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

A.1. Introduction/Motivation

Akey challenge in life science is the qualitative and
quantitative analysis of protein function and protein-protein interactions up
to dynamic protein networks involved in cellular logistics. This way of looking
at protein functions in cells has motivated significant efforts in the field of
protein labeling combined with high-resolution optical microscopy (1, 2).

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FAP technology

 

A.2. Theoretical
background

 

A.2.1.     
Malachite greenDC1 

Malachite green
(MG) is a fluorogenic dye or fluorogen, that shows fluorescence enhancement
upon binding to its engineered cognate protein, a fluorogen activating protein
(FAP)?.  MG shows strong fluorescence activation of 2,360-fold when it
binds to a specific RNA aptamer5. Binding of the dyes results
in structural restriction of rotations around a single bond within the
chromophores, inhibiting the vibrational deexcitation. Enhanced fluorescence of
such ‘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 be
specifically activated by expressed fluorogen activating proteins in both fixed
and living cells, by simple addition of dye to the media, and that
superresolution or conventional resolution images can be obtained by using low
or high concentrations of dye, respectively. 7 These protein domains can be fused to
targets of interest in living cells, providing the first generalizable example
of a molecularly specific Binding Activated Localization Microscopy probe.

 

 DC1Malachite green (MG) is a fluorogenic
dye that shows fluorescence enhancement upon binding to its engineered cognate
protein, a fluorogen activating protein (FAP)?.  Energy transfer donors
such as cyanine and rhodamine dyes have been conjugated with MG to modify the
spectral properties of the fluorescent complexes, where the donor dyes transfer
energy through Fo?rster resonance energy transfer to the MG complex resulting
in 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 of
the MG-?FAP complex has been enhanced using different energy-?transfer donors
like Cy3 and Rhodamine that donate into the primary absorbance band of the MG
dye.

The noncovalent equil. 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 mol. detection
and super-?resoln. imaging in fixed and living cells.  These sparse
labeling 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 to
its engineered cognate protein, a fluorogen activating protein (FAP)?.

 

 

Hexa-Cy3-MG (HCM) is a new light-harvesting dyedron
derivative of MG prepared by coupling six Cy3 molecules to one MG using
azide–alkyne cycloaddition chemistry to a highly decorated lysine linker
(Synthetic and characterization details in Supporting
Information), enabling the energy transfer from multiple Cy3 moieties to
significantly enhance the molecular brightness of the FAP/fluorogen complex (Supporting
Information Figure S1A), while maintaining a fluorogenic activation ratio
of ?330-fold
at 562 nm excitation.