Patent ID: 6562570
Filing Date: 2003-05-13
Classification: C12N,C12Q

Abstract:
A method for identifying sites on a target RNA which are accessible to pairing by antisense DNA, ribozymes or DNAzymes which comprises:a) incubating native or in vitro-synthesized target RNA with a random or semi-random ODN library and RNase H, or with a semi-random ribozyme or DNAzyme library, under hybridization conditions in a reaction medium which is a cell extract containing endogenous RNA-binding proteins, or which mimics a cell extract due to presence of one or more RNA-binding proteins, whereby any antisense ODN, ribozyme or DNAzyme within the library which is complementary to an accessisble site in the target RNA hybridizes to that site and the RNA is cleaved at that site; b) annealing to the target RNA an ODN primer P1 which is complementary to a region of the target RNA downstream from the portion of the target RNA molecule under study, and incubating the primed target RNA with a reverse transcriptase and dNTPs, whereby the RNA is copied into DNA from the 3â€² end of P1 to the site of cleavage created by the antisense ODN/RNase H, ribozyme or DNAzyme, creating a first strand DNA molecule with a 3â€² end that is complementary to the 5â€² end of the target RNA at the cleavage site; c) incubating the first strand DNA with guanosine triphosphate in presence of terminal deoxynucleotidyl transferase to add an (rG)2-4 tail on the 3â€² end of the DNA molecule; d) incubating the 3â€²-tailed DNA in the presence of a DNA ligase with a double-stranded ODN linker having a 3â€²C2-4 overhang on one strand, whereby the tail base pairs with the overhang and the other strand is ligated to the 3â€²-tailed DNA; e) mixing the ligated DNA with an ODN linker primer LP which is complementary to the strand of the linker which is ligated to the (rG)n tail and with a primer P2 which is complementary to the original target RNA in a region which corresponds to or is at least partially upstream from the region which is bound by P1, but it is downstream of the portion of the target RNA which is under study, and carrying out PCR in presence of a DNA polymerase and dNTPs to amplify DNA segments defined by primers LP and P2; and f) sequencing the PCR-amplified DNA to determine the cleavage site on the target RNA.