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.

The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen-activating protein (FAP) can produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and super-resolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved by using physiological buffers and cellular media, in which additives and switching buffers are not required to obtain super-resolution images. We evaluate the super-resolution properties and images obtained from a selected FAP clone fused to actin, and show that the photon counts per object are between those typically reported for fluorescent proteins and switching-dye pairs, resulting in 10–30 nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures.

Thrombin is the typical target in anticlotting therapy for many serious diseases such as heart attack and stroke. DNA aptamers are well-known thrombin inhibitors that prevent fibrinogen hydrolysis. We have discovered that exosite-targeting antithrombin aptamers enhance the activity of thrombin toward a small peptide substrate, Sar(N-methylglycine)-Pro-Arg-paranitroanilide, and that the activation of the enzyme by these aptamers is strongly inhibited by their complementary DNAs. Our study reveals that treatment with mixed aptamers or with a dual-aptamer construct led to an 8.6- or 7.8-fold enhancement in peptide hydrolysis relative to thrombin alone, a synergistic effect much higher than the activation observed with a monofunctional aptamer (1.5-fold for Apt27 or 2.7-fold for Apt15). In addition, we discovered that Apt27 is a biofunctional molecule for thrombin because of its activation effect. An enzyme kinetic study indicates that the binding of aptamers to exosites I and II significantly activates thrombin towards the peptide substrate, thus illustrating that binding of aptamers to exosites can allosterically regulate the active site of thrombin. Our study suggests the necessity of considering possible side effects when DNA aptamers are used for clinical applications involving the inhibition of thrombin-mediated clotting.