Selective cleavage of rna by means of eukaryotic rnase p and external guide sequence is derived

摘要

It has been discovered that any RNA can be targeted for cleavage by RNAase P from eukaryotic cells, for example, human HeLa cells, using a suitably designed oligoribonucleotide ('external guide sequence', or EGS) to form a hybrid with the target RNA, thereby creating a substrate for cleavage by RNAase P in vitro. There are two classes of EGS that can target RNA for cleavage by human RNAase P. These are prepared by transcription from a small DNA fragment that contains a sequence coding for an RNA that can assume a tRNA-like secondary structure. In the first class, the structure lacks at least the first thirteen nucleotides from the 5' terminus of the tRNA-like sequence, the anticodon stem and loop, the variable loop or part of the variable loop. The most efficient EGS with human RNAase P is the EGS in which the anticodon stem and loop was deleted. In the second class, the EGS has changes in both the equivalent of the T-loop and the anticodon stem of the tRNA-like segment of the EGS. Methods are also disclosed to randomly select and to express a suitable EGS in vivo to make a selected RNA a target for cleavage by the host cell RNAase P, thus preventing expression of the function of the target RNA. The methods and compositions should be useful to prevent the expression of disease-causing genes in vivo.