Artificial Ribonucleases: Efficient and Specific in Vitro Cleavage of Human c-raf-1 RNA

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Abstract
We report here the first successful sequence-specific cleavage of large RNA using artificial ribonucleases. A series of uniformly 2‘-methoxyethoxy-modified oligonucleotides bearing a europium complex were investigated as artificial ribonucleases specific for the human c-raf-1 mRNA. The efficiency and specificity of these oligonucleotide−metal conjugates to bind and to cleave 571 and 2977 nucleotides long c-raf-1 RNA transcripts in vitro in a sequence-specific manner is demonstrated. Quantitative analysis reveals a cleavage efficiency of 60−70% within 4 h at 37 °C. Precise mapping of cleavage sites using primer extension analysis shows that cleavage generally occurs at two or three major sites adjacent to the 3‘-end of the RNA target region. Cleavage is preferentially observed after purine nucleotides. This study demonstrates the potency of artificial ribonucleases targeting large, biologically relevant RNAs.