Selection and analytical applications of aptamers Camille L.A. Hamula, Jeffrey W. Guthrie, Hongquan Zhang, Xing-Fang Li, X. Chris Le Advances in systematic evolution of ligands by exponential enrichment (SELEX), a selection protocol for aptamers, have resulted in increased appl- ications of DNA and RNA aptamers in developing analytical techniques. We review recent developments in SELEX techniques as well as new aptamer- based bioanalytical applications. ª 2006 Elsevier Ltd. All rights reserved. Keywords: Affinity binding; Aptamer; Bioanalytical assay; Biosensor; Chiral separation; In vitro selection; Protein; SELEX 1. Introduction Nucleic acids possess desirable properties for use in analytical applications. Con- ventionally, they are used as probes that hybridize with and detect complementary target RNA and DNA sequences. In the last decade, short synthetic DNA and RNA sequences known as aptamers have been used as ligands to bind to non-nucleic acid targets with high specificity and affinity. Dissociation constants for typical aptamer binding are in the micromolar to low picomolar range [1,2]. This binding is not through sequence hybridization but through binding of stable, complex stem- loop and internal loop structures formed by the nucleic acids [1]. Aptamers are commonly evolved in vitro via a combinatorial chemistry technique known as systematic evolution of ligands by exponential enrichment (SELEX) [3]. They can be selected using a variety of targets from small molecules to whole organisms [4]. The ability to synthesize aptamers for a variety of targets has con- tributed to a wide range of analyses using aptamers. Another factor allowing the widespread application of aptamers to bioanalysis is that aptamers offer benefits for analytical applications when used in place of anti- bodies [5]. These benefits derive mainly from temperature stability and ease of aptamer production. Many of these analytical applications of aptamers, such as those based on chromatography, mass spectrometry, molecular beacons, and some label-free biosensors, have been reviewed previously [6–9], and will not be discussed further. This review focuses on recent develop- ments in SELEX analytical applications, as well as variations in SELEX techniques and the ramifications of such variations for aptamer characteristics. 2. SELEX techniques Fig. 1 shows the basic principle of most SELEX procedures [10]. Attempts to improve SELEX have led to the develop- ment of different techniques, delineated by variations in starting library, target molecule, and experimental protocol. These variations result in differences in aptamer affinity and specificity as well as selection efficiency. 2.1. Libraries A typical SELEX library comprises single- stranded RNA or DNA, in which a central region of randomized sequence is flanked on either side by fixed primer sequences for PCR and/or RT-PCR amplification. The first SELEX procedures used randomized single-stranded RNA libraries, in which the random sequence region was 100 nucleotides (nt) in length [1] and 8 nt [3], respectively. Since then, many other SELEX procedures have been carried out using randomized ssRNA or ssDNA libraries, typically containing 10 13 –10 15 different randomized sequences [11]. Although any length of randomized region Camille L.A. Hamula 1 , Jeffrey W. Guthrie 1 , Hongquan Zhang, Xing-Fang Li, X. Chris Le* Environmental Health Sciences, Department of Public Health Sciences and Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G3 1 These two authors contributed equally. *Corresponding author. Tel.: +1 780 492 6416; Fax: +1 780 492 7800 E-mail: xc.le@ualberta.ca Trends in Analytical Chemistry, Vol. 25, No. 7, 2006 Trends 0165-9936/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.05.007 681 0165-9936/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.05.007 681