A comparative thermodynamic study for both natural and artificial RNA/DNA–protein binary complexes

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Abstract

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.

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/RNA–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 RNA/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 Systematic 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.

Section snippets

Experimental

Aptamer 5′-CAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′ was synthesized on an Applied Biosystems 380B oligonucleotide synthesizer by phosphoroamidate method.

Human α-thrombin “Fluka” (Switzerland) had 600 NIH U/mg, α-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

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 complex has Kd=6.5±1.7 nM. TthS7 is

Acknowledgments

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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  • Label-free impedimetric thrombin sensor based on poly(pyrrole-nitrilotriacetic acid)-aptamer film

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    Citation Excerpt :

    After incubation for 30 min, the electrode was rinsed for 10 min with phosphate buffer under stirring and then incubated for 40 min with 20 μL of various concentrations of thrombin in 0.1 mol L−1 phosphate buffer (pH 7.4) containing 0.01 mol L−1 NaCl, 5×10−3 mol L−1 KCl, 1×10−3 mol L−1 MgCl2 (PBS). K+ ions were added in binding solution for stimulating the G–quartet assembly of aptamer (Spiridonova et al., 2002). The resulting electrodes are then washed under vigorous stirring for 10 min.

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