Wu, B. et al. Biophys. J. 102, 2936–2944 (2012).

RNA binding proteins can be used to image single RNA transcripts in living cells. In this approach, a phage coat protein (CP) is fused to a fluorescent protein (FP) and binds a transcript engineered to be recognized by the CP-FP fusion. Quantitative use of this approach remains challenging, however. In recent work, Wu et al. addressed some of the challenges. They reduced background from unbound CP-FP fusions, which bind to the target RNA as dimers, by constructing single-chain tandem dimers for two phage systems. They used fluorescence fluctuation spectroscopy to obtain brightness and diffusion information on these constructs in cells expressing the target RNA, thus yielding copy numbers for all diffusing species. Fluorescence fluctuation spectroscopy permits the quantitative study of engineered exogenous and endogenous transcripts in living cells.