Biomedical Engineering Reference
In-Depth Information
electrophoresis [42-44], microfluidic devices [45, 46], and so on.
With the automation of SELEX [47, 48], aptamers for multiple targets
can be quickly isolated in a high-throughput selection format.
The advantages of the use of the purified protein as target
molecule, such as low nonspecific binding and easy control of the
selection conditions, allow optimal enrichment of the sequences
specific to the target protein [49]. In the majority of cases, cell-specific
aptamers have been evolved to target soluble, purified proteins. For
example, for the evolution of the anti-PSMA RNA aptamer [27], a
purified fusion protein containing a modified extracellular form of
PSMA was immobilized on magnetic beads. Therefore, the bound
species were readily segmented from unbound sequences
a
simple washing step, and the bound species were recovered and
re-amplified for the next selection round. Similarly, 2
via
-F-modified RNA
aptamers targeting CD4 were generated by immobilizing soluble,
recombinant CD4 antigen onto sepharose-beads [28]. Additionally,
several 2
′
-F-modified anti-HIV-1 gp120 RNA aptamers have been
successfully identified by using either the BIAcore biosensor
′
system [50, 51] or conventional nitrocellulose filter methods [31].
As the most established isolation method, the cellulose filter-based
SELEX has succeeded in generating anti-EGFR aptamers [34] and
anti-TfR aptamers [36] highly specific for binding their purified
target proteins.
However, in some cases, aptamers raised against the purified
target protein were unable to recognize the same target in its
native conformation when the protein target is functionally part of
a multi-protein complex, is embedded in a physiological context,
or is subjected to a post-translational modifications [52]. For
example, RNA aptamers that were generated by using the purified
extracellular domain of human RTK Ret failed to bind to full-length
Ret expressed on the cell surface [53, 54]. Similarly, although
RNA aptamers indicated high binding affinity to their target
histidine-tagged EGFRvIII ectodomain, they didn't bind to the
full-length EGFRvIII protein that is extensively glycosylated when
expressed on the cell surface [55].
5.2.2
Whole Cell-Based SELEX Procedure
Recent advances in cell-based SELEX techniques have provided a
promising alternative approach for identifying aptamers that can
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