2
L. Hao and H. Gu
such as endogenous nucleic acid analysis, microfluidic cell separation, flow cytometry, nanoparticle-based sensing and detection, to improve the diagnostic effect. The
good biocompatibility of the aptamer makes it easy to penetrate into biological cells
for intracellular imaging detection. In the disease treatment, aptamer can be used as
either an antagonist, which specifically binds to target protein to inhibit the interaction
between proteins, or an activator to enhance the activity of the target protein. Furthermore, aptamer can be used for targeted delivery of drugs. Drugs can be combined
with aptamers through embedding, loading or nanomaterial wrapping. Then, targeted
drug delivery guided by aptamers is realized, which can not only enhance anti-tumor
ability but also reduce toxicity.
Aptamers are generated in vitro through a process called systematic evolution of
ligands by exponential enrichment (SELEX). SELEX technology was established
by Tuerk and Gold, and Ellington and Szostak [1, 2]. Typically, a selection cycle
starts with the incubation of the target with a DNA or RNA library, comprised of
approximately 10
12 –10
16 single-stranded random oligonucleotides with 20–80 bases
in length. The DNA or RNA sequences in the library consist of random region in
the middle and fixed sequences on both ends. The term “SELEX” refers to a process
that entails progressive separation of single-stranded DNAs or RNAs (ssDNA/RNA)
combinatorial single-stranded oligonucleotide library via repeated rounds of binding,
partitioning, and amplification [3–5].
1.2 Systematic Evolution of Ligands by EXponential
Enrichment (SELEX)
In a typical SELEX process, the targets are first incubated with the DNA library.
Subsequently, the target-binding oligonucleotides are separated from the unbound
ones. The bound DNA oligomers are then eluted and amplified by PCR. After several
rounds of selection, the resulting DNA sequences (aptamers) with high affinity and
specificity are enriched in the pool and sequenced (Fig. 1.1) [6]. In an early stage
of aptamer selection, RNA libraries are widely used due to the fact that RNA can
easily fold into complex 3D structures, which show higher affinity to the targets. The
differences in the majority of RNA SELEX protocols compared with DNA SELEX
include the requirement of the protection of RNA from RNAases, amplification by
T7 RNA polymerase and reverse transcription step before PCR. So, DNA libraries
are used more frequently now.
An initial DNA library (of typically 10
14 molecules) is incubated with the solid
support-bound target. Unbound DNA molecules are discarded while the active
species are recovered, amplified by PCR, and injected into subsequent rounds of
selection. The stringency of the selection protocol can be modulated by altering
physicochemical parameters such as concentration, pH, temperature, or buffer
composition. At the end of the protocol, the enriched population is sequenced and
the individual aptameric sequences are evaluated for their binding capability to the
target.
L. Hao and H. Gu
such as endogenous nucleic acid analysis, microfluidic cell separation, flow cytometry, nanoparticle-based sensing and detection, to improve the diagnostic effect. The
good biocompatibility of the aptamer makes it easy to penetrate into biological cells
for intracellular imaging detection. In the disease treatment, aptamer can be used as
either an antagonist, which specifically binds to target protein to inhibit the interaction
between proteins, or an activator to enhance the activity of the target protein. Furthermore, aptamer can be used for targeted delivery of drugs. Drugs can be combined
with aptamers through embedding, loading or nanomaterial wrapping. Then, targeted
drug delivery guided by aptamers is realized, which can not only enhance anti-tumor
ability but also reduce toxicity.
Aptamers are generated in vitro through a process called systematic evolution of
ligands by exponential enrichment (SELEX). SELEX technology was established
by Tuerk and Gold, and Ellington and Szostak [1, 2]. Typically, a selection cycle
starts with the incubation of the target with a DNA or RNA library, comprised of
approximately 10
12 –10
16 single-stranded random oligonucleotides with 20–80 bases
in length. The DNA or RNA sequences in the library consist of random region in
the middle and fixed sequences on both ends. The term “SELEX” refers to a process
that entails progressive separation of single-stranded DNAs or RNAs (ssDNA/RNA)
combinatorial single-stranded oligonucleotide library via repeated rounds of binding,
partitioning, and amplification [3–5].
1.2 Systematic Evolution of Ligands by EXponential
Enrichment (SELEX)
In a typical SELEX process, the targets are first incubated with the DNA library.
Subsequently, the target-binding oligonucleotides are separated from the unbound
ones. The bound DNA oligomers are then eluted and amplified by PCR. After several
rounds of selection, the resulting DNA sequences (aptamers) with high affinity and
specificity are enriched in the pool and sequenced (Fig. 1.1) [6]. In an early stage
of aptamer selection, RNA libraries are widely used due to the fact that RNA can
easily fold into complex 3D structures, which show higher affinity to the targets. The
differences in the majority of RNA SELEX protocols compared with DNA SELEX
include the requirement of the protection of RNA from RNAases, amplification by
T7 RNA polymerase and reverse transcription step before PCR. So, DNA libraries
are used more frequently now.
An initial DNA library (of typically 10
14 molecules) is incubated with the solid
support-bound target. Unbound DNA molecules are discarded while the active
species are recovered, amplified by PCR, and injected into subsequent rounds of
selection. The stringency of the selection protocol can be modulated by altering
physicochemical parameters such as concentration, pH, temperature, or buffer
composition. At the end of the protocol, the enriched population is sequenced and
the individual aptameric sequences are evaluated for their binding capability to the
target.