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that do not express the target protein, (2) positive selection by incubating recovered
unbound sequences with positive cells expressing the target protein, (3) recovery of
target-bound sequences, and finally (4) re-amplification of the recovered species. In
general, the number of cells, concentration of the library, ionic strength, incubation
time, washing times, and temperature can all be varied to provide more stringent
conditions favoring the selection of aptamers with the highest affinity.
The use of live cell-based SELEX to create aptamers that bind to target proteins
on the surface of live cells has many advantages. In contrast to purified protein-
based SELEX, it is not necessary to know the number or type of proteins on the cell
membrane surface [
49
]. Since the selection target is whole cells, a panel of aptamers
can be isolated against both known and unknown cell-surface proteins. Most impor-
tantly, the cell-surface proteins keep their native conformation throughout the selec-
tion process, thereby retaining their active biological functions. By using live
cell-based SELEX, many new aptamers have been successfully generated that are
capable of recognizing different cell populations, including lymphocytic leukemia
cells [
50,
51
] and rat glioblastoma cells [
52
]. Employing the cell-based SELEX
method, Tan and colleagues successfully isolated a panel of cell-specific ssDNA
aptamers with high binding affinities to CCRF-CEM cells (a T cell acute lympho-
blastic leukemia cell line) without the explicit knowledge of the cell's molecular
signature [
53
]. The selected aptamers are able to distinguish molecular differences
on cancer cells in patient samples. However, it should be noted that there are several
pitfalls with the cell-based selection procedures [
54
], for example, nucleic acids are
readily bound nonspecifically to dead cells, a problem which can impede effective
enrichment of target-specific sequences and even result in failure to obtain the
desired aptamers.
10.3
The Strategies of Aptamer-siRNA Targeting
Over the past few years, an increasing number of cell-specific RNA and DNA
aptamers have been successfully adopted for targeted delivery of the various mol-
ecules of interest both in cell culture and
in vivo
, such as anticancer drugs [
55-
64
] ,
toxins [
65
] , enzymes [
42
] , radionuclides [
66
] , virus [
67
] , and RNAi-based thera-
peutics (siRNAs, shRNAs) [
68
]. Despite these validated examples, only a few RNA
aptamers (PSMA, CD4, HIV-1 gp120, and CD30) have been demonstrated to have
competence for targeted siRNA/shRNA delivery. In this section, we mainly focus
on the application of cell-specific aptamers for cell-targeted RNA interference.
Several representative examples are discussed below.
10.3.1
Noncovalent Aptamer/siRNA Conjugates
In order to achieve targeted RNAi effects, the desired siRNA molecules can be con-
jugated with a cell-specific aptamer through either covalent conjugation or nonco-
valent assembly. The most established and best characterized aptamers for siRNA
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