Site-selective artificial ribonucleases: oligonucleotide conjugates containing multiple imidazole residues in the catalytic domain

Abstract

Design of site-selective artificial ribonucleases (aRNases) is one of the most challenging tasks in RNA targeting. Here, we designed and studied oligonucleotide-based aRNases containing multiple imidazole residues in the catalytic part and systematically varied structure of cleaving constructs. We demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part, the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide, and the type of anchor group, connecting linker structure and the oligonucleotide. Molecular modeling of the most active aRNases showed that preferable orientation(s) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker. The inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a stacking interaction with the neighbouring nucleotide residue. Altogether the obtained results show that dynamics factors play an important role in site-specific RNA cleavage. Remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct, which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond.

Scheme 1

Figure 1

Schematic representation of the catalytic structures of the site-selective artificial ribonucleases B-Im(N/m) and analysis of the conjugates. (a) Anchor groups used for attachment of dendrimeric RNA-cleaving constructs to oligonucleotide B 5′-phosphate. (b) Schematic representation of conjugates bearing four and eight imidazole residues and anchor groups of Type1, Type 2, and Type 3, obtained using precursor technology. Mass spectrometry data: 1: calculated mass 5523.30, found mass 5523.73; 2: 5566.80/5566.39; 3: 5692.89/5692.03; 4a: 6838.95/6838,65; 5: 6423.63/6425.03. (c) Analysis of the conjugates B-Im(N/m) by 12% polyacrylamide/8 M urea gel electrophoresis. 0.1 A₂₆₀ unit of each conjugate was applied on the gel; the conjugates were visualized in the gel by staining with Stains-All. Numbers on the top of the gel correspond to numeration of the conjugates in the Table 1 and Figure 1(b) and Supplementary Material Figure  1.

Figure 1

Schematic representation of the catalytic structures of the site-selective artificial ribonucleases B-Im(N/m) and analysis of the conjugates. (a) Anchor groups used for attachment of dendrimeric RNA-cleaving constructs to oligonucleotide B 5′-phosphate. (b) Schematic representation of conjugates bearing four and eight imidazole residues and anchor groups of Type1, Type 2, and Type 3, obtained using precursor technology. Mass spectrometry data: 1: calculated mass 5523.30, found mass 5523.73; 2: 5566.80/5566.39; 3: 5692.89/5692.03; 4a: 6838.95/6838,65; 5: 6423.63/6425.03. (c) Analysis of the conjugates B-Im(N/m) by 12% polyacrylamide/8 M urea gel electrophoresis. 0.1 A₂₆₀ unit of each conjugate was applied on the gel; the conjugates were visualized in the gel by staining with Stains-All. Numbers on the top of the gel correspond to numeration of the conjugates in the Table 1 and Figure 1(b) and Supplementary Material Figure  1.

Figure 2

Site-selective cleavage of yeast tRNA^Phe by the oligonucleotide conjugates bearing multiple imidazole residues in the catalytic part.

Figure 3

Kinetics of tRNA ^Phe cleavage by the conjugates: (a) tRNA cleavage by B-Im(4/1) (3) (rhombs), B-Im(4/2a) (4a) (squares), B-Im(4/3) (5), (asterisks), B-Im(8/4+1) (10) (triangles), and B-Im(24/4+2) (12) (crosses) under standard conditions at conjugates concentration 10 μM. (b) tRNA cleavage by B-Im(4/1) (3) at concentrations ranging from 0.5 to 50 μM.

Figure 4

Structure-activity correlations found for the conjugates B-Im(N/m). (a) Correlations between the number of imidazole groups in catalytic parts of conjugates and their catalytic activities. Cleaving data corresponds to incubation of the RNA with conjugates 1, 3, 6, 9, and 13 under standard conditions for 1 h. (b) Correlation between the type of anchor groups and catalytic activity of conjugates. Diagram presents data for tetraimidazole-containing conjugates 3, 4a, 5 (B-Im(4/m)) and octaimidazoles-containing conjugates 6, 7, 10 (B-Im(8/m)).

Figure 5

A fragment of the most favourable conformation, F4-Im(4/1), found for hybrid complex H- Im(4/1) between artificial ribonuclease B-Im(4/1) and tRNA^Phe where the close proximity of imidazole cleaving groups to the target sequence C⁶³-A⁶⁴ (green) is evident. Hydrogen bonds are shown in yellow.

Figure 6

A fragment showing one possible conformation (namely, final structure F9-Im(4/2a)) of B-Im(4/2a) (4a) found for the hybrid complex H-Im(4/2a). Distances between the cleaving groups and the sequence C⁶³-A⁶⁴ (green) are indicated. The dT (magenta) semistacking interaction with the dG¹fragment is also shown. Hydrogen atoms have been removed for easier visualization.

Figure 7

A fragment showing one possible conformation for the hybrid complex between artificial ribonuclease B-Im(4/2b) and tRNA. In this final conformation, F5-Im(4/2b), the imidazole cleaving groups are located distant from the target sequence. The dT linker fragment (magenta) shows interaction with the dG¹ residue in a semi-stacking interaction, possibly restricting the conformational freedom of this molecule. Hydrogen bonds are shown in yellow.