*2.1. Overview of DNA and RNA Capture-SELEX*

The classical procedure of the DNA Capture-SELEX selection method involves hybridization, immobilization, target-bound sequence elution, polymerase chain reaction (PCR) amplification, regeneration of single-stranded DNA (ssDNA), and sequencing (Figure 1A). Briefly, in the first step, a random ssDNA library is designed with a docking site that is complementary to the biotinylated capture oligonucleotides, and these ssDNAs are hybridized with capture-oligonucleotides to form biotinylated hybridized library via Watson-Crick base pairing. Next, the biotinylated library is immobilized onto SA-MBs, which function as solid support, and the loaded SA-MBs are then washed several times to remove the residual and non-specific sequences using a magnetic rack and generate a washed biotinylated library on the MBs (which comprise superparamagnetic iron oxide). Subsequently, a magnetic rack also used to discard supernatant (unbound or non-specific sequences), after which the counter-selection process is performed, wherein a counter target with a similar chemical structure and concentration as the positive targets, enhances the binding affinity and specificity of aptamers towards it positive small-molecule target [42]. Counter selection can be performed either in the last few rounds [39,43] or between the selected rounds [44,45] of Capture-SELEX, and the concentration of the counter target should be the same as the positive target in step 3 (Figure 1A). The negative selection is followed by positive selection. Next, an elution is performed; if the binding affinity between the target and aptamer is higher than that between the aptamer and the capture

oligonucleotide, the target-bound aptamer candidates are eluted. As the library sequences interact with the positive targets, only sequences with conformational changes due to the interaction can be eluted from the MBs [46].

In this review, we summarize two main strategies used to regenerate ssDNA from amplified dsDNA in step 6 (Figure 1A): the gel purification method [21,23,47] and the MB-based method [24]. A primer is designed and modified in order to identify the sense strand in the denaturing polyacrylamide gel, such as a reverse primer with poly-dA20 extension or a forward primer labeled with either fluorescein or the cyanine dye Cy3. The gel purification method involves denaturing polyacrylamide gel electrophoresis (PAGE), followed by extraction and purification. The MB-based method begins with PCR products with biotinylated reverse primers being incubated to facilitate their deposition onto MBs. The supernatant is then removed, and the non-biotinylated forward strands of dsDNA PCR products are released from the MBs by treatment with sodium hydroxide and then neutralized by treatment with an appropriate amount of tris(hydroxymethyl)aminomethane hydrochloride or hydrochloric acid. The regenerated ssDNA in each round is collected for the next round of selection and then prepared for sequencing after several rounds of selection. Currently, Capture-SELEX is mostly applied for the selection of DNA aptamers rather than RNA aptamers. RNA Capture-SELEX adopts the same selection strategies as DNA Capture-SELEX, except for including two more steps: transcription and reverse transcription (Figure 1). In addition, more rounds need to be performed in DNA Capture-SELEX (8–20 rounds) than in RNA DNA Capture-SELEX (6–12 rounds) to enrich the target-bound pool sufficiently.
