**1. Introduction**

Nucleic acid aptamers are small single-stranded oligonucleotides capable of adopting complex tertiary structures that allow noncovalent interactions with other molecules. Because aptamers closely interact with their targets, their structural features are essential for highly specific binding. The term *aptamer* was coined in 1990 from the Latin "aptus" meaning "fitting" and

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the Greek "meros" meaning "particle" [1]. Aptamers are generated through the iterative *in vitro* selection method called SELEX (systematic evolution of ligands by exponential enrich‐ ment) and are raised against a wide variety of molecular targets ranging from ions and macromolecules to whole organisms, including viruses, bacteria, yeast and mammalian cells [2]. Although many modifications to the SELEX method have been established to include new technologies and improve selection [3], the basic steps of SELEX remain immutable.

The SELEX method involves three well-defined steps [4]: the start point is the production of a synthetic oligonucleotide combinatorial library or oligonucleotide pool containing a central randomized region (15–70 nt) flanked by anchor sequences to allow polymerase chain reaction (PCR) amplification. The aleatory nature of the central region results in the production of an enormous pool of diverse oligonucleotides with diverse structures, thus providing the conformational variability necessary to produce moieties with binding capabilities for a desired target. The oligonucleotide pool can be directly used for SELEX to generate singlestranded DNA (ssDNA) aptamers, or as *in vitro* transcription template to produce an RNA pool to isolate RNA aptamers. Next, a selection procedure is performed based on the interac‐ tion properties of the library with the intended target. Only a very small fraction of the oligonucleotide pool tends to interact with the target, satisfying the selection criteria. Oligo‐ nucleotides that bind the target (aptamers) are recovered while the nonbound are removed through different strategies according to the nature of the aptamer–ligand complex (size, affinity, electric charge, hydrophobicity, etc.). In the final step, the recovered aptamers are amplified by PCR in order to regenerate a library with less variability but more affinity to the target that will be used in the next selection cycle. RNA pools are amplified by reverse transcription-coupled PCR (RT-PCR) and subsequent *in vitro* transcription before starting the next cycle.

The iterative selection cycles produce aptamers with high binding affinity to the target. Usually, a few cycles are required to isolate aptamers (4–20 cycles), but the precise number of cycles necessary for the isolation of highly specific aptamers depends on the selection criteria, the nature of the target and the type of library used. After the last selection cycle, aptamers are cloned and sequenced to obtain information on the individual oligonucleotides, which can be further characterized based on its ability to bind the target. It is common to observe conserved sequences or structures among the selected aptamers; these are indicative of efficient selection and may represent domains required for interaction.

Aptamer specificity is based on three-dimensional arrangements of a small number of contact points between the aptamer and its target, so the aptamer can achieve high selectivity to discriminate between two highly related molecules (i.e. enantiomers), or minimal structural differences such as the presence or absence of methyl or hydroxyl groups. The molecular recognition specificity and affinity level achieved by aptamers is comparable or even better than those of antibodies. These features place aptamers as an emerging class of molecules on their own with a huge range of diagnostic and therapeutic applications plus several advantages over antibodies including:

**•** Isolation by an *in vitro* process not dependent on animal cells or *in vivo* conditions. Therefore, the properties of aptamers can change on demand, and isolation can be manipulated to obtain aptamers with desirable properties for diagnosis. In addition, it allows aptamer isolation against toxins or poorly immunogenic molecules.


Since the development of SELEX, aptamers have been isolated against a wide diversity of targets such as amino acids [5, 6], antibiotics [7], nucleotides [8], enzymes [9], growth factors [10], mammalian cells [11], bacteria [12] and parasites [13]. Nowadays, some aptamers have even reached therapeutic applications in the clinic [14]. Furthermore, the first RNA aptamer for therapeutic purposes in humans (pegaptanib sodium or Macugen®) was approved by United States Food and Drug Administration (FDA) in 2004, as treatment for age-related macular degeneration (AMD) [15].
