Department of functioning of living systems

All publications (show selected)

Olga Doncova

Discovery of the novel protein encoded in mammalian mitochondrial DNA polymerase gene POL

Laboratory of bioinformatics approaches in combinatorial chemistry and biology

Mitochondrial DNA polymeraseis essential for mitochondrial DNA replication. It consists of two subunits. POLG gene, which codes for catalytic subunit of polymerase, is notorious for more than 200 mutations linked to devastating inherited diseases. It is proposed that POLG mutations lead to progressive accumulation of mitochondrial DNA mutations and, subsequently, to organism disfunction and death. Andreev and colleagues discovered an efficient CUG start-codon in 5’-leader of POLG mRNA which initiates translation of a long alternative reading frame overlapping with POLG ORF. The new open reading frame is rather long (260 triplets in humans) and is conserved in placental mammals. The new protein was named POLGARF (POLG Alternative Reading Frame). POLGARF accumulates in nucleoli, but upon certain mitogenic stimuli is cleaved and secreted outside of cells. It has been proposed that POLGARF evolved approximately 100-200 mln years ago by MIR transposon insertion into POLG gene, which allows alternative reading frame translation and evolution of POLGARF protein coding function.

Publications

  1. Loughran G, Zhdanov AV, Mikhaylova MS, Rozov FN, Datskevich PN, Kovalchuk SI, Serebryakova MV, Kiniry SJ, Michel AM, OConnor PBF, Papkovsky DB, Atkins JF, Baranov PV, Shatsky IN, Andreev DE (2020). Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF. Proc Natl Acad Sci U S A 117 (40), 24936–24946

Start codon context and occurrence of AUG codons in the beginning of protein coding open reading frames co-evolve

Laboratory of bioinformatics approaches in combinatorial chemistry and biology

Researchers from IBCh RAS together with their international collаborators discovered an evolutionary link between efficiency of start codons and the probability of AUG codon occurrence in the 5'-end parts of protein-coding sequences. When start codons are inefficient the next AUG codon is more likely to be found in the same reading frame. At the same time in case of highly efficient start codons, the next AUG codon is more likely to be found in alternative frames. They also have shown that weak initiation at the first starts is associated with the synthesis of shortened proteoforms as a result of initiation at the second starts.

Publications

  1. Benitez-Cantos MS, Yordanova MM, OConnor PBF, Zhdanov AV, Kovalchuk SI, Papkovsky DB, Andreev DE, Baranov PV (2020). Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context. Genome Res 30 (7), 974–984

Researchers from the Laboratory of Bioinformatics Approaches in Combinatorial Chemistry and Biology IBCH RAS, in collaboration with Irish colleagues, recently published a review in WIRES RNA that compiles the computational approaches, software tools, and data resources that have been developed over the last ten years for ribosome profiling data processing and analysis.

Publications

  1. Kiniry SJ, Michel AM, Baranov PV (2019). Computational methods for ribosome profiling data analysis. Wiley Interdiscip Rev RNA 11 (3), e1577

Researchers from the Laboratory of biocatalysis, Laboratory of proteolytic enzyme chemistry, Laboratory of bioinformatics approaches in combinatorial chemistry and biology, Laboratory of hormonal regulation proteins IBCh RAS together with their Russian and foreign colleagues showed that amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

Publications

  1. Terekhov SS, Eliseev IE, Ovchinnikova LA, Kabilov MR, Prjibelski AD, Tupikin AE, Smirnov IV, Belogurov AA, Severinov KV, Lomakin YA, Altman S, Gabibov AG (2020). Liquid drop of DNA libraries reveals total genome information. Proc Natl Acad Sci U S A 117 (44), 27300–27306

Genomic DNA i-motifs as fast sensors responsive to near-physiological pH microchanges

The group of molecular tools for living system studies

The design of robust sensors for measuring intracellular pH, based on the native DNA i-motifs (iMs) found in neurodegeneration- or carcinogenesis-related genes, is reported. Those iMs appear to be genomic regulatory elements and might modulate transcription in response to pH stimuli. Given their intrinsic sensitivity to minor pH changes within the physiological range, such noncanonical DNA structures can be used as sensor core elements without additional modules other than fluorescent labels or quenchers. We focused on several iMs that exhibited fast folding/unfolding kinetics. Using stopped-flow techniques and FRET-melting/annealing assays, we confirmed that the rates of temperature-driven iM-ssDNA transitions correlate with the rates of the pH-driven transitions. Thus, we propose FRET-based hysteresis analysis as an express method for selecting sensors with desired kinetic characteristics. For the leading fast-response sensor, we optimized the labelling scheme and performed intracellular calibration. Unlike the commonly used small-molecule pH indicators, that sensor was transferred efficiently to cell nuclei. Considering its favourable kinetic characteristics, the sensor can be used for monitoring proton dynamics in the nucleus. These results argue that the ‘genome-inspired’ design is a productive approach to the development of biocompatible molecular tools.

