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Biochemistry (Moscow), Vol. 67, No. 6, 2002, pp. 706 709. Translated from Biokhimiya, Vol. 67, No. 6, 2002, pp. 850 854. Original Russian Text Copyright © 2002 by Spiridonova, Kopylov.

NEWS IN BIOCHEMISTRY

DNA Aptamers as Radically New Recognition Elements for Biosensors
V. A. Spiridonova1* and A. M. Kopylov
1

1,2

Belozersky Institute of Physico Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939 3181; E mail: spiridon@genebee.msu.su 2 School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia Received November 1, 2001

Abstract--A fiber optic biosensor based on DNA aptamers used as receptors was developed for the measurement of throm bin concentration. Anti thrombin DNA aptamers were immobilized on silica microspheres, placed inside microwells on the distal tip on an imaging optical fiber, coupled to a modified epifluorescence microscope through its proximal tip. Thrombin concentration is determined by a competitive binding assay using a fluorescein labeled competitor. The biosensor is selective and can be reused without any sensitivity change. The thrombin limit of detection is 1 nM, sample volume is 10 µl, and assay time per sample is 15 min including the regeneration step. Key words: DNA aptamers, thrombin, glass fiber, biosensor, SELEX

The practical application of fundamental knowledge about DNA has been rapidly grown over the last decade. A development of high technology analytical methods based on immobilized DNA is now an effectively pro gressing field. The main achievement of microarray tech nology, so called DNA chips [1 3], is the ability to use the diversity of DNA libraries, amplified by polymerase chain reaction (PCR) to produce DNA probes for rapid analysis of thousands of genes and their mutant forms [4, 5], DNA polymorphism [6], for gene discovery and mon itoring of gene expression [7 9], i.e., to perform a multi ple genome microanalysis using a hybridization method. DNA chips are based on DNA fragments with a length of tens to hundreds of nucleotides, immobilized on an activated solid support1. Using the PIN micromanip ulator the robot transfers less than 1 nl of DNA solution from the microtiter plate well onto a corresponding microchip cell, with a droplet diameter of 150 µm. This procedure is performed by a robot, and some thousands of DNA probes can be deposited on a support of a few cm2. Thus, every cell contains a certain part of the genet ic information, and the integration of the DNA microchips within a single platform allows construction of a chip array with a full package of the genetic informa tion, available for any identification purpose. The development of microarrays based on DNA aptamers used as receptors can be seen as a logical con
* To whom correspondence should be addressed. 1 For more details see www.biochip.ru

tinuation of the DNA chip technology development, although the principle of target recognition is not based on hybridization, but is analogous to the immunochemi cal assay. Aptamers are antibody analogs in terms of both specificity and affinity, with an apparent advantage of the former to be reproduced by automated chemical synthe sis. Aptamers are single stranded oligonucleotides with a length of tens of nucleotides, obtained by SELEX tech nology, and exhibiting high affinity and specificity towards any defined recognition target. Aptamers are selected by SELEX technology (Systematic Evolution of Ligands by Exponential enrichment), which is discussed in detail in a number of reviews [10 22]2. Single stranded DNA aptamers have a very highly ordered tertiary structure, which allows them to form sta ble and specific complexes with different targets such as proteins, nucleic acids, low molecular weight com pounds, etc. The search for the aptamers begins with a selection of "random sequence" DNA library. Libraries are obtained by automated DNA synthesis with a gradient attachment of synthons using a mixture of nucleotides instead of single components. Usually, a randomized region is 30 to 60 nucleotides, flanked on both sides with a special DNA sequence for PCR amplification. The diversity of individual oligonucleotides in the random DNA libraries can be as high as of 1015 1018 molecules. A
2

The first issue of Current Opinion in Chemical Biology (1997) is focused on combinatorial chemistry including SELEX (see [19]).

0006 2979/02/6706 0706$27.00 ©2002 MAIK "Nauka / Interperiodica"


DNA APTAMERS AS RADICALLY NEW RECOGNITION ELEMENTS FOR BIOSENSORS principle of SELEX technology for DNA aptamers, con sisting of several consecutive selection cycles, is shown in Fig. 1. The key step is DNA binding with an immobilized ligand, e.g., protein. During this step, oligonucleotides with an affinity towards the target compound are selected from the tremendous number of molecules in the library. Unbound DNA molecules are eluted from the column, while bound aptamers are isolated from the complex. DNA aptamers are amplified by PCR, and a single chain DNA is obtained from the resulting DNA duplex using an asymmetric PCR, and then is used for a next selection cycle. Aptamers with a low affinity and specificity towards the target are displaced by that ones with high affinity and specificity in the following selection cycles. Basically, the DNA fraction obtained after several selection cycles is a mixture of aptamers with various affinities towards the target, but individual aptamers can be isolated by cloning and sequencing of the fraction. DNA molecules matching by the primary structure are identified among the isolated aptamers and used for the quantitative estimation of the aptamer affinity for the target. The stability of the com plexes is characterized by the apparent dissociation con stants (Kd). These values for the aptamer­protein com plexes vary within the 1 100 nM range, which is of simi lar affinity and specificity range as for antibody­antigen complexes. DNA aptamers towards the serine protease throm bin, which is a key enzyme involved in the blood coagula tion mechanism [23], were obtained [24 26] using SELEX technology. It should be mentioned that since thrombin is not a DNA binding protein, the possibility to elicit thrombin­aptamer complexes clearly demonstrates the power of SELEX technology. Bock et al. [24] have obtained a family of DNA aptamers capable of inhibiting thrombus formation in vitro. Primary structures of all isolated aptamers con tained a unique pentanucleotide motif with a characteris tic distribution of guanine residues, engaged in formation of G quartet structures similar to those found at the ends of telomeric DNA (PDB 156D). Macaya et al. [25] and Tsiang et al. [26] (see also [27, 28]) obtained other throm bin specific DNA aptamers that had more sophisticated architecture of G quartets with duplexes and additional hairpin loops and were able to form more stable complex es with thrombin. Values of apparent dissociation con stants for thrombin­aptamer complexes were within the nM to µM range. The tertiary structure of thrombin alone was deter mined by X ray analysis (PDB 1JOU). The structure of DNA aptamer was determined by NMR, revealing two G tetrads connected through two TT and one TGT loops (PDB 148D, see also [28]). The model of thrombin­aptamer complex was based on DNA­protein cross linking data (Fig. 2). Since they were first discovered, DNA aptamers have been considered for potential use as therapeutic agents
BIOCHEMISTRY (Moscow) Vol. 67 No. 6 2002
COMBINATORY DNA LIBRARY Binding Polymerase chain reaction (PCR) COLUMN WITH IMMOBI LIZED LIGAND

