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Ease of genetic encoding, labeling specificity, and high photostability are the most sought after qualities in a fluorophore for biological detection. Furthermore, many applications can gain from the fluorogenic nature of fluoromodules and the ability to turn on the same fluoromodules multiple times. Fluorogen-activating peptides (FAPs) bind noncovalently to their cognate fluorogens and exhibit enhanced photostability. Herein, the photostabilities of malachite green (MG)-binding and thiazole-orange-binding FAPs are compared under limiting- and excess-fluorogen conditions to establish distinct mechanisms for photostability that correspond to the dissociation rate of the FAP-fluorogen complex. FAPs with slow dissociation show evidence of dye encapsulation and protection from photo or environmental degradation and single-step bleaching at the single molecule level, whereas those with rapid dissociation show repeated cycles of binding and enhanced photostability by exchange of bleached fluorogen with a new dye. A combination of generalizable selection pressure based on bleaching, flow cytometry, and site-specific amino acid mutagenesis is used to obtain a modified FAP with enhanced photostability, due to rapid dissociation of the MG fluorogen. These studies shed light on the basic mechanisms by which noncovalent association can effect photostable labeling, and demonstrate novel reagents for photostable and intermittent labeling of biological targets.

Inefficiency of fluorescent labels imposes key limitations in fluorescence microscopy. Photobleaching, non-specific signal from multiple fluorophores, and lack of multiplexing and orthogonal labels impose major challenges in studying complex cellular systems, both at ensemble and single molecule levels. We have addressed these problems by exploiting the binding kinetics of fluorogen activating peptides that on binding to a non-fluorescent molecule (a fluorogen) exhibit fluorescence. Novel methods in flow cytometry and rational design of peptides enabled the elucidation of peptides and fluorogen pairs with varying off rates. Complexes with fast off rates (koff∼5E-3 s-1) can exchange a bleached fluorogen molecule with a fresh fluorogen molecule in solution. Since the free fluorogen is dark, we can image for extended time scales in the presence of excess fluorogen. For complexes with slow off rates (koff∼5E-5 s−1) and high affinity (Kd