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.