The main research direction of this Laboratory is the design of new proteins and nucleoprotein constructs as tools for medicine and biotechnology. Much of the work is focused on creating new tools for editing the genome of eukaryotic cells. During the last few years there has been a breakthrough in the engineering of living systems that is related to the improvement of three principal tools: zinc-finger nucleases, TAL effector nucleases (TALEN) and CRISPR/Cas9 (Urnov et al., 2010; Wright et al., 2014 Doudna & Charpentier, 2014). Additionally, the precise recombination technology known as "recombineering" enables easy editing of bacterial genomes (Leprince et al., 2012). Common drawbacks in all existing systems are their relatively low efficiency and the possibility of induction of non-targeted mutations.
In the coming years, explosive development in genomic editing technology is expected. Researchers are also attempting to edit the epigenome, by introducing or removing epigenetic methylation and histone modifications (Voigt & Reinberg, 2013), thus acquiring much more flexibility in the control over the cell phenotype. The laboratory addresses basic questions about the mechanisms of genome and epigenome modification: the mechanisms and regulation of genome methylation, active demethylation and other types of targeted modification, and the consequences of modifications temporarily present in DNA, RNA and mononucleotides as a result of spontaneous damage, specific modification by cellular systems, or purposeful introduction into cells.
Of the epigenetic mechanisms to date, two ways of stable activation and inactivation of genes have been studied in most detail. One is associated with changes in the state of chromatin condensation due to the covalent modification of histones and other proteins responsible for DNA packaging. The second relies upon covalent modification of the DNA itself, which changes its affinity for a variety of DNA-binding proteins (Bird, 2007). In higher eukaryotes, 5-methylcytosine and its oxidized derivative, 5-hydroxymethylcytosine (Branco et al., 2011) are the only modified DNA bases for which participation in the regulation of gene expression has been reliably shown. There is, however, data that other modifications of DNA and RNA, in particular oxidative ones, can also perform epigenetic functions in human cells (Moore et al., 2013; Zarakowska et al., 2014; Mikhed et al., 2015). Moreover, in recent years, it was discovered that epigenetic functions can be performed by noncanonical DNA structures (Cea et al., 2015), long non-coding RNAs (Kornfeld & Brüning, 2014, Peschansky & Wahlestedt, 2014), parts of coding and non-coding RNAs (Liu & Pan, 2015; Yue et al., 2015), and even non-standard RNAs and proteins arising from errors during transcription and translation (Gordon et al., 2015). Although much work has been devoted to mechanisms and the role of methylation of cytosine in the genome of higher eukaryotes, very little was known about the mechanisms of demethylation until recently when it has been shown to be based on targeted DNA damage and repair (Zharkov, Grin, 2012). The action mechanisms of the remaining epigenetic modifications remain largely unexplored.
Systems of enzymatic modification of DNA and RNA can be a rich source of tools for genomic engineering, medicine and biotechnology, as well as targets for new generations of drugs. Recent examples of translation research in active DNA modification include the inhibitors of DNA methyltransferases approved for cancer treatment in the mid-2000s (Stresemann and Lyko, 2008), cancer diagnostics methods based on DDNA methylation patterns (Van Neste et al., 2011) and the widespread use of genomic editing systems based on the endonucleases TALEN and CRISPR/Cas9 (Wright et al., 2014; Doudna & Charpentier, 2014).
In addition, the Laboratory works on the design of multifunctional nanoconstructs capable of recognizing several molecular targets and / or catalyzing several successive reactions, and on creating DNA-dependent enzymes with a given substrate specificity. Nanoconstructs for the selective elimination of individual cells, bacterial and viral particles from the blood, as well as nucleases with the desired substrate specificity for editing the cell genome and for rapid genotyping of human DNA, are the ultimate goals of this research. Today, the field of DNA nanoconstrucs is well established, yet the task of decorating them with protein components remains virtually unexplored with only isolated successful attempts (Delebecque et al., 2011; Simmel, 2012). The development of effeicient methods for assembling extended DNA/RNA-protein complexes would greatly enhance the capabilities of nanobiotechnology, including the creation of new drugs and diagnostic tools.
Head of the laboratory – Doctor of Biological Sciences, Dmitry Zharkov
