THEORETICAL DESIGN OF ANTISENSE RNA STRUCTURES SUBSTANTIALLY IMPROVESANNEALING KINETICS AND EFFICACY IN HUMAN-CELLS

The success of antisense therapeutics is not predictable despite their widespread use in biotechnology and molecular medicine. The relations hip between RNA structure and biological effectiveness is largely not understood; however, antisense RNA-mediated effects in vivo seem to be related to annealing kinetics in vitro. This study suggests that term inal unpaired nucleotides and overall flexibility of antisense RNA dir ected against the human immunodeficiency virus type 1 (HIV-1) are rela ted to fast RNA-RNA annealing in vitro as well as to strong inhibition of virus replication in human cells. Annealing rate constants of comp uter-selected antisense RNA species approach the values for natural an tisense RNA in the order of 10(s) M(-1)s(-1). When considering the unf avorable stability in cellular extracts of antisense RNA species that were found to anneal fast in vitro, an antisense effect against HIV-1 in human cells was observed that was 10- to 10,000-fold stronger than that measured for species predicted to anneal slowly. A computer-suppo rted structural design of antisense RNA can serve as a platform to det ermine RNA-RNA association in vitro and biological effectiveness in li ving cells.