Molecular Beacons: Powerful Tools for Imaging RNA in Living Cells

Figure 5

Approaches for RNA visualization in living cells. (a) In order to reduce the possibility of false-positive signals and also increase the specificity, one can design two shared stem MB having a pair reporters compatible with FRET, one to serve like a donor dye (red circle) and the other like a acceptor dye (green circle). With this strategy the sequence to be recognized increases its length and thus the specificity of the assay (observe that one MB needs to have the reporter at the 3′ end and the quencher at the 5′ end, in a not conventional attachment). After the MB design and synthesis the probe could be delivered using microinjection; in conjunction with other MB or fluorescent dyes could be used to measure RNA distribution at subcellular level. For example it is possible to use a specific fluorescent dye for the endoplasmic reticulum (ER) and also a MB designed to hybridize a given sequence of the ribosome. If a particular mRNA (based on the fluorescence of the MB) colocalizes with the ER dye and ribosome dye it means that this mRNA is translated in ribosomes at ER (b) or if the mRNA signal colocalizes only with the ribosome dye means that the mRNA translation occurs at free ribosomes (c); also it is possible that the mRNA translation takes place at both free and ER associated ribosomes. These are only some examples of the power of using MB in combination with other compartment or structure-specific dyes.