Aptamer-Mediated siRNA Targeting - RNA Interference from Biology to Therapeutics - page 207

10.2.1 Puri fi ed Membrane Protein-Based SELEX

As shown in Fig. 10.1a , a typical purified membrane protein-based SELEX proce-
dure consists of four main steps (1) binding to the target protein, (2) selection parti-
tioning, (3) recovery of target-bound sequences, and (4) re-amplification of
recovered species [ 34 ]. The critical step in aptamer selection is the selection parti-
tioning, which is to isolate target-bound sequences from unbound species in the
library through various approaches, including traditional bead, resin, membrane, or
chip-based segmentation approaches. Recently, novel isolation strategies have been
developed to accelerate the selection procedure and improve the efficiency of
aptamer selection. These include capillary gel electrophoresis [ 35- 37 ] , fl ow
cytometry, and micro fl uidic devices [ 38, 39 ] .
The purified membrane protein-based SELEX procedure has some advantages
over other methods, including low nonspecific binding and facile control of the
selection conditions [ 34 ]. Thus far, most of the cell-specific aptamers have been
generated using soluble, purified membrane proteins/receptors. For example, a tra-
ditional nitrocellulose membrane filter-based isolation method was employed to
generate anti-HIV-1 gp120 aptamers [ 40 ] , anti-EGFR aptamers [ 41 ] , and anti-TfR
aptamers [ 42 ]. In addition, aptamers targeting CD4 were produced by immobilizing
soluble, recombinant CD4 antigen onto Sepharose beads allowing elution of
unbound sequences and retention of bound species [ 43 ]. Lupold et al. immobilized
a purified fusion protein containing a modified extracellular form of PSMA on mag-
netic beads and successfully isolated two 2¢ - fl uoro-modi fi ed RNase-resistant RNA
aptamers (A-9 and A-10) with low nanomolar binding affinity [ 44 ] . Despite these
successes, efficient generation of new aptamers as cell-specific homing agents still
presents a major challenge due to the limited number of purified membrane pro-
teins. A target protein that is insoluble or only functionalizes in a native conforma-
tion or in a multi-protein complex is unavailable for the purified protein-based
SELEX procedure [ 45 ]. It was reported that some aptamers that were evolved to
bind to a soluble, purified membrane protein failed to recognize the same targets in
their native con fi rmation [ 46- 48 ]. For such proteins, the traditional purified protein-
based in vitro selection is not feasible.

10.2.2 Live Cell-Based SELEX

Live cell-based SELEX is the process in which live cells are used to select aptamers
for target recognition (Fig. 10.1b ) [ 31 ]. This method provides a promising alterna-
tive for the generation of aptamers that can recognize a particular target in its native
cell conformation [ 49 ]. In principle, this strategy relies essentially on the differ-
ences between the target cell population (positive cells) relative to a control cell
population (negative cells) which are used for counterselection. In similarity to the
purified protein-based SELEX, a live cell-based SELEX procedure also consists of
four main steps (1) counterselection by incubating the library with negative cells