In vitro selection supports the view of a kinetic control of antisense RNA-mediated inhibition of gene expression in mammalian cells

In principle, the steady-state concentrations of biomolecules in complex sy stems can be far from the thermodynamic equilibrium concentrations of indiv idual processes. This means that, in addition to thermodynamics, reaction k inetics may play an important role. This view is not fully reflected in com binatorial studies in biochemistry that focus on the selection of stably in teracting molecules reflected by high equilibrium constants. For kineticall y controlled processes in vivo, forward or backward reaction rates are crit ical but not necessarily an equilibrium state. Here we have studied the con trol of antisense RNA-mediated gene suppression in human cells on a general basis and in a way that excludes individual structure-specific influences. The complete antisense sequence space against the chloramphenicol acetyltr ansferase gene (cat) was generated and a kinetic selection technique was es tablished to enrich for fast annealing antisense species. Selected sub-popu lations showed successively faster annealing which was related to increased inhibition of cat gene expression in HeLa cells, providing strong evidence for the view that the suppression of gene expression by antisense RNA is c ontrolled kinetically regardless of specific RNA structures.