Biomedical Engineering Reference
In-Depth Information
selectively bind to a cell-surface antigen or a particular target cell
population [52, 56, 57]. In contrast to the purified protein-based
SELEX, the cell-based SELEX method is performed with whole living
cells, which ensures the native folded conformation or original
glycosylation pattern of the extracellular domains of target proteins.
Typically, this procedure includes two essential steps: (1) counter-
selection with nontargeted cells that do not express the target
molecules on the cell surface; and (2) positive-selection with the
targeted cells. By taking advantage of the molecular differences
between any two closely related cell populations, it is possible to
obtain aptamers against most antigens, even unknown targets or
multiprotein complexes if they are specially and selectively expressed
on or in the target cells [58].
Recently, several research groups have successfully adopted
this strategy to generate cell-specific aptamers. For example, CHO
(Chinese hamster ovary) cells that overexpress the recombinant
TGF-β (transforming growth factor) type III receptors have been used
as a target for the isolation of RNA aptamers against the TGF-β type
III receptor [59]. In this case, counter-selection has been conducted
with CHO cells lacking the target expression. Another example is a
cell-based DNA aptamer able to discriminate rat glioblastoma from
microglia cells [60]. Additionally, even with a lack of information
about the molecular targets, a panel of cell-specific DNA aptamers
has been successfully selected to bind to CCRF-CEM cells [35, 61].
They were capable of distinguishing T-cells and B-cells in patient
samples. One of the selected DNA aptamers, sgc8, identified to
target PTK7, has been demonstrated to be specifically taken up into
lymphoblastic leukemia T-cells
via
a receptor-mediated endocyosis
[62].
Although successful in individual cases, one of the most serious
drawbacks of cell-based SELEX is the nonspecific binding/uptake of
nucleic acids to dead cells, thereby delaying target-specific sequence
enrichment, even resulting in the failure of aptamer selection [63, 64].
Unfortunately, during the selection procedure, some steps or
conditions (for example, detachment of adherent cells, washing,
buffer concentrations, incubation times, etc.) might cause damage or
death to some fragile cells. Therefore, the careful elimination of dead
cell populations before incubation with aptamer library is crucial
and necessary. The complexity of target cell surface markers or the
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