1
Introduction of SELEX and Important SELEX Variants

Yiyang Dong1, Zhuo Wang2, Sai Wang3, Yehui Wu2, Yufan Ma2 and Jiahui Liu1

1 Beijing University of Chemical Technology, College of Life Science and Technology, Beijing, 100029, PR China

2 Beijing University of Chemical Technology, State Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Science, Beijing, 100029, PR China

3 Ocean University of China, College of Food Science and Engineering, Qingdao, 266003, PR China

1.1 SELEX

In 1990, from a randomly synthesized nucleic acid library composed of more than 1015 different sequences [1, 2], two laboratories led by Larry Gold and Jack William Szostak invented independently a technique for selection of aptamers or single-stranded functional oligonucleotides (DNA or RNA), which shows high affinity to their respective targets. As shown in Figure 1.1, the aptamers are developed by repeated selection and amplification processes [3]. This in vitro oligonucleotides selection scheme is termed as (Systematic Evolution of Ligands by EXponential Enrichment), and has become a general and powerful method for the discovery and isolation of nucleic acid aptamers for a rich variety of analytical applications.

Figure 1.1 Schematic drawing of SELEX procedures.

Source: Dong et al. 2013 [3]. Reprinted with permission of Taylor and Francis.

Mechanistically, the SELEX process starts with a single-stranded deoxyribonucleic acid () library generated by solid-phase synthesis using traditional phosphoramidite method, the library comprised of random sequences at the center flanked by defined primer binding sites at each 5´ and 3´ termini. The variety of the ssDNA library relies on the length of the random region. Around 1015 different sequences are contained in an initial ssDNA pool, which makes it appropriate for the existence of sequences specific for a target. Actually, the entire aptamer sequence library has only a short fraction that binds effectively to the target, which suggests that aptamer screening can be eventually accomplished within a short period of time [4]. It is noteworthy that long random sequences can provide higher structural complexity, which is vital in isolating aptamers with high affinity [5]. In an early stage of aptamer selection, RNA libraries are widely used due to the fact that RNA can easily fold into complex 3D structures which show higher affinity to the targets. However, RNA aptamers are much more expensive than DNA aptamers, and they are relatively difficult to be modified in SELEX cycles [6]. Furthermore, stems and loops can be generated when ssDNA folds into a 3D configuration [7]. So DNA libraries are used more frequently now.

The essential steps of a typical SELEX process include binding, partitioning/eluting, amplification, and identification. Normally, due to limited resolution and efficiency, many cycles of SELEX rounds need to be carried out to obtain a good result. In the first step, a random DNA or RNA library is incubated with the target to ensure definite binding of some oligonucleotides with the target, while others will be removed in the following steps. In the partitioning/eluting step, unbound oligonucleotides can be removed on the basis of the different molecular weights of nucleotide-target complexes and nucleic acids. For example, a chromatographic column packed with the target-immobilized beads can be applied for the separation of oligonucleotides binding with the target molecules. Besides chromatography, other methods can also be used to separate unbound, weakly bound, or elute bound oligonucleotides, for example, filtration, heating, the change of ionic strength or pH, the addition of denaturing substances such as urea, sodium dodecyl sulfate (), or ethylenediaminetetraacetic acid [8-10]. In this step, target molecules are interacted with either free nucleic acid or separable nucleic acid immobilized on a certain substrate [11]. However, it is difficult to elute strongly bound oligonucleotides from targets, which may restrict the isolation of the aptamers with extremely high affinity. Because of this, high affinity of aptamers are commonly obtained by SELEX with free-form target molecules [12, 13]. In some cases, it is difficult to remove unbound oligonucleotides from the free target-oligonucleotide complex. So, low-affinity targets are appropriate for SELEX with free-form target molecules. After partitioning/eluting, the next step is to amplify the bound oligonucleotides by (polymerase chain reaction) with primers for aptamer screening. The amplification will generate a new population of oligonucleotides for the next round of SELEX. Commonly, 10-20 amplifications are needed [14]. After repeated cycles of selection and amplification, the nucleotide pool becomes enriched, while the affinity between nucleotide and aptamers becomes higher because low- or no-affinity oligonucleotides are removed in previous cycles. The progress of SELEX can be monitored by the quantification of target-bound oligonucleotides among the pools of incubated nucleotides at each round of SELEX [15]. The selection is stopped when oligonucleotides bound to the target are fully dominant in the pool of oligonucleotides or significant enhancement of target-bound oligonucleotides cannot be observed during two or three successive SELEX rounds. These selected oligonucleotides are subject to amplification. Subsequently, the sequences of individually selected oligonucleotides are identified by cloning and sequencing of the selected clones, and the number of different aptamer sequences screened by the SELEX process depends on the stringency of the selection conditions and target characteristics thereof.

