Laboratory of genetically encoded molecular tools

Department of Biophotonics

Head: Konstantin Lukyanov

Fluorescent proteins, photoactivatable fluorescent proteins, genetically encoded fluorescent sensors, live cell labeling, genetically encoded photosensitizers

Laboratory works on development of novel fluorescent tags and methods of fluorescence labeling of biological objects. The main focus is on the fluorescent proteins of GFP family, and also on other methods of noninvasive visualization of structures and processes in live cells. These methods are widely used in biomedical research enabling deciphering the molecular mechanisms of various normal and pathological phenomena and facilitating preclinical drug screening.

We are fully equipped for gene-engineering works, mammalian cell culture works, fluorescence and laser scanning confocal microscopy, and optical spectroscopy.

Laboratory collaborates with many groups of the Institute: Total Synthesis Lab on studying physic-chemical properties of fluorescent protein chromophores and developing novel methods of fluorescence labeling using their analogs; Molecular technologies laboratory on development of novel fluorescent sensors; Laboratory of X-ray study on analysis of 3D structure of fluorescent proteins and structure-guided changes of their spectral properties; Laboratory of molecular bases of embryogenesis on application of fluorescent tools for developmental studies.

Also, Laboratory collaborates with Institute of Biomedical Technologies (Nizhny Novgorod Medical State Academy) on novel methods of super-resolution fluorescence microscopy and on fluorescence imaging of mouse tumor models; Laboratory of Physical Biochemistry (A.N. Bach Institute of Biochemistry) on fluorescence lifetime imaging microscopy of biological models; Kyril Solntsev (Georgia Institute of Technology, Atlanta, GA) on ultrafast spectroscopy of fluorescent proteins; laboratory of Anna Krylov (University of Southern California, Los Angeles, CA) on studying physic-chemical processes in fluorescent proteins; laboratory of Vladislav Verkhusha (Albert Einstein College of Medicine, Bronx, NY) on development of novel fluorescent proteins; laboratory of Jens Meiler (Vanderbilt University, Nashville, TN) on computer modeling of fluorescent proteins.

Laboratory (Laboratory of Biophotonics in 2009-2020) was established in 2009 by branching out of the Laboratory of Molecular Technologies for Biology and Medicine headed by Sergey Lukyanov; our team is working with fluorescent proteins since 1999.

Novel fluorescent proteins

GFP and related fluorescent proteins are widely used as genetically encoded tags for visualization of proteins and cell populations in live systems. We use site-directed and random mutagenesis to create new variants of fluorescent proteins. This approach allows to generate proteins with usual spectral properties due to new chromophore structures or altered amino acid environment of the chromophore. In addition, we are working on improvement of fluorescent proteins characteristics such as brightness and photostability that are important for their practical applications.

Chudakov DM, et al. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev. 2010, 90, 1103-63.

Pletnev VZ, et al. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine. Acta Crystallogr D Biol Crystallogr. 2013, 69, 1850-60.

Sarkisyan KS, et al. Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime. Biophys J. 2015, 109, 380-9.

Mishin AS, et al. Novel uses of fluorescent proteins. Curr Opin Chem Biol. 2015, 27, 1-9.

Sarkisyan KS, et al. Local fitness landscape of the green fluorescent protein. Nature. 2016, 533, 397-401.

Bozhanova NG, et al. Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores. ACS Chem Biol. 2017 Jul 21;12(7):1867-1873.

Povarova NV, et al. Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models. Int J Mol Sci. 2017 Jul 12;18(7). pii: E1503. doi: 10.3390/ijms18071503.

Bozhanova NG, et al. Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal. Chem. Sci., 2017, 8, 7138-7142.


Novel genetically encoded sensors

We are working on new fluorescent protein-based sensors for various regulatory activities, which enable quantitative visualization of the target events in live cells. For example, we recently developed ratiometric sensor for nonsense-mediated mRNA decay (NMD) and far-red sensor for caspase-3.

Gurskaya NG, et al. Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins. Nucleic Acids Res. 2012, 40, e57.

Pereverzev AP, et al. Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep. 2015, 5, 7729.

Zlobovskaya OA, et al. Genetically encoded far-red fluorescent sensors for caspase-3 activity. Biotechniques. 2016, 60, 62-8.

Sergeeva TF, et al. Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis. Biochim Biophys Acta. 2017, 1864(3):604-611.

