Mapping of accessible sites for oligonucleotide hybridization on hepatitisdelta virus ribozymes

Semi-random libraries of DNA 6mers and RNase H digestion were applied to se arch for sites accessible to hybridization on the genomic and antigenomic H DV ribozymes and their 3' truncated derivatives. An approach was proposed t o correlate the cleavage sites and most likely sequences of oligomers, memb ers of the oligonucleotide libraries, which were engaged in the formation o f RNA-DNA hybrids. The predicted positions of oligomers hybridizing to the genomic ribozyme were compared with the fold of polynucleotide chain in the ribozyme crystal structure, The data exemplified the crucial role of targe t RNA structural features in the binding of antisense oligonucleotides. It turned out that cleavages were induced if the bound oligomer could adapt an ordered helical conformation even when it required partial penetration of an adjacent double-stranded region, The major features of RNA structure dis favoring hybridization and/or RNase H hydrolysis were sharp turns of the po lynucleotide chain and breaks in stacking interactions of bases. Based on t he predicted positions of oligomers hybridizing to the antigenomic ribozyme we chose and synthesized four antisense DNA 6mers which were shown to dire ct hydrolysis in the desired, earlier predicted regions of the molecule.