707

BOUND DNA MOLECULES

Elution

NON BOUND DNA MOLECULES

Fig. 1. The principle of SELEX technology for DNA aptamers is based on a number of repetitive binding cycles of the DNA library containing a random sequence to a target compound.

[20, 29, 30], and only recently they also have been adopt ed as new recognition elements for biosensors [31] and flow cytometry (see references in [20]). The first report on this application was published by Lee and Walt from Tafts University USA [32]. They used anti thrombin DNA aptamers provided by E. Ellington (Center for Aptamer Research, Indiana University, USA), one of the leaders in SELEX technology development. Aptamers were covalently immobilized on the surface of non porous silica microspheres of 3 µm diameter, according to Ferguson et al. [33]. A six carbon atom spacer with an active end amino group was used to modify the 5 end of 15 mer DNA aptamer GGTTGGTGTGGTTGG, and the resulting derivative was activated by cyanuric chlo ride. Hydrophilic microspheres with surface silanol group (Si OH) [34] displayed a low degree of nonspecific hydrophobic thrombin binding. Microspheres were first reacted with (3 aminopropyl)triethoxysilane, then with glutaraldehyde and polyethylenimine (PEI). PEI ensures high bead capacity and provides a spacer arm, which in combination with six carbon atom spacer enhances the steric accessibility of immobilized aptamer to thrombin. Microbeads with immobilized DNA aptamers were placed into microwells (3 µm diameter) that were fabri cated by etching of the distal tip of the imaging fiber (500 µm diameter) with hydrofluoric acid. Microwell vol ume was 10 µl. The proximal tip of the optical imaging fiber was coupled to a modified extension of epifluores cence microscope [35]. A solution of fluorescein labeled thrombin (3000 NIH units/mg, Sigma, USA) was pipet ted onto the microwells of the image fiber with embedded microspheres. Fluorescein labeled thrombin (F throm bin) was synthesized by a reaction of thrombin with fluo rescein succinimide. F thrombin was selectively bound by the microspheres with immobilized aptamers, and light emitted by a fluorescent label was registered by the


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SPIRIDONOVA, KOPYLOV thrombin to the blank microspheres was below 5%. F thrombin limit of detection was 10 nM. Specificity of aptamer coated beads for F thrombin binding was assessed with bovine serum albumin. The most convenient assay format of thrombin analysis for routine medical purposes is, however, a com petitive binding assay. Optimal F thrombin concentration for thrombin analysis was found to be 200 nM. The method exhibited good results over nM to µM thrombin concentration range, thus matching the thrombin physi ological concentration range in blood plasma, which is within 5 500 NIH units/ml [36, 37]. Hence, the method can be applied for thrombin quantification in real world blood samples. Assay time per sample is 15 min including the regeneration step: thrombin binding by the aptamer microbeads was complete in 7 8 min, and the regenera tion was performed in 5 6 min. No change of biosensor parameters was detected over 8 h operation time. The biosensor can be reused many times without loss of sensi tivity. Storage stability of the microspheres with immobi lized aptamers was over 3 months. Competitive F thrombin binding assay is analogous to traditional Enzyme Linked Immunosorbent Assay (ELISA) and can be performed using standard ELISA microtiter plates [38]. Moreover, the present investigation demonstrates new possibilities for multi analyte detec tion, which is governed entirely by the affinity and speci ficity of the DNA aptamers1. Authors would like to thank Professor S. M. Strukova for useful discussions and senior scientist I. N. Kurochkin for a careful reading of the manuscript. Financial support from the projects funded by the Russian Foundation for Basic Research (grant Nos. 01 04 48603, 01 04 48643, 99 04 39072), Ministry of Science (grant No. 415/99), and Universities of Russia (grant No. 99 1700) is greatly acknowledged.

Fig. 2. Model of the tertiary structure for thrombin (PDB 1JOU)­aptamer DNA (PDB 148D) complex. An arrow shows the contact determined by the chemical cross linking method [27].

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DNA APTAMERS AS RADICALLY NEW RECOGNITION ELEMENTS FOR BIOSENSORS
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