Theoretical and experimental selection parameters for HBV-Directed antisense RNA are related to increased RNA-RNA annealing

Annealing kinetics of antisense species against two different target regions of the hepatitis B virus (HBV) were measured by kinetic in vitro selection. Individual association rates were related to energies calculated for local sequence segments and predicted structures of the complete pregenomic target RNA. A relationship between the presence of external loops and joint sequences with fast pairing was observed whereas internal loops did not favor fast RNA-RNA annealing. The findings were used to predict a fast-annealing HBV-directed antisense oligodeoxyribonucleotide that turned out to pair with its target RNA at an association rate constant of k = 9.2 x 10⁴ M^-1 S^-1, which is substantially faster than the annealing rates of artificial antisense RNA so far included in in vitro selection assays.