The SELEX Process

SELEX is an in vitro selection process for producing high specificity and affinity nucleic acid aptamers against a target of choice. The general selection process begins with a synthetic random ssDNA or RNA pool and aim at the enrichment of binding sequences. In a complete random pool, one of the parameters that determined the number of different representatives is the length of the random region. In theory, a longer random sequence space can generate more complex structures, however, the overall yield of the longer pools falls off quickly and one may argue that longer random sequence results in less complete coverage of sequence space than shorter random sequence tracts. The advantage of minimizing the aptamer to the smallest possible size is that it is important for downstream ease of solid-phase synthesis and to limit costs associated with manufacture of the resultant aptamer. In general, pools used for the in vitro selection span less than 100 nucleotides in random sequence positions with a diversity of up to 1016 _{different molecules (Conrad et al., 1996; Pollard et al., 2000). In most cases of} selection against proteins, which are not normally thought to bind to nucleic acids, completely random sequence pools are useful for the isolation of novel binding species without prior knowledge of functional nucleic acid sequences or structural motifs.

Figure 1.9 shows nucleic acid pools used for in vitro selection experiments. Each oligonucleotide composes of a core random sequence flanked by defined constant sequences at the 5’ and the 3’ end, which are necessary for polymerase chain reaction amplification. An RNA pool can be generated from DNA template through the transcription process if the T7 RNA polymerase promoter is included in the primer.

Primer containing T7 Promoter

5’-AAGCATCCGCTGGTTGAC---N40---GATCTTGGACCCTGCGAA-3’ DNA template

Reverse primer

Forward primer

5’-AAGCATCCGCTTGAC---N40---GATCTTGGACCCTGCGAA-3’

Reverse primer

Figure 1.9 Examples of N40 pool. The T7 RNA polymerase promoter (delineated in bold) is incorporated in a primer necessary for reverse transcription process. It is possible to add enzyme

restriction sites in the primer region to facilitate the cloning.

CTAGAACCTGGGACGCTT-5’

CTAGAACCTGGGACGCTT-5’ 5’-GCTAATACGACTCACTATAAAGCATCCGCTGGTTGAC

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In vitro Selection of Aptamers

Conventional SELEX for DNA aptamer selection (Figure 1.10) involves the repetition of five steps:

 Binding of oligonucleotides to targets

 Partition involving the separation of ligands that bind to targets from an unbound oligonucleotide pool

 Elution of bound oligonucleotides

 PCR amplification in which the number of binding sequences will be increased  Generation of ssDNA or RNA which is necessary for the next round of SELEX

process

The number of rounds needed for the manual selection of high affinity and high selectivity aptamers are typically more than ten rounds. Each round takes approximately a day or two.

Figure 1.10 The conventional in vitro aptamer selection. Nucleic acid starting pools are as large as 1016 unique sequences. Iterative round of selection are carried out aiming at an enrichment of specific and tight binding aptamers to their molecular targets.

To start the selection, the pool of nucleic acids is incubated with the molecule of interest. The target can be immobilized or free in solution. In an initial round, a long incubation time and less stringent conditions, especially a higher protein to oligonucleotide ratio, are applied to the selection. This is necessary to reduce the risk of eliminating low abundance strong binders in the early rounds (He et al., 1996). More stringent conditions, such as decreasing the protein to oligonucleotide ratio, changing the buffer, reaction volume and incubation time, are required as the number of rounds increase. Following incubation, the partitioning of non-binding sequences from bound sequences can be achieved by separation procedures. This is one of the most crucial steps as separation efficiency is a key factor in selection performance. The poorer the separation efficiency, the larger the number of repeated selection operations required, making conventional aptamer screening a time and labor consuming process. Several separation techniques used in SELEX include nitrocellulose membrane filtration (Tuerk and Gold, 1990; Morris et al., 1998; Pileur et al., 2003; Noma and Ikebukuro, 2006), affinity chromatography (Ellington and Szostak, 1990; Cheng et al., 2008), magnetic separation (including Ni-NTA and streptavidin coated magnetic beads) (Murphy et al., 2003; Stoltenburg et al., 2005; Barton et al., 2009; Bing et al., 2010a; Gong et al., 2012), electrophoresis (mobility shift and CE) (Tsai and Reed, 1998; Mosing et al., 2005; Li et

al., 2007) or simply through centrifugation in case of cell-SELEX (Sefah et al., 2010).

After the separation step, bound oligonucleotides are recovered by eluting them from the target molecules and then amplifying to increase the copy number of recovered pool for subsequent selection rounds. Using the optimum number of PCR cycles helps to reduce the formation of PCR artifacts due to self-internal priming and over- amplification. Following amplification where dsDNA products are generated, ssDNA is prepared by removing the antisense strand, for example, by  exonuclease digestion.

