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Structure and Interaction of Biomolecules at Surfaces

Doc. RNDr. Miroslav Fojta, CSc.
Research Group Leader

THEMATIC RESEARCH FOCUS

RESEARCH AREAS

  • Interactions of natural and chemically modifi ed biopolymers with electrodes, relations between biopolymer structure and its electrochemical, interfacial and electrocatalytic properties
  • Novel techniques of biopolymer labelling
  • Effects of DNA and/or protein chemical modifi cation on the biopolymer molecular recognition features
  • Development of novel bioanalytical/bioelectroanalytical and diagnostic tools

MAIN OBJECTIVES

  • Investigations of the behaviour of natural and chemically modifi ed biomacromolecules at electrically charged surfaces linked to the development of novel electrochemical biosensors and bioassays.
  • Development of novel electrochemical biosensors, bioassays and diagnostic approaches.

CONTENT OF RESEARCH

Nucleic acids and proteins are electrochemically active species due to the presence of reducible or oxidisable moieties (nucleobases, side groups of some amino acids). In addition to intrinsic biopolymer responses, electroactive markers have been utilised to obtain reversible electrode processes at less extreme potentials than those produced by DNA or protein itself. Electrochemical detection has been utilised for various analytical applications, including detection of DNA damage, sequence-specifi c DNA sensing, probing DNA interactions with drugs, etc. Considerable progress has also been achieved in the development of novel ways of working (detection) electrodes applicable in the area of biopolymer sensing. Recent literature refl ects a remarkable boom in the development of electrochemical biosensors and bioassays, with a considerable contribution by the group. However, in contrast to a huge number of studies utilising simple (e.g. oligonucleotide) models for the development of DNA sensors and testing their performance, studies demonstrating application of electrochemical sensing on more “real” systems (such as large DNA or protein molecules) are still rather scarce.

Interactions of nucleic acids, proteins and their components with/at electrically charged surfaces will be studied in detail with respect to nucleotide/aminoacid composition/sequence, secondary, tertiary or higher-order structures. We will focus on elucidating the mechanisms of structural changes in the surface-confi ned biopolymers (DNA unwinding, the formation of non-B DNA structures, protein folding/unfolding) on applying an external electric fi eld, as well as on the characterisation of the surface structure and electric fi eld eff ects on the interaction between a surface-confi ned molecule and its counterpart in solution.

Novel techniques of biopolymer labelling will be developed to design novel bioanalytical and diagnostic tools. We will use various techniques, including enzymatic incorporation of labelled nucleotides into nucleic acids using enzymes and direct chemical modification of natural nucleic acids and their synthetic analogues using oxoosmium complexes. Modified nucleic acids will be studied in detail concerning the effects of sitespecific, terminal or global modification on their molecular recognition features (such as DNA hybridisation, mismatch sensitivity, protein-DNA recognition). Sensitive electrochemical or other detection techniques will be developed using label-specific analytical signals.

KEY RESEARCH EQUIPMENT

PLANNED RESEARCH INFRASTRUCTURE

Technology Units

  • Modified nucleic acids in biosensing
  • Joint laboratory of bioelectrochemistry and electrochemical biosensors

CURRENT RESEARCH INFRASTRUCTURE

The research group is currently equipped with or has access to standard facilities for bacterial and cell culturing, nucleic and protein isolation, purification and characterisation, performing biochemical studies of DNA structure and DNA-protein interactions (cultivators, biohazard box, deep freezer, centrifuges, FPLC, electrophoreses, phosphorimager, documentation systems, PCR cycler, etc.). Currently available equipment for electrochemical analysis and surface characterisation (multimode electrochemical analyzers, electrode systems) needs upgrade and expansion to allow expansion of the group’s research within CEITEC.

MAIN PROJECTS

  • DNA labeling with redox markers for electrochemical sensing. Applications in analysis of nucleotide sequences and molecular diagnostics (IAA400040901), Academy of Sciences of the Czech Republic, 2009-2013, M. Fojta, Institute of Biophysics AS CR, M. Hocek, Institute of Organic Chemistry and Biochemistry AS CR.
  • Construction of novel functional nucleic acids for applications in chemical biology, catalysis and self assembly (GA203/09/0317), Czech Science Foundation, 2009-2013, M. Hocek, Institute of Organic Chemistry and Biochemistry AS CR, M. Fojta, Institute of Biophysics AS CR.
  • Novel electrochemical sensors and sensing techniques for the analysis of nucleic acids structure and interactions (GAP206/11/1638), Czech Science Foundation, 2011-2015, M. Fojta, Institute of Biophysics AS CR, B. Yosypchuk, J. Heyrovsky Institute of Physical Chemistry AS CR.
  • Centre of Biophysical Chemistry, Bioelectrochemistry and Bioanalysis. New Tools for Genomics, Proteomics and Biomedicine (LC06035), Ministry of Education, Youth and Sports, 2006-2011, M. Fojta, Institute of Biophysics AS CR, J. Barek, Charles University in Prague, J. Pazourek, Masaryk University, K. Vytřas, University of Pardubice, B. Vojtěšek, Masaryk Memorial Cancer Institute.

SELECTED PUBLICATIONS

  • PIVONKOVA, H., HORAKOVA, P., FOJTOVA, M., FOJTA, M. Direct Voltammetric Analysis of DNA Modifi ed with Enzymatically Incorporated 7-Deazapurines. Anal. Chem. 2010, 82(16), p. 6807-6813.
  • VRABEL, M., HORAKOVA, P., PIVONKOVA, H., KALACHOVA, L., CERNOCKA, H., CAHOVA, H., POHL, R., SEBEST, P., HAVRAN, L., HOCEK, M., FOJTA, M. Base-Modifi ed DNA Labeled by [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) Complexes: Construction by Polymerase Incorporation of Modifi ed Nucleoside Triphosphates, Electrochemical and Luminescent Properties, and Applications. Chem. Eur. J. 2009, 15(5), p. 1144-1154.
  • FOJTA, M., BILLOVA, S., HAVRAN, L., PIVONKOVA, H., CERNOCKA, H., HORAKOVA, P., PALECEK, E. Osmium tetroxide, 2,2 ‚-bipyridine: Electroactive marker for probing accessibility of tryptophan residues in proteins. Anal. Chem. 2008, 80(12), p. 4598-4605.
  • CAHOVA, H., HAVRAN, L., BRAZDILOVA, P., PIVONKOVA, H., POHL, R., FOJTA, M., HOCEK, M. Aminophenyl- and nitrophenyl-labeled nucleoside triphosphates: Synthesis, enzymatic incorporation, and electrochemical detection. Angewandte Chemie-international edition. 2008, 47(11), p. 2059-2062.
  • FOJTA, M., KOSTECKA, P., TREFULKA, M., HAVRAN, L., PALECEK, E. Multicolor electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes. Anal. Chem. 2007, 79(3), p. 1022-1029.
 

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