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Bioelectrochemistry 5676 (2002) xxx xxx www.elsevier.com/locate/bioelechem

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Shor t communication

V. Spiridonova*, T. Rassokhin, A. Golovin, E. Petrova, T. Rohzdestvensky, Yu. Pakhomova, A. Kopylov
A.N. Belozersky Institute of Physico-Chemical Biolog, Laboratory Building ``A'', and Chemistry Department, Moscow State University, Vorobyevy Gory, 119899 Moscow, Russian Federation Received 1 June 2001; received in revised form 14 November 2001; accepted 19 November 2001

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Abstract

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In recent years, Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique has been developed into a fast growing field. In contrast to traditional recognition elements, like antibody, our interests focus on novel molecular recognition elements based on nucleic acids, which are of value both for the therapy and biosensors. A comparative study of thermodynamic for both natural and artificial RNA/DNA protein complexes would establish bases for a specificity of complex formation. In particular, we have shown that aptamers could be used for a direct measuring of thrombin enzymatic activity in a solution. D 2002 Published by Elsevier Science B.V.

Keywords: RNA/DNA protein interactions; Ribosome; SELEX; RNA/DNA aptamers; Thrombin; Enzymatic activity

1. Introduction

Wide-scale molecule testing requires a development of fast and easy-to-use devices, like biosensor. The devices intimately couple a biological recognition element, interacting with a target analyte, with a physical transducer that translated the bio-recognition event into a useful signal. In contrast to traditional recognition elements, like antibody, our interests focus on novel molecular recognition elements based on nucleic acids, which are of value for biosensors. The unique network structure of single-stranded DNA and RNA provides a ground for affinity and specificity; and recognition is a basic principle of DNA / R NA protein interactions. The parameters of these interactions could be described in terms of thermodynamic, i.e. dissociation constant (Kd). A comparative study of thermodynamic for both natural and artificial R NA / DNA-protein complexes would establish bases for a specificity of complex formation. Aptamer is a single-stranded oligonucleotide that could specifically bind with high affinity and specificity to a selected target molecule, for example drug, protein, etc. Aptamers are discovered by in vitro selection process known as Sys-

tematic Evolution of Ligands by EXponential enrichment (SELEX) [1,2]. Generally, SELEX-derived aptamers could be considered as functional analogues of monoclonal antibodies. A huge library of single-stranded oligonucleotides, differing in nucleotide sequence, represents an array of differently shaped molecules with different affinities for a different areas of a given target protein. An initial template is obtained by automatic chemical synthesis of DNA fragments, and each has a random sequence of 30 60 nt. Several rounds of selection yield a fraction enriched in aptamers, with relative binding several orders of magnitude higher compared to the initial library. Finally, an aptamer pool is cloned, individual aptamers are sequenced, and their affinity for a ligand estimated, providing data for choosing the best aptamers (``winners''). This paper describes a power of DNA aptamer to be used for a direct measurement of thrombin enzymatic activity in solution.

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A comparative ther modynamic st udy for both nat ural and ar tif icial R NA/DNA protein binar y complexes

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2. Experimental Aptamer 5 V-CA G T C CGT GGTAG GGCAG GT TG G GGTGACT-3V was synthesized on an Applied Biosystems 380B oligonucleotide synthesizer by phosphoroamidate method.

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*

Corresponding author. Tel.: +7-095-939-3149; fax: +7-095-939-3181. E-mail address: Spiridon@genebee.msu.su (V. Spiridonova).

1567-5394/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 1567-5394(02)00028-2

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concentration is far greater than that of titrated DNA aptamer.

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3. Results and discussion As an example of natural RNA protein interaction study, the complexes of prokaryotic ribosomal protein S7 with different RNA has been chosen. In vitro EcoS7 is able to bind to both Escherichia coli 16S rRNA fragment and E. coli S12 S7 intercistronic region. It allows to study a property of a single protein to recognize two different RNA, by measuring apparent dissociation constants (Kd) by nitrocellulose (NC) filter-binding assay. The EcoS7 16S rRNA fragment

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Fig. 1. A secondary structure of DNA aptamer (A), and a tertiary structure model of the aptamer thrombin complex (B) [8].

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Human a-thrombin ``Fluka'' (Switzerland) had 600 NIH U/mg, a-human thrombin ``Renam'' (Russia) had 4500 NIH U/mg. Binding assays were carried out by nitrocellulose filter partitioning [8]. Buffer was 20 mM Tris Ac pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2. The dissociation constant is defined as the protein concentration at a half value of isotherm flattening, provided that protein

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Fig. 2. Isotherms of thrombin binding to the DNA aptamer under different conditions. Complex formation was done at 37 °C for 15 min. Circles and triangles graph -- thrombin activity 4500 NIH U/mg, squares graph-- thrombin activity 600 NIH U/mg. 20 mM Tris Ac, pH 7,6; 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1mM CaCl2. 20 mM Tris Ac, pH 7,6; 100 mM NaCl, 1 mM MgCl2.

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complex has Kd = 6.5 F 1.7 nM. TthS7 is able to bind to Eco16S rRNA with Kd =36 F 6 nM, which is high enough for a heterologous complex. SELEX technique, applied to S12 S7 intercistronic fragment (67 ns) of str mRNA [3], has yielded aptamers to TthS7 with nucleotide sequences different from the natural variant. The aptamer has Kd =50 F 8 nM, which is very close to the value of heterologous complex. The results of the structural thermodynamic study of the natural RNA protein complexes provide a solid base to extend the approach to artificial complexes. Thrombin is a multifunctional serine protease with both pro-coagulant and anti-coagulant functions, therefore has a high medical significance [4]. The number of authors selected the different families of DNA aptamers for thrombin [5 8]. One of the best aptamers is 5V-CAGTCCGTGGTAGGGCAGGTTGGGGTGACT3V, which forms two G-quartets with an additional DNA duplex (Fig. 1) [8]. The aptamer binds to the protein with Kd = 0.5 nM. Fig. 2A shows the aptamer binding isotherms for thrombin, obtained by NC filter binding assay. The buffer has the same salt components as blood: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2. Two different samples of thrombin have been used: from ``Fluka'' -- 600 NIH U/mg, and from ``Renam'' (Russia) -- 4.500 NIH U/mg. It turned out that DNA aptamer binding to thrombin with high enzymatic specific activity is 20 times higher than Fluka thrombin. Therefore, it has been clearly demonstrated that the values of Kd depend on enzymatic activity of thrombin and not on total protein concentration. The same correlation was found for some different buffer conditions (Fig. 2B), despite the absence of K+, which stimulates G-quartet assembly of the aptamer. Therefore, the DNA aptamer could be used for a direct measuring of thrombin enzymatic activity in solution. When the manuscript has been already applied we have learned that Lee and Walt [9] have applied DNA aptamer for fiber-optic biosensor for thrombin.

Acknowledgements The work is supported by grants RFBR 01-04-48603, RFBR-NSFC 99-04-39072, Ministry of Science and technology 415/19, University of Russia 991700, Moscow Government 1.1.125.

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References

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