For the discovery of aptamer as a novel molecular recognition biochemical element, conventional SELEX is inherently an in vitro screening protocol of elegant simplicity, independent of animal or cell lines, which applies three principles of evolution - heredity, variation, and selection pressure [16]. Various aptamers, e.g. tetracycline aptamer [17] and thrombin aptamer [4], were successfully developed with iterative experimental rounds of incubation with ssDNA or RNA library, partitioning from the unbound, amplification of the binders, affinity characterization, and sequence identification, respectively.

However, the efficiency of conventional SELEX in the discovery of aptamers is somewhat low in terms of its cost-effectiveness, unsatisfactory specificity, limited partition capability, the necessity of a foreknowable targetability, difficult predictability, and inadequate stability or cross-linking capability, etc. Hence, different upgraded SELEX variants, i.e. negative SELEX (counter SELEX, subtractive SELEX), one-round SELEX, capillary electrophoresis ()-SELEX, microfluidic-SELEX, cell-SELEX, HTS-SELEX or in silico-SELEX, post-SELEX or in chemico-SELEX, auto-SELEX, primer-free SELEX, genomic SELEX, photo-SELEX, qPCR-SELEX, and so on were developed to further enhance the selection efficiency or analytical functionality, accordingly.

1.2 Negative SELEX and Its Analogs

In order to ensure the specificity of the aptamer recognition with target analytes solely, negative SELEX (counter SELEX, or subtractive SELEX), was frequently applied to remove nonspecific binding or erroneous recognition with structurally similar compounds of target analytes, and this is especially the case when target molecules of low abundance are in complex matrices, such as cell lysates, whole blood, or other body fluids.

To our knowledge, the first negative SELEX practice was reported by Ellington and Szostak in 1992, when they successfully developed DNA aptamers against small organic dye molecules. To specifically enrich the DNA pool with aptamer candidates, a non-cognate dye precolumn was suspended over a cognate dye column, then a chemically synthesized DNA pool with an estimated complexity of 2-3 × 1013 different sequences were loaded onto the precolumn, and the precolumn retained most nonspecific bound sequences when washed with one column volume buffer. The selectivity in percent DNA bound after negative selection can be improved from 2.2, 1.5, 0.8 to 19.0, 7.0, 4.8, using affinity resin cibacron blue (), reactive green 19 (GR), and reactive blue 4 (B4) coupled to cross-linked agarose beads, respectively [18].

On the basis of a similar scheme, Takahashi et al. [19] successfully developed an isogenic cell-SELEX with a counterselection strategy to generate RNA aptamers toward cell surface protein, say, integrin alfa-V (ITGAV), a major transmembrane receptor widely expressed in almost all the cells and closely associated with human diseases such as cancers and pulmonary fibrosis. As illustrated in Figure 1.2, gene of interest () overexpressed human cell line HEK293 cells were used for positive selection, while GOI knockdown cells as mock cells by microRNA-mediated silencing were used for counterselection, a 100-fold difference in the expressing...