Research directions
Among the most important efforts in the laboratory is creating novel molecular instruments and technologies based on fluorescent proteins and targeted at studying and manipulating biological processes. The modern methods for analysing complex genomes and transcriptomes, namely substractive hybridization, cDNA normalization, genomic DNA normalization, etc. are also being developed in our lab.
Main results
1998—1999. The genes encoding novel fluorescent proteins, homologous to Green Fluorescent Protein (GFP) were cloned from Anthozoa (coral polyps) species. These novel fluorescent proteins were found to be of different fluorescence colors, from blue to red. Diversity of colors made them useful markers for multicolor labeling of gene expression, protein-protein interactions and signaling processes in cell.
1998—2001. A new highly efficient method for rapid amplification of full-length cDNA (Step-Out PCR) was developed. Today, this method is the most practical way to obtain full-length cDNA. It is widely used in many laboratories around the world. Assay kit SMART-RACE based on this method is commercially available from Clontex, USA.
2002—2005. A new enzyme, designated Duplex-Specific Nuclease (DSN) was isolated and characterized in collaboration with the laboratory of Marine Biochemistry of the Pacific Institute of Bioorganic Chemistry (Vladivostok, Russia). This enzyme was found to specifically cleave double-strand DNA. Based on the unique properties of DSN, we have developed original technologies for detection of single nucleotide mutations (SNPs) in DNA samples and for creation of normalized cDNA libraries.
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—2006. The first genetically encoded fluorescent sensor for hydrogen peroxide was created. This sensor, designated HyPer, is characterized with high specificity and sensitivity and can be used for in vivo detection of hydrogen peroxide in different cell compartments.
2005—2007. A panel of perfected 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.
Also discovery of fluorescent proteins in corals, creation of photoswitching fluorescent proteins technology and invention of genetically encoded photosensitizer were named among the main achievements of the Russian Academy of Sciences in the field of physico-chemical biology of years 1999, 2003 and 2005, respectively.
