A.1. protein (FAP)?. MG shows strong fluorescence activation

A.1.

Introduction/MotivationAkey challenge in life science is the qualitative andquantitative analysis of protein function and protein-protein interactions upto dynamic protein networks involved in cellular logistics. This way of lookingat protein functions in cells has motivated significant efforts in the field ofprotein labeling combined with high-resolution optical microscopy (1, 2).FAP technology A.2. Theoreticalbackground A.2.

1.     Malachite greenDC1 Malachite green(MG) is a fluorogenic dye or fluorogen, that shows fluorescence enhancementupon binding to its engineered cognate protein, a fluorogen activating protein(FAP)?.  MG shows strong fluorescence activation of 2,360-fold when itbinds to a specific RNA aptamer5. Binding of the dyes resultsin structural restriction of rotations around a single bond within thechromophores, inhibiting the vibrational deexcitation. Enhanced fluorescence ofsuch ‘molecular rotors’ has also been reported for an antibody-dye complex,although with much smaller increases. 6 It was also demonstrated that these fluorogens can bespecifically activated by expressed fluorogen activating proteins in both fixedand living cells, by simple addition of dye to the media, and thatsuperresolution or conventional resolution images can be obtained by using lowor high concentrations of dye, respectively.

7 These protein domains can be fused totargets of interest in living cells, providing the first generalizable exampleof a molecularly specific Binding Activated Localization Microscopy probe.  DC1Malachite green (MG) is a fluorogenicdye that shows fluorescence enhancement upon binding to its engineered cognateprotein, a fluorogen activating protein (FAP)?.  Energy transfer donorssuch as cyanine and rhodamine dyes have been conjugated with MG to modify thespectral properties of the fluorescent complexes, where the donor dyes transferenergy through Fo?rster resonance energy transfer to the MG complex resultingin binding-?conditional fluorescence emission in the far-?red region.Malachite Green(MG) is a fluorogen which when bound to Fluorogen Activating Peptide (FAP)emits in the far-?red region.  The brightness and excitation properties ofthe MG-?FAP complex has been enhanced using different energy-?transfer donorslike Cy3 and Rhodamine that donate into the primary absorbance band of the MGdye.

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The noncovalent equil. activation of a fluorogenic malachite green dye and its cognatefluorogen-?activating protein (FAP) can produce a sparse labeling distributionof densely tagged genetically encoded proteins, enabling single mol. detectionand super-?resoln.

imaging in fixed and living cells.  These sparselabeling conditions are achieved by control of the dye concn. in the milieu,and do not require any photoswitching or photoactivation.Malachite green(MG) is a fluorogenic dye that shows fluorescence enhancement upon binding toits engineered cognate protein, a fluorogen activating protein (FAP)?.  Hexa-Cy3-MG (HCM) is a new light-harvesting dyedronderivative of MG prepared by coupling six Cy3 molecules to one MG usingazide–alkyne cycloaddition chemistry to a highly decorated lysine linker(Synthetic and characterization details in SupportingInformation), enabling the energy transfer from multiple Cy3 moieties tosignificantly enhance the molecular brightness of the FAP/fluorogen complex (SupportingInformation Figure S1A), while maintaining a fluorogenic activation ratioof ?330-foldat 562 nm excitation.