Publications

  1. Turaev AV, Isaakova EA, Severov VV, Bogomazova AN, Zatsepin TS, Sardushkin MV, Aralov AV, Lagarkova MA, Pozmogova GE, Varizhuk AM (2020). Genomic DNA i-motifs as fast sensors responsive to near-physiological pH microchanges. Biosens Bioelectron , 112864

Phenoxazine-based scaffold for designing G4-interacting agents

Laboratory of ligand-receptor interactions,  The group of molecular tools for living system studies

G-quadruplexes (G4) represent one class of non-canonical secondary nucleic acid structures that are currently regarded as promising and attractive targets for anti-cancer, anti-viral and antibacterial therapy. We probe a new i-clamp-inspired phenoxazine scaffold for designing G4-stabilizing ligands. The length of the protonated aminoalkyl tethers (‘arms’) of the phenoxazine-based ligand was optimized in silico. Two double-armed ligands differing in the relative orientation of their arms and one singlearmed ligand were synthesized. The two-armed ligands significantly enhanced the thermal stability of the G-quadruplex structures (increasing the melting temperature by up to 20 °C) and displayed G4 selectivity over duplex DNA. The ligands look promising for biological studies and the phenoxazine scaffold could be a starting point for designing new G4-interacting compounds.

Publications

  1. Tsvetkov VB, Varizhuk AM, Lizunova SA, Nikolenko TA, Ivanov IA, Severov VV, Belyaev ES, Shitikov EA, Pozmogova GE, Aralov AV (2020). Phenoxazine-based scaffold for designing G4-interacting agents. Org Biomol Chem 18 (31), 6147–6154

Shиненный с G-квадруплексами, увеличивает флуоресценцию синтетических аналогов хромофора GFPort Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues

Group of chemistry of heterocyclic compounds,  The group of molecular tools for living system studies

Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.

How hyperthermia enhances effects of chemo- and radiotherapy

Laboratory of molecular oncology

Victoria Shender and Georgy Arapidi, employees of the Laboratory of Molecular Oncology, together with colleagues from the Department of Molecular Biology of the Biological Faculty of Moscow State University, participated in a study published in the journal Cells in 2020 (DOI: 10.3390 / cells9061423). The paper deciphers the molecular mechanisms of how hyperthermia, used to enhance the effect of chemotherapy and radiotherapy, can affect the sensitivity of cancer cells to this traditional type of treatment. It turned out that heating leads to "sticking" of transcription factors and proteins involved in the organization of the chromatin structure, regulation of transcription and replication, which makes DNA repair less efficient, thus leading to the death of transformed cells.

Publications

  1. Luzhin AV, Avanesyan B, Velichko AK, Shender VO, Ovsyannikova N, Arapidi GP, Shnaider PV, Petrova NV, Kireev II, Razin SV, Kantidze OL (2020). Chromatin Trapping of Factors Involved in DNA Replication and Repair Underlies Heat-Induced Radio- and Chemosensitization. Cells 9 (6),

Integrative analysis of miRNA and mRNA sequencing data reveals potential regulatory mechanisms of ACE2 and TMPRSS2.

Laboratory of microfluidic technologies for biomedicine

The development of novel approaches for regulating the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) is becoming increasingly important within the context of the ongoing COVID-19 pandemic since these enzymes play a crucial role in cell infection. We searched for putative ACE2 and TMPRSS2 expression regulation networks mediated by various miRNA isoforms (isomiR) across different human organs using publicly available paired miRNA/mRNA-sequencing data from The Cancer Genome Atlas (TCGA) project. As a result, we identified several miRNA families targeting ACE2 and TMPRSS2 genes in multiple tissues. In particular, we found that lysine-specific demethylase 5B (JARID1B), encoded by the KDM5B gene, can indirectly affect ACE2/TMPRSS2 expression by repressing transcription of hsa-let-7e/hsa-mir-125a and hsa-mir-141/hsa-miR-200 miRNA families which are targeting these genes.

 

Figure: Scheme of the interaction network consisting of JARID1B, let-7e/miR-125a, and miR-141/miR-200 miRNA families and ACE2/TMPRSS2 enzymes.

Publications

  1. Nersisyan S, Shkurnikov M, Turchinovich A, Knyazev E, Tonevitsky A (2020). Integrative analysis of miRNA and mRNA sequencing data reveals potential regulatory mechanisms of ACE2 and TMPRSS2. PLoS One 15 (7), e0235987

Regulatory T cells in patients with early untreated rheumatoid arthritis

Laboratory of molecular oncology

Yu.P. Rubtsov, an employee of the Laboratory of Molecular Oncology. together with specialists from the Institute of Rheumatology named after Nasonova published in the journal Biochimie the results of a study of the size and phenotype of regulatory T cells in patients with rheumatoid arthritis. This clinical study showed that:

  • Early patients with untreated rheumatoid arthritis have a significant defect in blood Treg;
  • Decrease in the number of Tregs in patients with limited expression of ISOS, CD40L and PD-L1 on the cell surface;
  • Treatment with methotrexate (MT) restores the amount and expression of Treg activation markers;
  • Surface expression of CTLA4 can post to assess treatment response MT.

