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. 1. Introduction The idea of site-selective artificial ribonucleases which are capable of cleaving any particular RNA sequence in vitro and in vivo, is a very attractive approach as, apart from being useful tools in molecular biology, these chemical ribonucleases are anticipated to be helpful for target validation, and even for the development of potential antiviral or anticancer therapeutics. The idealized site-selective artificial ribonuclease would be a compound that is easily synthesized, chemically stable, targeted to any chosen RNA sequence, and highly efficient in cleaving of the phosphodiester bonds. The oligonucleotide-based artificial ribonucleases seem to meet the majority of these criteria due to the fact that they can be directed to almost any desired RNA region in a site specific-manner by controlling the sequence of the oligonucleotide recognition part. Virtually all types of the reactive groups, which are known to catalyze RNA scission, have been exploited for design and preparation of artificial ribonucleases [1–3]. Conjugates of oligonucleotides and RNA cleaving groups can be synthesized using two main approaches: incorporation of a catalytic
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