Charles R. Cantor, Sequenom, Incorporated, San Diego, CA, USA

Detecting specific RNAs in living cells
Past attempts to detect specific RNAs in living cells have required either injecting in RNA-complementary molecular beacons, or genetically engineering natural RNAs to include hundreds of binding sites for fluorescent RNA binging proteins. Such methods may seriously compromise the validity of results seen because they have the potential to seriously perturb normal cell function. We have recently developed alternative methods that provide high contrast detection of natural RNAs in living cells with little if any risk of perturbation. These detect two adjacent RNA sequences and use the proximity of these sequence to assemble functional fluorescent proteins from initially separated non-fluorescent halves. Such RNA-mediated protein complementation opens up a range of possibilities for exploring the location and function of RNAs in living cells and for manipulating cells based on the presence of specific expressed RNA sequences. Examples in bacteria, yeast, and mammalian cells will be described. The system shows fast response permitting kinetic information to be obtained, not just static localization.