Selective self-assembly of compact discrete DNA nanostructures

Laboratory of molecular design and synthesis

Production of small discrete DNA nanostructures containing covalent junctions requires reliable methods for the synthesis and assembly of branched oligodeoxynucleotide conjugates. This study reports an approach for self-assembly of hard-to-obtain primitive discrete DNA nanostructures – “nanoethylenes”, dimers formed by double-stranded oligonucleotides using V-shaped furcate blocks. We scaled up the synthesis of V-shaped oligonucleotide conjugates using pentaerythritol-based diazide and alkyne-modified oligonucleotides using copper(I)-catalyzed azide-alkyne cycloaddition and optimized the conditions for “nanoethylene” formation. Next, we designed “nanoethylene”-based “nanomonomers” containing pendant adaptors. They demonstrated smooth and high-yield spontaneous conversion into the smallest cyclic product, DNA tetragon aka “nano-methylcyclobutane”. Formation of DNA nanostructures was confirmed using native polyacrylamide gel electrophoresis and atomic force microscopy, and additionally studied by molecular modeling. The proposed facile approach to discrete DNA nanostructures using precise adaptor-directed self-assembly expands the toolkit for the realm of DNA origami.

Publications

  1. Brylev VA, Ustinov AV, Tsvetkov VB, Barinov NA, Aparin IO, Sapozhnikova KA, Berlina YY, Kokin EA, Klinov DV, Zatsepin TS, Korshun VA (2020). Toehold-Mediated Selective Assembly of Compact Discrete DNA Nanostructures. Langmuir 36 (49), 15119–15127

The efficacy of fluorescent hybridization assays is often limited by low signal-to-background ratio of the probes that can be partially overcome by sophisticated signal amplification methods. Deep understanding of the mechanisms of fluorescence quenching and energy transfer in complex DNA probes, choice of optimal donor/acceptor pairs along with rational design can significantly enhance the performance of DNA probes. We propose novel FRET dual DNA probes with the excimer-forming pyrene pair as a donor and sulfo-Cy3 dye as an acceptor. The probes demonstrated remarkable 75-fold enhancement of sulfo-Cy3 fluorescence upon target capturing. Stokes shift up to 220 nm minimizes fluorescence crosstalk. Time-correlated single-photon counting revealed two excited states of pyrene excimer wherein only one is directly involved in the resonance energy transfer to sulfo-Cy3. Optimized DNA probes demonstrated high sensitivity with excellent signal-to-background ratio which were applied for visualization of 18S rRNA by fluorescent in situ hybridization in HEK293T cells.

IBCh RAS together with their Russian and foreign colleagues showed that amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

Publications

  1. Terekhov SS, Eliseev IE, Ovchinnikova LA, Kabilov MR, Prjibelski AD, Tupikin AE, Smirnov IV, Belogurov AA, Severinov KV, Lomakin YA, Altman S, Gabibov AG (2020). Liquid drop of DNA libraries reveals total genome information. Proc Natl Acad Sci U S A 117 (44), 27300–27306

Selective Eradication of Staphylococcus aureus by the Designer Genetically Programmed Yeast Biocontrol Agent

Laboratory of biocatalysis,  Laboratory of bioinformatics approaches in combinatorial chemistry and biology,  Laboratory of proteolytic enzyme chemistry

Staphylococcus aureus is a common human pathogen that is particularly often associated with antibiotic resistance. The eradication of this ubiquitous infectious agent from its ecological niches and contaminated surfaces is especially complicated by excessive biofilm formation and persisting cells, which evade the antibacterial activity of conventional antibiotics. Here, we present an alternative view of the problem of specific S. aureus eradication. The constitutive heterologous production of highly specific bacteriolytic protease lysostaphin in yeast Pichia pastoris provides an e_cient biocontrol agent, specifically killing S. aureus in coculture. A yeast-based anti-S. aureus probiotic was efficient in a high range of temperatures and target-to-effector ratios, indicating its robustness and versatility in eliminating S. aureus cells. The efficient eradication of S. aureus by live lysostaphin-producing P.pastoris was achieved at high scales, providing a simple, biocompatible, and cost-effective strategy for S. aureus lysis in bioproduction and surface decontamination. Future biomedical applications based on designer yeast biocontrol agents require evaluation in in vivo models. However, we believe that this strategy is very promising since it provides highly safe, effcient and selective genetically programmed probiotics and targeted biocontrol agents.

Acid-sensitive channels ASIC1 are a molecular target of three-finger toxins from black mamba venom (Dendroaspis polylepis) - mambalgins. Mambalgins effectively inhibit homo- and heteromeric receptors containing the ASIC1a subunit; however, the possibility of their use as antitumor agents has not been previously studied. It was shown that ASIC1a mRNA is expressed in cells of chronic myelogenous leukemia and gliomas, but not in normal cells. Mambalgin-2 inhibited the growth of U251 MG and A172 glioma cells with EC50 in the nanomolar range, without affecting the proliferation of normal astrocytes. Interestingly, mambalgin-2 mutants did not affect the growth of glioma cells, indicating ASIC1a as the main molecular target of mambalgin-2 in glioma cells. Mambalgin-2 caused cell cycle arrest, inhibited phosphorylation of cyclin D1 and cyclin-dependent kinases (CDK), and induced apoptosis in glioma cells. In addition, mambalgin-2 inhibited the growth of the primary cell culture obtained from a patient with glioblastoma. Our data point on mambalgin-2 as a promising prototype of drugs for the targeted treatment of oncological diseases, and channels containing the ASIC1a subunit as a new target of targeted therapy.

Knockdown of the α5 chain of laminins is associated with partial differentiation of tumor cells

Laboratory of microfluidic technologies for biomedicine

The interaction of tumor cells with the components of the extracellular matrix can effect on the rate of disease progression and metastasis. The most important components of this matrix are laminins, heterotrimeric glycoproteins consisting of one α-, one β- and one γ-chain (αβγ). Laminins are involved in the regulation of all the most important processes of living cell by interacting with receptors on the cell surface. In our work, we studied the effect of endogenous expression of the α5 laminin chain on colorectal cancer cells. Knockdown of the α5 chain has been shown to be associated with partial dedifferentiation of tumor cells, apparently by changing the activity of the Wnt signaling pathway and the mTORC1 complex. In addition, ER stress signaling pathways were activated in the cells, which contributed to an increase in the sensitivity of cells to 5-fluorouracil.

Figure: Scheme of the changes that occurred in colon cancer cells in response of the endogenous expression of the α5 laminin chain. Proteins for which there is a decrease or increase in the expression of their genes are highlighted in yellow and green, respectively.

Publications

  1. Maltseva DV, Raigorodskaya MP, Zgoda VG, Tonevitsky EA, Knyazev EN (2020). Intracellular Transport of Ribosome-Inactivating Proteins Depends on Annexin 13. Dokl Biochem Biophys 494 (1), 219–221
  2. Maltseva DV, Raigorodskaya MP, Tikhonova OV, Knyazev EN, Tonevitsky EA (2020). Relationship between the Expression Level of PSMD11 and Other Proteasome Proteins with the Activity of Ricin and Viscumin. Dokl Biochem Biophys 493 (1), 198–200
  3. Maltseva D, Raygorodskaya M, Knyazev E, Zgoda V, Tikhonova O, Zaidi S, Nikulin S, Baranova A, Turchinovich A, Rodin S, Tonevitsky A (2020). Knockdown of the α5 laminin chain affects differentiation of colorectal cancer cells and their sensitivity to chemotherapy. Biochimie 174, 107–116
  4. Nikulin SV, Knyazev EN, Maltseva DV, Sakharov DA, Gerasimenko TN (2019). Use of impedance spectroscopy to assess the effect of laminins on the in vitro differentiation of intestinal cells. Biotekhnologiya 35 (6), 102–107
  5. Maltseva DV, Raigorodskaya MP, Tsypina IM, Turchinovich A, Zgoda VG, Nikulin SV (2019). Participation of laminin α5-Chain in the regulation of colorectal cancer cell differentiation. Biotekhnologiya 35 (6), 3–11
  6. Shkurnikov M, Nikulin S, Nersisyan S, Poloznikov A, Zaidi S, Baranova A, Schumacher U, Wicklein D, Tonevitsky A (2019). LAMA4-Regulating miR-4274 and Its Host Gene SORCS2 Play a Role in IGFBP6-Dependent Effects on Phenotype of Basal-Like Breast Cancer. Front Mol Biosci 6, 122
  7. Maltseva DV, Raigorodskaya MP, Belyakova GA, Turchinovich AA (2019). Effect of endogenous expression of the laminin α5 chain on chemotherapy resistance of colorectal cancer cells. Biotekhnologiya 35 (5), 29–35
  8. Knyazeva EA, Knyazev EN, Gerasimenko TN, Kindeeva OV, Maltseva DV, Turchinovich A, Sergievich AA (2019). Laminin 521 alters the SNAI1, ZNF708 and GRN gene expression in BeWo b30 cells and creates physiological conditions for the placental barrier. Biotekhnologiya 35 (5), 87–93
  9. Raigorodskaya MP, Turchinovich A, Tsypina IM, Zgoda VG, Nikulin SV, Maltseva DV (2019). Laminin 521 Modulates the Сytotoxic Effect of 5-Fluorouracil on Colorectal Cancer HT29 Cells. Biotekhnologiya 35 (6), 73–79
  10. Knyazev EN, Nyushko KM, Alekseev BY, Samatov TR, Shkurnikov MY (2015). Suppression of ITGB4 Gene Expression in PC-3 Cells with Short Interfering RNA Induces Changes in the Expression of β-Integrins Associated with RGD-Receptors. Bull Exp Biol Med 159 (4), 541–545
  11. Maluchenko NV, Moisenovich MM, Egorova SG, Gusarova VY, Agapov II, Balashova TA, Tonevitskii AG (1999). Analysis of major antigen determinants of mistletoe lectin I. Biofizika 44 (6), 1020–1021

Transcription factor Foxp1 plays important role in Treg

Laboratory of molecular oncology

Regulatory T cells (Treg) is subpopulation of T helper lymphocytes which possesses immune suppressive properties. Treg are critical in protection from excessive immune response and autoimmunity. Unique functions of Treg are defined by transcription factor Fop3 that controls Treg-specific expression of genes. Dr. Yury Rubtsov from the Dept. of functioning of living systems in cooperation with colleagues from the Memorial Sloan-Kettering Cancer Center (New York City) studied a role of Foxp3-related protein, Foxp1, specifically in the murine Treg. In the absence of Foxp1, Treg displayed distorted Foxp3 chromatin binding which led to decreased sensitivity of the cells to IL-2 and diminished suppressive capacity. Results of the study are published in Nature Immunology.

Publications

  1. Konopacki C, Pritykin Y, Rubtsov Y, Leslie CS, Rudensky AY (2019). Transcription factor Foxp1 regulates Foxp3 chromatin binding and coordinates regulatory T cell function. Nat Immunol 20 (2), 232–242

Integrator is a key component of human telomerase RNA biogenesis

Laboratory of molecular oncology

Telomeres are special DNA-protein structures that are located at the ends of linear eukaryotic chromosomes. The telomere length determines the proliferation potential of cells. Telomerase is a key component of the telomere length maintenance system. While telomerase is inactive in the majority of somatic cells, its activity determines the clonogenic potential of stem cells as a resource for tissue and organism regeneration. Reactivation of telomerase occurs during the process of immortalization in the majority of cancer cells. Telomerase is a ribonucleoprotein that contains telomerase reverse transcriptase and telomerase RNA components. The RNA processing mechanism of telomerase involves exosome trimming or degradation of the primary precursor. Recent data provide evidence that the competition between the processing and decay of telomerase RNA may regulate the amount of RNA at the physiological level. We show that termination of human telomerase RNA transcription is dependent on its promoter, which engages with the multisubunit complex Integrator to interact with RNA polymerase II and terminate transcription of the human telomerase RNA gene followed by further processing.

Publications

  1. Rubtsova MP, Vasilkova DP, Moshareva MA, Malyavko AN, Meerson MB, Zatsepin TS, Naraykina YV, Beletsky AV, Ravin NV, Dontsova OA (2019). Integrator is a key component of human telomerase RNA biogenesis. Sci Rep 9 (1), 1701
  2. Konopacki C, Pritykin Y, Rubtsov Y, Leslie CS, Rudensky AY (2019). Transcription factor Foxp1 regulates Foxp3 chromatin binding and coordinates regulatory T cell function. Nat Immunol 20 (2), 232–242

Benzothiazole-based cyanines as fluorescent “light-up” probes for duplex and quadruplex DNA

The group of molecular tools for living system studies

Analogs of benzothiazole orange (BO) with one, two or three methylbenzothiazolylmethylidene substituents in the 1-methylpyridinium ring were obtained from the respective picolinium, lutidinium or collidinium salts. Fluorescence parameters of the known and new dyes in complexes with various DNA structures, including G-quadruplexes (G4s) and i-motifs (IMs), were analyzed. All dyes efficiently distinguished G4s and ss-DNA. The bi- and tri-substituted derivatives had basically similar distributions of relative fluorescence intensities. The mono-substituted derivatives exhibited enhanced sensitivity to parallel G4s. All dyes were particularly sensitive to a G4 structure with an additional duplex module (the thrombin-binding aptamer TBA31), presumably due to a distinctive binding mode (interaction with the junction between the two modules). In particular, BO showed a strong (160-fold) enhancement in fluorescence quantumyield in complex with TBA31 compared to the free dye. The fluorescence quantum yields of the 2,4-bisubstituted derivative in complex with well-characterized G4s from oncogene promoters were in the range of 0.04e0.28, i.e. comparable to those of ThT. The mono/bi-substituted derivatives should be considered as possible light-up probes for G4 formation.

