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A Reaction-Diffusion Model to Study RNA Motion by Quantitative Fluorescence Recovery after Photobleaching

José Braga ^*, James G. McNally  and Maria Carmo-Fonseca ^*

^* Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal; and Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Correspondence: Address reprint requests to José Braga, Institute of Molecular Medicine, Faculty of Medicine, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal. Tel.: 351-21-7999411; Fax: 351-21-7999412; E-mail: josebraga{at}fm.ul.pt.

Fluorescence recovery after photobleaching (FRAP) is a powerful technique to study molecular dynamics inside living cells. During the past years, several laboratories have used FRAP to image the motion of RNA-protein and other macromolecular complexes in the nucleus and cytoplasm. In the case of mRNAs, there is growing evidence indicating that these molecules assemble into large ribonucleoprotein complexes that diffuse throughout the nucleus by Brownian motion. However, estimates of the corresponding diffusion rate yielded values that differ by up to one order of magnitude. In vivo labeling of RNA relies on indirect tagging with a fluorescent probe, and here we show how the binding affinity of the probe to the target RNA influences the effective diffusion estimates of the resulting complex. We extend current reaction-diffusion models for FRAP by allowing for diffusion of the bound complex. This more general model can be used to fit any fluorescence recovery curve involving two interacting mobile species in the cell (a fluorescent probe and its target substrate). The results show that interpreting FRAP data in light of the new model reconciles the discrepant mRNA diffusion-rate values previously reported.

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