Kost LA, Nikitin ES, Ivanova VO, Sung U, Putintseva EV, Chudakov DM, Balaban PM, Lukyanov KA, Bogdanov AM. Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor. PLoS One. 2017 Sep 1;12(9):e0184225.


Photoconversions of fluorescent proteins

Photoactivatable fluorescent proteins (PAFP) are used for tracking movements of proteins, organelles and cells in live systems, as well as for super-resolution fluorescence microscopy. Earlier, our team developed several PAFPs, such as KFP1, PS-CFP and Dendra, which were among the world's first members of this protein type. Currently, we continue to work on development of new PAFPs and PAFP-based techniques.

In 2009 we discovered oxidative photoconversion of green fluorescent proteins based on electron transfer from the chromophore to an external molecule of electron acceptor. We are studying mechanisms of this phenomenon and development of methods of its practical applications (for example, for enhancement of fluorescent protein photostability).

In collaboration with Nizhny Novgorod State Medical Academy we work on development of novel labels and methods of super-resolution fluorescence microscopy (PALM/STORM single molecule localization microscopy).

Chudakov DM, et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol. 2003, 21, 191-4.

Gurskaya NG, et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol. 2006, 24, 461-5.

Bogdanov AM, et al. Green fluorescent proteins are light-induced electron donors. Nat Chem Biol. 2009, 5, 459-61.

Bogdanov AM, et al. Cell culture medium affects GFP photostability: a solution. Nat Methods. 2009, 6, 859-60.

Mamontova AV, et al. Influence of cell growth conditions and medium composition on EGFP photostability in live cells. Biotechniques. 2015, 58, 258-61.

Bogdanov AM, et al. Turning On and Off Photoinduced Electron Transfer in Fluorescent Proteins by π-Stacking, Halide Binding, and Tyr145 Mutations. J. Am. Chem. Soc. 2016, 138, 4807-4817.

Acharya A, et al. Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing? Chem Rev. 2017 Jan 25;117(2):758-795.

Klementieva NV, et al. Green-to-red primed conversion of Dendra2 using blue and red lasers. Chem Commun (Camb). 2016 Nov 18;52(89):13144-13146.

Klementieva NV, et al. Intrinsic blinking of red fluorescent proteins for super-resolution microscopy. Chem Commun (Camb). 2017 Jan 10;53(5):949-951.


Genetically encoded photosensitizers

Phototoxic fluorescent proteins (the first such protein, KillerRed, was developed by us in 2006) produce reactive oxygen species (ROS) upon light illumination. We are developing this optoginetic technology, which makes it possible to induce oxidative stress in target cell compartments, inactivate proteins and kill specific cell populations using light.  

Bulina ME, et al. A genetically encoded photosensitizer. Nat Biotechnol. 2006, 24, 95-9.

Lukyanov KA, et al. Fluorescent proteins as light-inducible photochemical partners. Photochem Photobiol Sci. 2010, 9, 1301-6.

Serebrovskaya EO, et al. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed. J Biomed Opt. 2014,19, 071403.

Sarkisyan KS, et al. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PLoS One. 2015, 10, e0145287.

2002—2006. A panel of photoactivated fluorescent proteins with different types of light-induced spectral transitions was introduced: nonflourescent-to-red (KFP1), blue-to-green (PS-CFP), and green-to-red (Dendra). These newly developed instruments KFP1, PS-CFP and Dendra were applied for precise photolabeling of cells, cell organelles, and proteins and subsequent tracking of the labeled object. The new tools were also used for monitoring the target protein degradation in an individual cell in real time using fluorescence confocal microscopy.

2005—2006. The first genetically encoded photosensitizer was created. This phototoxic red fluorescent protein named KillerRed can be used for precise light-induced destruction of proteins and cell killing.

2005—2007. A panel of improved fluorescent proteins for practical applications was created using the methods of directed molecular evolution. Particularly, there were obtained red and far-red fluorescent proteins, exceeding all known analogs in brightness. Bright far-red fluorescent proteins open up new prospects in whole-body fluorescent imaging technology.

2005—2008. First syntheses of chromophores of red fluorescent proteins (asFP595, Kaede, zFP538) and their structural analogs were performed. This work revealed various aspects of structure-properties relationship in this group of chromophores and allowed to propose promising amino-acid substitutions in fluorescent proteins to obtain variants with novel spectral properties.


Konstantin Lukyanov

Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map