Structural rearrangements in RNA on the binding of an antisense oligonucleotide: implications for the study of intra-molecular RNA interactions and the design of cooperatively acting antisense reagents with enhanced efficacy.

We show that binding of an antisense oligonucleotide can lead to considerable changes in the target mRNA structure. The approaches described here are not only useful in the study of intra-molecular interactions in RNAs but can also be used to design oligonucleotides that facilitate binding of other antisense reagents. Such "cooperatively acting" antisense reagents have the potential to overcome several problems faced in their use, for example, low efficacy and non-specificity. To provide proof-of-principle, radiolabelled cyclin B5 transcript, a model mRNA, was hybridised with an antisense oligonucleotide array. An oligonucleotide sequence was selected from the array hybridisation data and was used in an RNase H/oligonucleotide library (dN12) assay to assess its ability to enhance cleavage of target RNA. This oligonucleotide ("facilitator") greatly enhanced cleavage of B5 RNA at a neighbouring site. The precise position and sequence of this "new" site was determined by further hybridisation of RNA-facilitator mixture to the B5 antisense array. Antisense oligonucleotides designed from the new region were used in combination with the facilitator in a cell-free system. The presence of the facilitator considerably enhanced cleavage of B5 RNA with these oligonucleotides. These approaches may be useful in designing antisense reagents against sequences of specific interest, such as, gene fusion sites, splice variants, mutant alleles and tightly structured RNA sites.