Site-specific RNA cleavage by imidazole-containing polyamine compounds is described in the absence of free imidazole. Varying the position of the imidazole and the structure of the polycationic chain could lead to a novel compound that triggers a unique and specific cleavage of the target RNA. When two imidazoles are present, cleavages occur at a concentration one order of magnitude lower than when there is only one imidazole. Adding an imidazole buffer to the mono-imidazole derivatives might reduce their respective concentrations required for cleavage.
The imidazole rings of catalytic residues His12 and His119 in the catalytic site of RNase A serve, respectively, as base and acid catalysts and the protonated side chain of Lys41 stabilizes the pentacoordinated phosphorous intermediate during transition state (24
). Our experimental results are consistent with a cooperative participation of one of the amino group of the polyamine chain, predominantly protonated at physiological pH, as proton source, and the imidazole moiety, which is partially unprotonated, as base. The polycationic moiety is also involved in binding the negatively charged ribose-phosphate backbone. Indeed, when the imidazole ring of conjugate 1
is superimposed onto His12 from the active site of RNase A crystal structure in complex with cytidine 3′-monophosphate (25
), the polycationic backbone has sufficient length and flexibility to superimpose one of its protonated amino groups on a nitrogen atom of the imidazole ring of His119 (Fig. A). The same observations have been noticed with compounds 2
. For 3
, although the bond requirement is fulfilled, binding of the polycationic chain onto the RNA may place the imidazole ring too far away from the ribose-phosphate backbone. Alternatively, the longer distance between the imidazole and the protonable nitrogens might prevent RNA catalysis. The distance between one of the two protonated nitrogens of the spermidine chain and the imidazole ring of 4
is minimal, but still sufficient to trigger RNA hydrolysis. The number of cleavages is probably reduced because a shorter polyamine with fewer protonated nitrogens binds the RNA at fewer locations, including sites 1 and 3, but not 2 and 4 (Fig. B). When norspermine replaces spermidine (compound 5
), only A38 from site 3 is cleaved, indicating that both the position of the polyamine moiety and the distance between a protonated nitrogen and an imidazole have been optimized to induce a single cleavage site. Nevertheless, intra-molecular catalysis is not proven, and inter-molecular catalysis cannot be excluded, especially at such concentrations. 15
N NMR spectroscopy was used to map the interactions between natural polyamines and total tRNA from E.coli
and have shown that the internal -NH2+
- groups bind more strongly to tRNA than the external -NH3+
). Thus, substitutions of the secondary amines in conjugates 4, 6 and 7 might affect their binding to tRNAPhe
Figure 6 (A) Superimposing 1 and 4 (yellow-blue) onto RNaseA active site (red) in complex with cytidine 3′-monophosphate (green). (B) tRNAPhe tertiary structure (blue backbone following the ribose-phosphate chain) with the cleavage sites (red), magnesium (more ...)
Yeast tRNAPhe structure has density for a spermine in the major groove of the TΨC stem, as well as accurate information on the location of Mg2+ binding sites. The location of the cleavage sites triggered by conjugates 1–7 is indicated [Fig. ; the 2′-OH from the attacked riboses (r) and the hydrolyzed phosphates (p) are displayed]. The cleavage sites are clustered into four areas (sites 1–4). In site 1, two cleavage sites are opposite since the sugar-phosphate backbone loops back between U8 and C13; in that cleft, there are Mg2+ binding sites that could be occupied by some of the protonated nitrogens of the polycationic chain. Nearby sites 2 and 3, Mg2+ binding sites could be favored anchoring points for the protonated nitrogens of the conjugates. Site 4 is close to the binding site of spermine, suggesting that the triamine moiety might bind at a similar location; alternatively, there are Mg2+ binding sites within the T- and D-loops that could be displaced by protonated nitrogens of the polycationic chain. Three additional Mg2+ binding sites are in the acceptor stem that prevents hydrolysis intermediate state formation.
In conclusion, novel RNaseA mimics that do not depend upon free imidazole for RNA cleavage were designed, synthesized and tested on an RNA whose detailed atomic structure is known. One conjugate cleaves the RNA target at a unique location within one of its main functional domains, without using sequence-specific recognition through base pairing. There is correlation between the location of the magnesium binding sites from the RNA target and the cleavages sites. Further investigations of other imidazole derivatives are currently in progress in our laboratories.