Selective activation of two sites in RNA by acridine-bearing oligonucleotides for clipping of designated RNA fragments.

Artificial enzymes for selective scission of RNA at two designated sites, which are valuable for advanced RNA science, have been prepared by combining lanthanide(III) ion with an oligonucleotide bearing two acridine groups. When these modified oligonucleotides form heteroduplexes with substrate RNA, the two phosphodiester linkages in front of the acridines are selectively activated and preferentially hydrolyzed by lanthanide ion. This two-site RNA scission does not require any specific RNA sequence at the scission sites, and the length of clipped RNA fragment is easily and precisely controllable by changing the distance between two acridine groups in the modified oligonucleotide. The two-site scission is also successful even when the substrate RNA has higher-order structures. By using these two-site RNA cutters, RNA fragments of predetermined length were obtained from long RNA substrates and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Single nucleotide polymorphisms in homozygous and heterozygous samples were accurately and easily detected in terms of the difference in mass number. Multiplex analyses of in vitro transcripts from human genome were also successful.