title: Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins.
abstract: Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20-30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments.
authors: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Cheryl A
authors: CA
authors: Telmer
authors: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Richa
authors: R
authors: Verma
authors: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Haibing
authors: H
authors: Teng
authors: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Susan
authors: S
authors: Andreko
authors: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Leann
authors: L
authors: Law
authors: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Marcel P
authors: MP
authors: Bruchez
mesh_terms: D002451:Cell Compartmentation; D005456:Fluorescent Dyes / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D013347:Subcellular Fractions / Q000378:metabolism*; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes
references: J Am Chem Soc. 2012 Jan 25;134(3):1623-9
references: 22224915
references: J Am Chem Soc. 2010 Aug 18;132(32):11103-9
references: 20698676
references: Arch Biochem Biophys. 2001 Feb 15;386(2):251-60
references: 11368349
references: Biochim Biophys Acta. 1993 Jul 25;1150(1):89-97
references: 8334141
references: PLoS One. 2011;6(3):e17896
references: 21479270
references: J Cell Biol. 2006 Nov 20;175(4):579-93
references: 17116750
references: Chembiochem. 2011 Sep 19;12(14):2217-26
references: 21793150
references: Angew Chem Int Ed Engl. 2011 Sep 5;50(37):8750-2
references: 21793145
references: J Biol Chem. 1990 May 25;265(15):8808-16
references: 2160469
references: J Am Chem Soc. 2013 Jan 9;135(1):242-51
references: 23252842
references: Chem Biol. 2008 Feb;15(2):128-36
references: 18291317
references: Nat Methods. 2005 Apr;2(4):255-7
references: 15782216
references: Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):10914-9
references: 20534555
references: J Am Chem Soc. 2012 Jun 13;134(23):9589-92
references: 22642566
references: Histochem J. 2001 Feb;33(2):65-9
references: 11432641
references: J Am Chem Soc. 2010 Jul 14;132(27):9301-9
references: 20568758
references: ACS Chem Biol. 2014 Mar 21;9(3):606-12
references: 24471525
references: Proc Natl Acad Sci U S A. 2013 Apr 9;110(15):5858-63
references: 23479651
references: J Am Chem Soc. 2010 Nov 3;132(43):15099-101
references: 20936809
references: Science. 1998 Jul 10;281(5374):269-72
references: 9657724
references: J Am Chem Soc. 2008 Sep 24;130(38):12620-1
references: 18761447
references: Angew Chem Int Ed Engl. 2012 May 14;51(20):4838-42
references: 22461279
references: Chembiochem. 2010 Mar 22;11(5):646-8
references: 20175182
references: Biopolymers. 2012;98(3):234-8
references: 22782565
references: Nat Methods. 2012 Jul;9(7):671-5
references: 22930834
references: J Am Chem Soc. 2012 Feb 29;134(8):3720-8
references: 22239252
references: Methods Mol Biol. 2007;356:195-208
references: 16988404
references: Angew Chem Int Ed Engl. 2009;48(48):9164-6
references: 19876989
references: Nat Methods. 2013 Aug;10(8):751-4
references: 23770755
references: Nat Methods. 2009 May;6(5):355-8
references: 19363494
references: J Mol Biol. 2013 Nov 15;425(22):4595-613
references: 23978698
references: Nat Biotechnol. 2003 Jan;21(1):86-9
references: 12469133
references: Mol Biotechnol. 2013 Jul;54(3):829-41
references: 23242633
references: J Am Chem Soc. 2013 Aug 21;135(33):12360-5
references: 23927377
references: PLoS One. 2012;7(5):e37551
references: 22649538
references: Org Lett. 2011 Aug 19;13(16):4160-3
references: 21786765
references: Trends Cell Biol. 2007 Sep;17(9):456-64
references: 17825565
references: J Am Chem Soc. 2011 May 4;133(17):6745-51
references: 21473619
references: Bioconjug Chem. 2010 Dec 15;21(12):2320-6
references: 20961132
references: J Am Chem Soc. 2012 Aug 22;134(33):13692-9
references: 22873118
references: J Am Chem Soc. 2009 Sep 16;131(36):12960-9
references: 19737016
references: Biochim Biophys Acta. 1992 Feb 26;1119(2):218-24
references: 1311608
references: J Biomol Screen. 2014 Sep;19(8):1220-6
references: 24820110
references: Nat Cell Biol. 2010 Mar;12(3):273-7
references: 20154681
references: Am J Physiol Cell Physiol. 2011 Apr;300(4):C723-42
references: 21209361
references: Bioconjug Chem. 2009;20(10):1843-1847
references: 20976031
references: J Biol Chem. 1990 May 25;265(15):8817-22
references: 2160470
references: IUBMB Life. 2009 Nov;61(11):1029-42
references: 19859977
references: Nat Chem. 2013 Feb;5(2):132-9
references: 23344448
references: J Biol Chem. 1997 May 23;272(21):13555-61
references: 9153202
references: Chemistry. 2011 Jul 18;17(30):8342-9
references: 21656862
references: Protein Sci. 2011 Sep;20(9):1509-19
references: 21714025
references: Nat Biotechnol. 2008 Feb;26(2):235-40
references: 18157118
references: Chemphyschem. 2014 Mar 17;15(4):687-95
references: 24194371
references: J Am Chem Soc. 2002 May 29;124(21):6063-76
references: 12022841
references: Nat Methods. 2014 Jul;11(7):731-3
references: 24859753