Artificial ribonucleases: efficient and specific in vitro cleavage of human c-raf-1 RNA.
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 2,977 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 degrees 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.
Canaple
Laurence
L
Universität Bern, Departement für Chemie und Biochemie, Bern.
Hüsken
Dieter
D
Hall
Jonathan
J
Häner
Robert
R
D005063:Europium / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D018716:Molecular Mimicry; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D012313:RNA / Q000378:metabolism*; D012260:Ribonucleases / Q000138:chemical synthesis* / Q000494:pharmacology; D013379:Substrate Specificity
D009841:Oligonucleotides; D005063:Europium; D012313:RNA; D019908:Proto-Oncogene Proteins c-raf; D012260:Ribonucleases