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 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.
Patzel
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V
Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, Heidelberg, Germany.
Sczakiel
G
G
D020029:Base Pairing / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018075:RNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics
D016372:RNA, Antisense; D018075:RNA, Complementary; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase