The group of molecular tools for living system studies

Department of functioning of living systems

Head: Andrey Aralov, Ph.D.
Baruh238@mail.ru+7(926)6062910

RNA, DNA, G-quadruplex, i-motif, antisense oligonucleotides, probes, pH sensors, modification, fluorescence

The main research areas of the group include:

  • design and synthesis of nucleotide modifications for fine tuning the properties of canonical and non-canonical secondary nucleic acids structures;
  • development of nucleotide modifications suitable for antisense and RNAi technologies for regulating gene expression;
  • synthesis and screening of probes/ligands for detecting and fine tuning physicochemical parameters of noncanonical secondary nucleic acids structures; 
  • development and study of the properties of metal-binding modifications within oligonucleotides;
  • study of the physicochemical properties of modifications that arise in living organisms when exposed to mutagenic factors; 
  • design and synthesis of antiviral compounds.

All publications (show selected)

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Andrey Aralov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 51, office. 454
  • Phone: +7(926)6062910
  • E-mail: Baruh238@mail.ru

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

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

In collaboration with Laboratory of ligand-receptor interactions

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

In collaboration with Group of chemistry of heterocyclic compounds

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.

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

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

Molecular tools for stabilizing non-canonical nucleic acids secondary structures

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

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

In collaboration with Laboratory of molecular design and synthesis

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