheme 1

Synthesis of the phosphoramidite of the PyC3 spacer. Regents and conditions: (a) p-anisaldehyde, TsOH, CH₂Cl₂, 12 h, 83%. (b) NaH, 1-bromomethylpyrene, THF/toluene, 48 h, 77%. (c) TsOH, MeOH/CH₂Cl₂, 30 h, 30%. (d) Dimethoxytrityl chloride, triethylamine, DMAP, CH₂Cl₂, 12 h, 65%. (e) N, N'-diisopropyl 2-cyanoethyl phosphoramidic chloride, N, N'-diisopropylethylamine, CH₂Cl₂, 2 h, 100%.

Figure 3

Cleavage of DNA/RNA duplexes in trans-(±)-cyclohexane-1,2-diamine (0.5 M), 20 mM NaCl, and 1 mM Tris-EDTA (37°C, pH 8.0, 18 h). Lane 1, RNA 1 in the absence of the catalyst. Lane 2, RNA 1 in the presence of the catalyst. Lane 3, duplex 1/2, Lane 4, duplex 1/7, Lane 5, duplex 1/8.