Our cells' molecules form an intricate network of interactions. Today's techniques, however, can only be used to measure individual molecular reactions outside the cells. Since molecular concentrations are much higher in cells than in the laboratory, scientists suspect that the kinetics of molecular reactions in living cells differ substantially from external probes. We expected the cellular reaction speed to be higher," confirms LMU biophysicist Professor Dieter Braun. "However, our novel optical approach showed that depending on the length of the strands the coupling of DNA-strands inside living cells can be both faster and slower than outside." Data yielded from living cells are highly valuable for the development of models to understand the complex interactions as well as pathological processes in biological cells. Braun and his team now plan to probe a variety of molecular reactions in living cells, visualizing the heartbeat of cellular networks. (PNAS online, 14 November 2009)
In their work, the scientists investigated the hybridization the coupling and de-coupling of two DNA-strands, which they introduced into living cells. To determine the reaction time constant they used an infrared laser to induce temperature oscillations of different frequencies in the cell and measured the concentration of the reaction partners, namely of coupled and de-coupled DNA. At low frequencies, these concentrations followed the temperature oscillations, whereas at higher frequencies they experienced a phase delay and oscillated with diminished amplitude. Both delay time and amplitude decrease, were evaluated to obtain the reaction time constant.
The team determined the concentrations using the so-called fluorescent energy transfer (FRET), which takes place between two chromophores at a certain spatial distance. They applied a FRET pair to the DNA-strands such that energy transfer occurred only if the strands were coupled. The chromophores were e
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