Publications

  1. Turaev AV, Tsvetkov VB, Tankevich MV, Smirnov IP, Aralov AV, Pozmogova GE, Varizhuk AM (2019). Benzothiazole-based cyanines as fluorescent “light-up” probes for duplex and quadruplex DNA. Biochimie 162, 216–228

H3K36 Methylation and the Chromodomain Protein Eaf3 Are Required for Proper Cotranscriptional Spliceosome Assembly

Laboratory of membrane bioenergetics

Spliceosome assembly takes place in the context of the chromatin environment, suggesting that the state of the chromatin may affect splicing. The molecular details and mechanisms through which chromatin affects splicing, however, are still unclear. Here, we show a role for the histone methyltransferase Set2 and its histone modification, H3K36 methylation, in pre-mRNA splicing through high-throughput sequencing. Moreover, the effect of H3K36 methylation on pre-mRNA splicing is mediated through the chromodomain protein Eaf3. We find that Eaf3 is recruited to intron-containing genes and that Eaf3 interacts with the splicing factor Prp45. Eaf3 acts with Prp45 and Prp19 after formation of the precatalytic B complex around the time of splicing activation, thus revealing the step in splicing that is regulated by H3K36 methylation. These studies support a model whereby H3K36 facilitates recruitment of an “adapter protein” to support efficient, constitutive splicing. Leung et al. demonstrate that H3K36 trimethylation facilitates efficient pre-mRNA splicing through the association of chromodomain protein Eaf3. Eaf3 binds to methylated H3K36 at intron-containing genes to stabilize association of the splicing factor Prp45 and regulate proper cotranscriptional spliceosome assembly.

Publications

  1. Leung CS, Douglass SM, Morselli M, Obusan MB, Pavlyukov MS, Pellegrini M, Johnson TL (2019). H3K36 Methylation and the Chromodomain Protein Eaf3 Are Required for Proper Cotranscriptional Spliceosome Assembly. Cell Rep 27 (13), 3760–3769.e4

Fluorescent Yin-Yang probes for RT-qPCR detection of low copy HIV RNA

Laboratory of molecular design and synthesis

Nucleic acids labeled with a fluorophore/quencher pair are widely used as probes in biomedical research and molecular diagnostics. We synthesized novel DNA molecular beacons double labeled with the identical dyes (R6G, ROX and Cy5) at 5′- and 3′-end, studied their photophysical properties, and demonstrated that fluorescence quenching by formation of the homo dimer exciton in such molecular beacons allows using them in homogeneous assays. Further, we developed and evaluated homo Yin-Yang DNA probes labeled with identical dyes and used them for detection of low copy HIV RNA by RT-qPCR. They demonstrated improved sensitivity (LLQ: 10 vs 30 copies mL-1) in comparison to commercially available Abbott RealTime HIV-1 kit based on VIC-BHQ dyes both for model mixtures (naive human plasma with added deactivated HIV-1 virus) and for preliminarily confirmed 36 clinical samples (4 vs 1 positive ones for low-copy samples). The research was performed in collaboration with ScolTech, Research Institute of Epidemiology, and other institutions.

Publications

  1. (book) Kireev DE, Farzan VM, Shipulin GA, Korshun VA, Zatsepin TS (2020). RT-qPCR Detection of Low-Copy HIV RNA with Yin-Yang Probes. Methods Mol Biol 2063, 27–35
  2. Farzan VM, Kvach MV, Aparin IO, Kireev DE, Prikazchikova TA, Ustinov AV, Shmanai VV, Shipulin GA, Korshun VA, Zatsepin TS (2019). Novel homo Yin-Yang probes improve sensitivity in RT-qPCR detection of low copy HIV RNA. Talanta 194, 226–232

Antiviral compounds based on 5-(perylen-3-ylethynyl)uracil scaffold

Group of Cross-Linking Enzymes,  Laboratory of molecular design and synthesis

Rigid amphipathic fusion inhibitors (RAFIs) are potent antivirals based on a perylene core linked with a nucleoside moiety. Sugar-free analogues of RAFIs, 5-(perylen-3-ylethynyl)uracil-1-acetic acid and its amides were synthesized using combined protection group strategy. Compounds appeared to have low toxicity on porcine embryo kidney (PEK) or rhabdomiosarcoma (RD) cells together with remarkable activity against enveloped tick-borne encephalitis virus (TBEV): EC50 values vary from 0.077 μM to subnanomolar range. Surprisingly, 3-pivaloyloxymethyl (Pom) protected precursors showed even more pronounced activity. The propargylamide of N3-Pom-protected 5-(perylen-3-ylethynyl)uracil acetic acid, a universal precursor, was used in CuAAC click reaction for the synthesis of several derivatives including three ramified molecules with high activities against tick borne encephalitis virus (TBEV). Pentaerythritol-based polyazides were used for the assembly of molecules containing 2…4 antiviral 5-(perylen-3-ylethynyl)uracil scaffolds, the first examples of polyvalent perylene antivirals. Four prepared compounds, including one ramified cluster, showed remarkable 1…3 nM EC50 values against TBEV in cell culture. The results suggest that the carbohydrate moiety of RAFI nucleosides does not play a crucial role in their antiviral action, and biological activity of the 5-(perylen-3-ylethynyl)uracil scaffold can be effectively modulated by substituents in positions 1 and 3. The high antiviral activity of new compounds, coupled with low toxicity advocate their potential role in antiviral therapy. The antiviral evaluations were performed in Chumakov Institute of Poliomyelitis and Viral Encephalitides.