In case of RNA aptamers, an additional step is required to reverse transcribed RNA to cDNA. The cDNA sequences were then used as templates for PCR amplification and then were transcribed back into an RNA pool for the next round. By iterative rounds of selection, partition and amplification, groups of sequences having high affinity and selectivity are enriched while non-specific sequences or weak binders are removed. Cloning and sequencing of the enriched pool is then performed in order to generate individual sequences for further investigation of their binding ability.

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In vitro Selection of Aptamers

A negative selection can be applied to remove any non-specific binding aptamers that bind the solid support material in the absence of target. This process will sieve non- target dependent binding molecules from the population where their accumulation would be problematic by overrunning a selected population (Jhaveri and Ellington, 2000).

The repetitive rounds of selection are a time and labor consuming process and this is an obstacle to generating aptamers. The requirement to increase the throughput and speed of selection is challenging. Therefore, an automated platform has been developed to achieve fast, reproducible and parallelized selection of multiple targets under multiple conditions (Cox et al., 1998; Cox and Ellington, 2001; Eulberg et al., 2005)

The first automatic system for in vitro selection was set up in the Ellington lab aiming at the selection of anti-protein aptamers. Their first platform was used to select RNA sequences that bound oligo-dT captured on magnetic beads using Beckman Biomek 2000 pipetting robot. Integration of magnetic-based separation to several devices to assist the automation and performing the selection under optimized biological methods yielded RNA that contained repeated adenosine sequences in the random region (Cox et

al., 1998). The system was then adapted to the selection of RNA aptamers that bound

hen-egg white lysozyme (Cox and Ellington, 2001). Several modifications were carried out from the previous platform in order to efficiently recover the highest affinity aptamers, the most important of which was the use of membrane filtration for the partitioning rather than magnetic bead capture. Some other modifications such as direct amplification of bead-bound nucleic acids, progressive reduction of thermal cycler numbers (rather than programmed as a constant number) and providing appropriate conditions for anti-protein selection by omitting some chemicals which may interfere with the binding were carried out. The anti-lysozyme aptamer obtained from 12 rounds in 2 days of automated selection formed a complex with lysozyme that had a dissociation constant of 31nM and also inhibited its catalytic activity. Although the rate of throughput exceeds that of manual selection by a factor of 10-100 and also shows the ability to run parallel selection, the authors commented that other proteins may not be as easy a target as lysozyme and leaving out the quality control step routinely used in manual selection, such as size selection of the products, may result in the accumulation of artifacts, which could lead to selection failure (Cox and Ellington, 2001).

The accumulation of artifacts rarely occurs in well-executed manual selections in which important factors including choice of selection buffer, partitioning stringency, amplification and monitoring enrichment can be adjustable. At some point during selection, the experimenter needs to make a decision of when to increase stringency using which methods and when to stop the selection, for example. Those inputs affect the efficiency and outcome of selection and therefore are considered as advantages of the manual selection. To mimic manual selection, the automatic system must be more flexible. Eulberg and his colleague (2005) designed a flexible automatic selection protocol that offered the flexibility to adjust selection stringency and allowed online monitoring. However, they presented partial automation for the whole selection process by running the first three rounds manually. The reason for starting the selection manually was to assure that the selection process started with all possible sequences in the pool. The first significant improvement from the Cox’s platform was the development of fully automated semi-quantitative PCR protocol which allowed the user to monitor the status of the selection, to prevent over-amplification and thus to allow for optimal adjustment of selection parameters for the subsequent rounds. The second improvement was the development of automation in RNA purification using ultracentrifugation in order to purify nucleic acid and allow buffer change. This robotic system performed more slowly than the previous platform. However, the improvement of flexibility would provide more successful opportunity of selection (Eulberg et al., 2005).

In recent years, not only have automatic systems for aptamer selection been developed but also many modified SELEX methods have gained huge interest for generating new approaches to selection (Stoltenberg et al. 2007; McKeague and DeRosa, 2012). All of these variants aimed to reduce the number of selection rounds needed to generate high affinity ligands or to change aptamer properties. The consequences of performing selection for only a few rounds are then economically relevant where we can obviously save time, reagent and energy while still having a great opportunity to achieve the selection. Although automated systems have been developed to the point of having flexibility and versatility, every single step necessary for repetitive cycles remains unchanged. Examples of alternatives and new variants of SELEX are described in the next section.

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In vitro Selection of Aptamers