20
L. Hao and H. Gu
cells as targets as well. Compared with protein-based SELEX, cell-based selection
can be performed on molecular signature without prior knowledge of the whole cell.
When molecular recognition of cancer cells is needed, it is not necessary to know
about the amount or type of proteins on the cell membrane. The selection process
itself can distinguish between different types of cells, resulting in a specific type of
cancer cells that can only bind to the aptamer, rather than normal cells or other types
of cancer cells [68]. Moreover, it is feasible to perform whole cell selection in the
presence of many receptor proteins on the surface of the cell membrane. So, we can
select a set of aptamer probes, which can reveal the molecular characteristics of the
target cancer type. This is the main advantage of cell-SELEX for cancer diagnosis
and clinical analysis.
Currently, cell-SELEX is commonly utilized in aptamer selection for cancer study.
A cultured precursor T-cell acute lymphoblastic leukemia (ALL) cell line, CCRFCEM, has been used as the target. Negative selection is performed with a B-cell
line derived from human Burkitt’s lymphoma as a negative control by adding a
Ramos selection process to exclude possible binding of DNA sequences to common
molecules on the surface of leukemic cells [69]. In this work, CCRF-CEM cells are
incubated with ssDNAs. The cell surface-binding sequences are eluted by heating
after they are washed, and interact with excess Ramos cells afterward. The sequences
still free in the supernatant were amplified by PCR to form the starting pool and can
be selected for the next round.
Several types of cancer cells have been successfully used in the cell-SELEX
process to filter out novel aptamer probes. These aptamers show superior affinity and
excellent specificity. However, it should be noted that the number and duration of
cell-SELEX selection are longer than conventional SELEX, and cell-SELEX usually
takes the risk of failure to damage fragile cells [69].
1.3.6.2 HTS SELEX
In classical SELEX, the PCR products in the last round of selection are usually
cloned and sequenced by classic Sanger sequencing method. However, this step is
time-consuming and laborious, and the number of the obtained sequences is generally
<100, which is not necessarily representative of the whole population of aptamers.
Because of PCR bias, the most frequent sequences of the final round are not always
the aptamers with high affinity. In other words, some high-affinity aptamers may not
be enriched. These infrequent aptamers may be missed due to limited clone number
or inefficient cloning. Recent years, in order to avoid this kind of cloning bias,
high-throughput sequencing technology (HTS), which is also called next-generation
sequencing technology (NGS), and bioinformatics analysis technology are introduced to the SELEX procedure. HTS can read at least millions of sequences in
a sequencing experiment, which can effectively reduce cloning bias. More importantly, HTS SELEX enables visualization of dynamic changes among millions of
sequences throughout selection. So, it is possible to identify high-affinity aptamers
during a much earlier selection round, which is more time efficient. Fewer rounds of
L. Hao and H. Gu
cells as targets as well. Compared with protein-based SELEX, cell-based selection
can be performed on molecular signature without prior knowledge of the whole cell.
When molecular recognition of cancer cells is needed, it is not necessary to know
about the amount or type of proteins on the cell membrane. The selection process
itself can distinguish between different types of cells, resulting in a specific type of
cancer cells that can only bind to the aptamer, rather than normal cells or other types
of cancer cells [68]. Moreover, it is feasible to perform whole cell selection in the
presence of many receptor proteins on the surface of the cell membrane. So, we can
select a set of aptamer probes, which can reveal the molecular characteristics of the
target cancer type. This is the main advantage of cell-SELEX for cancer diagnosis
and clinical analysis.
Currently, cell-SELEX is commonly utilized in aptamer selection for cancer study.
A cultured precursor T-cell acute lymphoblastic leukemia (ALL) cell line, CCRFCEM, has been used as the target. Negative selection is performed with a B-cell
line derived from human Burkitt’s lymphoma as a negative control by adding a
Ramos selection process to exclude possible binding of DNA sequences to common
molecules on the surface of leukemic cells [69]. In this work, CCRF-CEM cells are
incubated with ssDNAs. The cell surface-binding sequences are eluted by heating
after they are washed, and interact with excess Ramos cells afterward. The sequences
still free in the supernatant were amplified by PCR to form the starting pool and can
be selected for the next round.
Several types of cancer cells have been successfully used in the cell-SELEX
process to filter out novel aptamer probes. These aptamers show superior affinity and
excellent specificity. However, it should be noted that the number and duration of
cell-SELEX selection are longer than conventional SELEX, and cell-SELEX usually
takes the risk of failure to damage fragile cells [69].
1.3.6.2 HTS SELEX
In classical SELEX, the PCR products in the last round of selection are usually
cloned and sequenced by classic Sanger sequencing method. However, this step is
time-consuming and laborious, and the number of the obtained sequences is generally
<100, which is not necessarily representative of the whole population of aptamers.
Because of PCR bias, the most frequent sequences of the final round are not always
the aptamers with high affinity. In other words, some high-affinity aptamers may not
be enriched. These infrequent aptamers may be missed due to limited clone number
or inefficient cloning. Recent years, in order to avoid this kind of cloning bias,
high-throughput sequencing technology (HTS), which is also called next-generation
sequencing technology (NGS), and bioinformatics analysis technology are introduced to the SELEX procedure. HTS can read at least millions of sequences in
a sequencing experiment, which can effectively reduce cloning bias. More importantly, HTS SELEX enables visualization of dynamic changes among millions of
sequences throughout selection. So, it is possible to identify high-affinity aptamers
during a much earlier selection round, which is more time efficient. Fewer rounds of