Molecular tools for stabilizing non-canonical nucleic acids secondary structures

The group of molecular tools for living system studies

Ligands and nucleic base modifications for stabilizing non-canonical nucleic acids secondary structures have been developed.

Publications

  1. Schönrath I, Tsvetkov VB, Zatsepin TS, Aralov AV, Müller J (2019). Silver(I)-mediated base pairing in parallel-stranded DNA involving the luminescent cytosine analog 1,3-diaza-2-oxophenoxazine. J Biol Inorg Chem 24 (5), 693–702
  2. (book) Zatsepin TS, Varizhuk AM, Dedkov VG, Shipulin GA, Aralov AV (2019). Oligonucleotide Primers with G8AE-Clamp Modifications for RT-qPCR Detection of the Low-Copy dsRNA. Methods Mol Biol 1973, 281–297
  3. Tsvetkov VB, Zatsepin TS, Turaev AV, Farzan VM, Pozmogova GE, Aralov AV, Varizhuk AM (2019). DNA i-Motifs With Guanidino-i-Clamp Residues: The Counterplay Between Kinetics and Thermodynamics and Implications for the Design of pH Sensors. Comput Struct Biotechnol J 17, 527–536

Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation

Laboratory of membrane bioenergetics

Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133+ PN signature, transition to a CD109+ MES subtype in a C/EBP-β-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133−/CD109+ cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM.

Publications

  1. Minata M, Audia A, Shi J, Lu S, Bernstock J, Pavlyukov MS, Das A, Kim SH, Shin YJ, Lee Y, Koo H, Snigdha K, Waghmare I, Guo X, Mohyeldin A, Gallego-Perez D, Wang J, Chen D, Cheng P, Mukheef F, Contreras M, Reyes JF, Vaillant B, Sulman EP, Cheng SY, Markert JM, Tannous BA, Lu X, Kango-Singh M, Lee LJ, Nam DH, Nakano I, Bhat KP (2019). Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation. Cell Rep 26 (7), 1893–1905.e7

New antiviral nucleoside derivatives for inhibiting the reproduction of varicella-zoster virus and tick-borne encephalitis virus

Laboratory of molecular design and synthesis,  The group of molecular tools for living system studies

A series of analogues of potent antiviral perylene nucleoside dUY11 with methylthiomethyl (MTM), azidomethyl (AZM) and HO-C1-4-alkyl-1,2,3-triazol-1,4-diyl groups at 3`-O-position as well as the two products of copper-free alkyne-azide cycloaddition of the AZM derivative were prepared and evaluated against tick-borne encephalitis virus (TBEV). Four compounds showed EC50 ≤10 nM, thus appearing the most potent TBEV inhibitors to date. Moreover, these nucleosides have higher lipophilicity (clogP) and increased solubility in aq. DMSO vs. parent compound dUY11.

Phenoxazine scaffold is widely used to stabilize nucleic acid duplexes, as a part of fluorescent probes for the study of nucleic acid structure, recognition, and metabolism etc. We present the synthesis of phenoxazine-based nucleoside derivatives and their antiviral activity against a panel of structurally diverse viruses: enveloped DNA herpesviruses varicella zoster virus (VZV) and human cytomegalovirus, enveloped RNA tick-borne encephalitis virus (TBEV), and non-enveloped RNA enteroviruses. Studied compounds were effective against DNA and RNA viruses reproduction in cell culture. 3-(2’-Deoxy-β-D-ribofuranosyl)-1,3-diaza-2-oxophenoxazine proved to be a potent inhibitor of VZV replication with superior activity against wild type than thymidine kinase deficient strains (EC50 0.06 and 10 µM, respectively). This compound did not show cytotoxicity on all the studied cell lines. Several compounds showed promising activity against TBEV (EC50 0.35-0.91 µM), but the activity was accompanied with pronounced cytotoxicity. These compounds may be considered as a good starting point for further structure optimization as antiherpesviral or antiflaviviral compounds.

Publications

  1. Proskurin GV, Orlov AA, Brylev VA, Kozlovskaya LI, Chistov AA, Karganova GG, Palyulin VA, Osolodkin DI, Korshun VA, Aralov AV (2018). 3′-O-Substituted 5-(perylen-3-ylethynyl)-2′-deoxyuridines as tick-borne encephalitis virus reproduction inhibitors. Eur J Med Chem 155, 77–83

Ultrasharp probes for biomolecules atomic force microscopy

Group of electron microscopy

During the last years electron microscopy group has been working on the efficient use of atomic force microscopy for visualization of biomacromolecules. Previously, we have determined empirically the conditions for obtaining nanostructures on the tips of standard atomic force microscope probes, which improve the resolution of the method by an order of magnitude (from 15-20 nm to ~ 2 nm). In 2018, we completed a study of the physical properties of these structures, explaining the growth mechanisms and efficiency in microscopy. According to the results of work in 2018, an article was published in the journal Ultramicroscopy, included in 1 quartile. In 2017, 2 papers were presented at conferences.

