Hydrolysis of bulged nucleotides in hybrids formed by RNA and imidazole-derivatized oligo-2'-O-methylribonucleotides

Abstract

In order to enhance the efficacy of small antisense molecules, we examined a series of antisense oligonucleotides derivatized with functional groups designed to enable them to hydrolyze their RNA target. Solid phase synthetic methods were used to prepare imidazole-derivatized antisense oligo-2'-O-methylribonucleotides. Upon binding, these oligonucleotides create internal bulged bases in the target RNA that serve as sites for hydrolysis. We observed that an oligonucleotide derivatized with a side chain containing two imidazole groups was capable of hydrolyzing 58% of its RNA target when incubated with the target for 48 hours at 37°C and physiological pH.

Figure 1

RNA-N/oligo-2′-O-methylribonucleotide hybrid duplexes used to test susceptibility of bulged RNA bases to imidazole hydrolysis (Panel A). UV thermal denaturation profile of RNA-2/Oligo-1 (Panel B). The melting temperatures of the hybrid duplexes are shown in the table.

Figure 2

Susceptibility of bulged nucleotides to hydrolysis in imidazole buffer. Duplexes formed between 100 nM 5′-[³²P]-labeled RNA-1 and 10 μM oligo-1 (Panel A) or oligo-2 (Panel B) were incubated at 37°C in buffer containing 1.0 M imidazole, pH 7.0 and 0.5 mM EDTA for the times indicated. The reactions were analyzed on a 20% denaturing polyacrylamide gel.

Figure 3

Molecular models showing the position of the cleaving moieties relative to the bulged nucleotides when these groups are attached to the 5′-end and an internal position in the oligonucleotide (Panel A) or when both groups are attached at internal position in the oligonucleotide (Panel B). The models were generated using Hyperchem 8.0 modeling software.

Figure 4

Synthesis of imidazole-conjugated oligo-2′-O-methylribonucleotides. Abbreviations: control pore glass (CPG); 2′-t-butyldimethylsilyl (TBS); tetra-n-butylammonium fluoride (TBAF); dimethoxytrityl (DMT); carbonyldiimidazole (CDI); 1,4-diaminobutane (DAB).

Figure 5

Hydrolysis of RNA-1 by imidazole-conjugated oligonucleotides. The structures of the hybrids formed between RNA-1 and the imidazole-conjugated oligonucleotides are shown in Panel A. A phosphorimage of the gel used to analyze reactions between 10 μM oligo-3, or oligo-5 and 100 nM RNA-1 incubated in buffer containing 50mM MOPS pH 7.5, 100 mM sodium chloride, 0.5 mM EDTA for 24 hrs is shown in Panel B. Lane B: RNA-1 target partially digested with sodium bicarbonate at 90 °C. Lane C: RNA-1 incubated in pH 7.5 buffer for 24 hrs. Note: All lanes were run on the same gel. Irrelevant lanes were deleted for clarity.

Figure 6

Synthesis of oligo-7. Abbreviations: polystyrene (PS); 2′-t-butyldimethylsilyl (TBS); dimethoxytrityl (DMT); triethylamine trihydrofluoride (TEA:THF); carbonyldiimidazole (CDI); tris(2-aminoethyl)-amine (TAEA).

Figure 7

Hydrolysis of RNA by oligo-7. Panel A shows the structure of the oligo-7 and the duplexes formed with RNA-1 or RNA-2. 10 μM Oligo-7 and 100 nM 5′-[³²P]-labeled RNA-1 (Panel B) or RNA-2 (Panel C) were incubated at 37 °C in buffer containing 50mM MOPS pH 7.5, 100 mM sodium chloride, 0.5 mM EDTA. Aliquots were withdrawn at the times indicated and subjected to PAGE on a 17% sequencing gel. Lane B: RNA target partially digested with sodium bicarbonate at 90 °C; Lane T₁: RNA-2 target partially digested with RNase T₁; Lane C₁: RNA-1 incubated in buffer for 96 hrs; Lane C₂: 10 μM unconjugated oligo-7 and 100 nM RNA-1 incubated in buffer for 96 hrs; Lane C₃: RNA-2 incubated in buffer for 96 hrs; Lane C₄: 10 μM unconjugated oligo-7 and 100 nM RNA-2 incubated in buffer for 96 hrs.

Figure 8

Hydrolysis of RNA-1 or RNA-2 by oligo-7 in the presence of Mg²⁺. 10 μM Oligo-7 and 100 nM 5′-[³²P]-labeled RNA-1 (Panel A) or RNA-2 (Panel B) were incubated at 37 °C in buffer containing 50mM MOPS pH 7.5, 100 mM sodium chloride, 0.5 mM EDTA, 20 mM Mg²⁺. Aliquots were withdrawn at various times and subjected to PAGE on an 17% sequencing gel. Lane C₁: RNA-1 incubated in buffer for 96 hrs; Lane B: RNA-2 target partially digested with sodium bicarbonate at 90 °C; Lane T₁: RNA-2 target partially digested with RNase T_1; Lane C₂: RNA-2 incubated in buffer for 96 hrs. Note, in Panel A the wells for lanes 48 and 96 were deeper than for C₁ and T₀ which accounts for the difference in mobility of the full length RNA in these lanes.