Mini-ILL RNases, methods for changing specificity of RNA sequence cleavage by mini-ILL RNases, and uses thereof

摘要

The object of the invention is a Mini-Ill RNase with amino acid sequence comprising an acceptor part, and a transplantable a4 helix, and a transplantable a5b-a6 loop, which form structures of a4 helix and a5b-a6 loop, respectively, in the Mini-Ill RNase structure, wherein the fragments which form structures of a4 helix and a5b-a6 loop, respectively, correspond structurally to respective structures of a4 helix and a5b-a6 loop formed by amino acid sequence fragments 46-52 and 85-98, respectively, of Mini-Ill RNase from Bacillus subtilis shown in SEQ ID NO: 1, wherein the said Mini- Ill RNase exhibits sequence specificity in dsRNA cleavage being dependent only on a ribonucleotide sequence of the substrate, and independent from an occurrence of secondary structures in the substrate's structure, and independent from a presence of other assisting proteins, and wherein the Mini-Ill RNase is not the Mini-Ill protein from Bacillus subtilis of SEQ ID NO: 1, nor SEQ ID NO: 1 with D94R mutation. The invention also relates to a method of obtaining a chimeric Mini-Ill RNase, a Mini-Ill RNase encoding construct, a cell with a Mini-Ill RNase encoding gene, use of Mini-Ill RNase for dsRNA cleavage, as well as a method of dsRNA cleavage depending only on a ribonucleotide sequence.