Synthesis takes place using the method of gas-phase chemical deposition, activated by plasma. Statistical analysis of electron microscopy images shows that the growth rate of branched nanostructures is about 100 nm / hour. The tip radius of the obtained structures is 1-2 nm. The results of the study of the probes obtained by the method of Raman scattering show that the branched structures consist of amorphous carbon, apparently, similar to glassy carbon, since they have high mechanical characteristics.

Publications

  1. Obraztsova EA, Basmanov DV, Barinov NA, Klinov DV (2018). Carbon Nanospikes: Synthesis, characterization and application for high resolution AFM. Ultramicroscopy 197, 11–15

The structure of the two components of the lipopeptide antibiotic crystallomycin from a sample obtained 60 years ago has been established. The identity of the components of two crystallomycin components to these of aspartocin (the structure of which has been elucidated recently) has been found. The antibiotic exhibits Ca2+ -dependent activity against gram-positive bacteria. The conformations of crystallomycin 2 in solution were investigated using NMR.

The amino acid 4-chloro-L-kinurenin, previously found in natural products only once, was found in the peptide antibiotic INA-5812. We first described the fluorescent properties of 4-chloro-L-kinurenin and its use as an energy donor for the excitation of other fluorophores.

The structure of two new macrolide antibiotics, astolides A and B, has been established using various 2D NMR techniques. Astolide molecules contain simultaneously a membrane-active polyol macrolide and a redox-active naphthoquinone residue as aglycones. The presence of a hydroxyl group at position 18 dramatically changes the spectrum of biological activity in comparison with the known analogues – antifungal activity increases and cytotoxicity reduces.

Publications

  1. Alferova VA, Shuvalov MV, Suchkova TA, Proskurin GV, Aparin IO, Rogozhin EA, Novikov RA, Solyev PN, Chistov AA, Ustinov AV, Tyurin AP, Korshun VA (2018). 4-Chloro-l-kynurenine as fluorescent amino acid in natural peptides. Amino Acids 50 (12), 1697–1705
  2. Alferova VA, Novikov RA, Bychkova OP, Rogozhin EA, Shuvalov MV, Prokhorenko IA, Sadykova VS, Kulko AB, Dezhenkova LG, Stepashkina EA, Efremov MA, Sineva ON, Kudryakova GK, Peregudov AS, Solyev PN, Tkachev YV, Fedorova GB, Terekhova LP, Tyurin AP, Trenin AS, Korshun VA (2018). Astolides A and B, antifungal and cytotoxic naphthoquinone-derived polyol macrolactones from Streptomyces hygroscopicus. Tetrahedron 74 (52), 7442–7449
  3. Jiang ZK, Tuo L, Huang DL, Osterman IA, Tyurin AP, Liu SW, Lukyanov DA, Sergiev PV, Dontsova OA, Korshun VA, Li FN, Sun CH (2018). Diversity, novelty, and antimicrobial activity of endophytic actinobacteria from mangrove plants in Beilun Estuary National Nature Reserve of Guangxi, China. Front Microbiol 9 (MAY), 868
  4. Tyurin AP, Alferova VA, Paramonov AS, Shuvalov MV, Malanicheva IA, Grammatikova NE, Solyev PN, Liu S, Sun C, Prokhorenko IA, Efimenko TA, Terekhova LP, Efremenkova OV, Shenkarev ZO, Korshun VA (2018). Crystallomycin revisited after 60 years: Aspartocins B and C. Medchemcomm 9 (4), 667–675

The role of protein regulators of splicing in the intercellular communication of tumor cells

Group of Cross-Linking Enzymes,  Laboratory of membrane bioenergetics

A novel mechanism of communication between tumor cells mediated by intercellular transport of spliceosomes has been demonstrated. Investigation of clinical samples from patients with ovarian cancer, as well as primary cultures of human glioblastoma cells, allowed us to observe that, upon induction of apoptosis, many tumor cells secrete into the extracellular environment various components of the spliceosome, a ribonucleoprotein complex that splices RNA molecules in eukaryotes. During the early stages of apoptosis the spliceosomes are transported from the nucleus to the cytoplasm, and then are secreted inside membrane vesicles (exosomes). These vesicles can be captured by neighboring tumor cells, and the splicing proteins contained therein affect mRNA splicing in the recipient cells. This leads to the formation of different protein isoforms and the acquisition of a more aggressive and treatment-resistant phenotype of surviving tumor cells. One of the key players of this process was identified as the RBM11 protein, which is readily transferred between glioblastoma cells and regulates alternative splicing of important oncogenes such as MDM4 and CyclinD1.