IN-VITRO SELECTION OF ANTISENSE OLIGONUCLEOTIDES TARGETED TO A HAIRPIN STRUCTURE

Antisense oligonucleotides are widely used to selectively prevent pre-RNA splicing, mRNA translation or cDNA synthesis from om a retvoviral RNA template. However, intramolecular folding of the RNA chain can sequester the target sequence into a stable structure. Consequently, the antisense effect can be greatly reduced or even abolished. Hydrogen donor and acceptor sites due still available on nucleic acid bases involved in secondary structures. However, the rational design of antisense sequences able to recognize the three dimensional array of these sites is not available. We used an in vitro selection. procedure to fish out aptastrucs, i. e., oligomers, able (''apte'') to bind to a structure A population of randomly synthesized oligonucleotides was mixed with the structure of interest and oligodeoxynucleotide sequences bound to the target were selected and amplified. The selection involves the destruction of the unbound candidates by a restriction enzyme. This procedure can be used-both for RNA and DNA target structures and does not require the purification of the bound oligonucleotides at each cycle of selection. Several cycles of selection-amplification, followed by cloning and sequencing, allowed us to identify three oligonucleotides able to form a complex with a DNA hairpin. Due to the sequence of the selected candidates, these aptastruc-hairpin complexes involve very likely non-canonical interactions between the two partners.