The creation of artificial ribonucleases is one of the most challenging approaches towards RNA targeting. Their design has normally been based on imitation of the active site of natural ribonucleases by chemical conjugation of hydrolytically active groups that normally constitute the catalytic domain of such RNases. The conjugation of such cleaving constructs to antisense oligonucleotides could potentially produce artificial ribonucleases capable of cleaving their RNA targets in a sequence-specific manner. We have applied a multidisciplinary approach involving extensive biophysical analysis (UV-visible, fluorescence, CD and NMR spectroscopies), along with biochemical assays and high-level computational methods, to design and investigate the action of various types of metal-independent, oligonucleotide-based artificial ribonucleases.

Two main aspects will be reported in this study. The first explores the performance of bis-imidazole-containing artificial ribonucleases possessing an additional phenazinium anchor group to enhance affinity of the cleaving groups to the RNA target and improve their hybridisation properties via additional stacking and electrostatic interactions with nucleotide residues. The second aspect is the exploitation of the molecular modelling (Figure 1) to evaluate structural and dynamics properties of studied artificial ribonucleases containing an additional anchor groups to determine the structural basis of difference in their hydrolytic activity and reveal factors affecting the cleavage potential.


Figure 1: Fragment of final conformation of artificial ribonuclease hybridized to its target, showing a possible orientation of the imidazole cleaving groups and location of phenazinium anchor.