Sequence-specific artificial ribonucleases. I. Bis-imidazole-containing oligonucleotide conjugates prepared using precursor-based strategy

Abstract

Antisense oligonucleotide conjugates, bearing constructs with two imidazole residues, were synthesized using a precursor-based technique employing post-synthetic histamine functionalization of oligonucleotides bearing methoxyoxalamido precursors at the 5'-termini. The conjugates were assessed in terms of their cleavage activities using both biochemical assays and conformational analysis by molecular modelling. The oligonucleotide part of the conjugates was complementary to the T-arm of yeast tRNA(Phe) (44-60 nt) and was expected to deliver imidazole groups near the fragile sequence C61-ACA-G65 of the tRNA. The conjugates showed ribonuclease activity at neutral pH and physiological temperature resulting in complete cleavage of the target RNA, mainly at the C63-A64 phosphodiester bond. For some constructs, cleavage was completed within 1-2 h under optimal conditions. Molecular modelling was used to determine the preferred orientation(s) of the cleaving group(s) in the complexes of the conjugates with RNA target. Cleaving constructs bearing two imidazole residues were found to be conformationally highly flexible, adopting no preferred specific conformation. No interactions other than complementary base pairing between the conjugates and the target were found to be the factors stabilizing the 'active' cleaving conformation(s).

Scheme 1.

Scheme 1.

Figure 1

Imidazole-containing oligonucleotide conjugates. (A) Schematic representation of oligonucleotide conjugates B-R(1/1), B-R(1/2) and B-R(2). Cleaving constructs R(1/1), R(1/2) and R(2) comprise bis-imidazole cleaving groups, flexible linker and anchor groups (shown by solid boxes) covalently attached to the addressed oligonucleotide B via the 5′-phosphate group. (B) Schematic representation of the terminal modifiers (M) bearing methoxyoxaloamido (MOX) precursor groups along with a phosphoramidite moiety. (C) Synthesized modifiers were introduced onto the 5′-terminus of the synthetic oligodeoxyribonucleotide at the last step of an automated synthesis, resulting in the formation of the MOX-oligonucleotide precursor. The MOX precursor groups were then post-synthetically derivatized with histamine to yield oligonucleotide conjugate, bearing the imidazole-containing catalytic construct at the 5′ end.

Figure 2

Cleavage of tRNA^Phe by oligonucleotide conjugates B-R(1/1) and B-R(1/2). (A) Cloverleaf structure of yeast tRNA^Phe and the target sequence for oligonucleotide B-based artificial ribonucleases B-R(n) [R(n) is the RNA-cleaving construct shown in Figure 1). Arrows indicate the sites of tRNA cleavage by the oligonucleotide conjugates. (B) RNase H and imidazole-buffer probing of the tRNA^Phe structure in the complex with oligonucleotide B. Autoradiograph of 12% polyacrylamide/8 M urea gel after separation of [3′-³²P]tRNA^Phe and cleavage products. Lanes: C, intact tRNA; L and T, tRNA partial cleavage by 2 M imidazole buffer, pH 7.0 and by RNase T1 under denaturing conditions, respectively; 1 and 4, tRNA cleaved by RNase H (0.25 U) or 2 M imidazole buffer in the absence of oligonucleotide B at 20°C for 2 and 12 h, respectively; 2 and 3, tRNA cleaved by RNase H (0.25 and 0.5 U, respectively) in the complex with oligonucleotide B (5 × 10⁻⁶ M) at 20°C for 2 h; 5 and 6, tRNA hydrolyzed by 2 M imidazole buffer in the complex with oligonucleotide B (5 × 10⁻⁶ M) at 37°C for 12 and 18 h, respectively. Solid lines indicate oligonucleotide B complementary region. (C) Autoradiograph of 12% polyacrylamide/8 M urea gel. Cleavage reactions were performed as described in Materials and Methods. Lanes: C1, tRNA^Phe incubated in the reaction buffer at 37°C for 5 h; C2, tRNA^Phe incubated in the reaction buffer in the presence of 1 × 10⁻⁶ M oligonucleotide B at 37°C for 5 h; C3, tRNA^Phe incubated in the reaction buffer in the presence of 1 × 10⁻⁶ M cleaving construct R(2) at 37°C for 5 h; C4, tRNA^Phe incubated in the reaction buffer in the presence of an equimolar mixture of oligonucleotide B and cleaving construct R(2) at 37°C for 5 h; L, ladder produced by 2 M imidazole buffer; T, partial digest with RNase T1; lanes 1–6, incubation of tRNA^Phe with 5 × 10⁻⁷ M conjugate B-R(1/1); lanes 7–12, incubation of tRNA^Phe with 1 × 10⁻⁶ M conjugate B-R(1/1); lanes 13–18, incubation of tRNA^Phe with 5 × 10⁻⁶ M conjugate B-R2(1/2) each for 0.5, 1, 2, 3, 4 and 6 h, respectively.

Figure 3

Influence of concentration of conjugates B-R(1/1) (curve 1) and B-R(1/2) (curve 2) on the efficiency of tRNA^Phe cleavage: [3′-³²P]tRNA^Phe (5 × 10⁻⁷ M) was incubated at 37°C for 4 h in 50 mM imidazole buffer, pH 7.0, containing 200 mM KCl, 0.1 mM EDTA and 100 μg/ml RNA carrier in the presence of conjugate. Curve 3 represents the extent of binding of parent oligonucleotide B to tRNA^Phe under the same conditions (see Figure 2B).

Figure 4

The kinetics of tRNA^Phe cleavage with oligonucleotide conjugates B-R(n). (A) The kinetics of tRNA^Phe cleavage by conjugate B-R(1/1) at 10.0, 5.0, 1.0 and 0.5 μM (curves 1a, 1b, 1c and 1d, respectively), and conjugates B-R(1/2) and B-R(2) at 10.0 μM (curves 2 and 3, respectively). Cleavage reactions were performed at 5 × 10⁻⁷ M [3′-³²P]tRNA^Phe in 50 mM imidazole buffer, pH 7.0, containing 200 mM KCl, 1 mM EDTA, 100 μg/ml RNA carrier at 37°C. (B) The solid lines represent the fitting of experimental data [curves 1a, 1c and 1d in (A)] for the tRNA^Phe cleavage with conjugate B-R(1/1) to a three-state model of consecutive first-order reactions (Scheme 1).

Figure 5

A fragment of F5-R(1/1) final structure representing one of the probable conformations of the B-R(1/1) molecule, and showing the possible orientation of the cleaving groups. The C₆₃–A₆₄ cleavage site is shown in cyan; distances (Å) between the imidazole groups and target atoms of cleavage site are indicated.