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Nucleic Acids Research, 2000, Vol. 28, No. 13 2462-2466
© 2000 Oxford University Press

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

Volker Patzel1 and Georg Sczakiel1,2,*

1Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany and 2Medizinische Universität zu Lübeck, Institut für Molekulare Medizin, Ratzeburger Allee 160, D-23538 Lübeck, Germany

In principle, the steady-state concentrations of biomolecules in complex systems can be far from the thermodynamic equilibrium concentrations of individual processes. This means that, in addition to thermodynamics, reaction kinetics may play an important role. This view is not fully reflected in combinatorial studies in biochemistry that focus on the selection of stably interacting molecules reflected by high equilibrium constants. For kinetically controlled processes in vivo, forward or backward reaction rates are critical but not necessarily an equilibrium state. Here we have studied the control 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 acetyltransferase gene (cat) was generated and a kinetic selection technique was established to enrich for fast annealing antisense species. Selected sub-populations 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 controlled kinetically regardless of specific RNA structures.

* To whom correspondence should be addressed at: Medizinische Universität zu Lübeck, Institut für Molekulare Medizin, Ratzeburger Allee 160, D-23538 Lübeck, Germany. Tel: +49 451 500 2730; Fax: +49 451 500 2729; Email: sczakiel@dkfz-heidelberg.de


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