12
L. Hao and H. Gu
electronic, thermodynamic and mechanical properties, GO has attracted more attention. GO can adsorb single-strand oligonucleotides, including ssDNA and RNA, by
π−π stacking interaction between the hexagonal cells of GO and the ring structures
in the DNA bases. But dsDNA cannot be adsorbed by GO because its bases are
wrapped in the double helix structure. Relying on this property, the Park et al. [38]
group of Korea University first reported a non-immobilized GO-SELEX in 2012.
As shown in Fig. 1.8a, after incubating ssDNA library with the target adipokines
“Nampt,” whose blood plasma levels are attributed to obesity and obese related
metabolic diseases such as type 2 diabetes, mellitus or cardiovascular diseases, GO
solution was added into the mixture. The free unbound ssDNAs in the solution got
adsorbed on GO, while ssDNAs bound to target remained in solution. Then, centrifugation was carried out to separate the ssDNAs bound GO from the supernatant
with ssDNAs bound to the target. In 2014, based on the previous GO SELEX, this
research group [39] further developed a multiple GO SELEX. After the initial three
rounds of selection against mixed targets and the subsequent two individual rounds
of selection against each different target, specific aptamers for three different types
of pesticides, tebuconazole, inabenfide and mefenacet, were successfully obtained
(Fig. 1.8b). Furthermore, a couple of flexible multitarget aptamers, which can bind
with two or three different targets, were also found. Since then, GO-SELEX and its
derived methods have been used to select aptamers against virus, biotoxin, metal ion,
antibiotic, endocrine-disrupting chemicals [40–45], etc.
GO-SELEX is an immobilization-free SELEX without any fixing operation, either
for target molecules or for oligonucleotide libraries. So, it will not change the original
conformation of the target, and it can also avoid the steric hindrance of the immobilized target and the nonspecific binding to a solid substrate [46]. However, there are
some defects for GO-SELEX. Single-strand oligonucleotides can self-desorb from
the surface of GO, which reduces the screening efficiency. Furthermore, if the small
molecular target can be absorbed by GO, GO-SELEX cannot be used for screening.
1.3.4 Improving the Specificity of the Aptamer
1.3.4.1 Negative SELEX
In the screening of aptamer against low molecular weight target, the target molecules
are usually attached to the immobilization matrixes, including beads, nitrocellulose membrane, microwell plate, etc. These immobilization matrixes can help and
facilitate separation operations. However, during the incubation process, some of
the sequences might bind to the immobilization matrixes, resulting in nonspecific
aptamers. In order to ensure the specificity of the aptamer recognition with target
analytes solely, negative SELEX was frequently applied to eliminate such possibility.
To our knowledge, the first negative SELEX practice was reported by Ellington
and Szostak [47], when they successfully developed DNA aptamers against small
organic dye molecules. After three selection cycles, they incubated the library with
L. Hao and H. Gu
electronic, thermodynamic and mechanical properties, GO has attracted more attention. GO can adsorb single-strand oligonucleotides, including ssDNA and RNA, by
π−π stacking interaction between the hexagonal cells of GO and the ring structures
in the DNA bases. But dsDNA cannot be adsorbed by GO because its bases are
wrapped in the double helix structure. Relying on this property, the Park et al. [38]
group of Korea University first reported a non-immobilized GO-SELEX in 2012.
As shown in Fig. 1.8a, after incubating ssDNA library with the target adipokines
“Nampt,” whose blood plasma levels are attributed to obesity and obese related
metabolic diseases such as type 2 diabetes, mellitus or cardiovascular diseases, GO
solution was added into the mixture. The free unbound ssDNAs in the solution got
adsorbed on GO, while ssDNAs bound to target remained in solution. Then, centrifugation was carried out to separate the ssDNAs bound GO from the supernatant
with ssDNAs bound to the target. In 2014, based on the previous GO SELEX, this
research group [39] further developed a multiple GO SELEX. After the initial three
rounds of selection against mixed targets and the subsequent two individual rounds
of selection against each different target, specific aptamers for three different types
of pesticides, tebuconazole, inabenfide and mefenacet, were successfully obtained
(Fig. 1.8b). Furthermore, a couple of flexible multitarget aptamers, which can bind
with two or three different targets, were also found. Since then, GO-SELEX and its
derived methods have been used to select aptamers against virus, biotoxin, metal ion,
antibiotic, endocrine-disrupting chemicals [40–45], etc.
GO-SELEX is an immobilization-free SELEX without any fixing operation, either
for target molecules or for oligonucleotide libraries. So, it will not change the original
conformation of the target, and it can also avoid the steric hindrance of the immobilized target and the nonspecific binding to a solid substrate [46]. However, there are
some defects for GO-SELEX. Single-strand oligonucleotides can self-desorb from
the surface of GO, which reduces the screening efficiency. Furthermore, if the small
molecular target can be absorbed by GO, GO-SELEX cannot be used for screening.
1.3.4 Improving the Specificity of the Aptamer
1.3.4.1 Negative SELEX
In the screening of aptamer against low molecular weight target, the target molecules
are usually attached to the immobilization matrixes, including beads, nitrocellulose membrane, microwell plate, etc. These immobilization matrixes can help and
facilitate separation operations. However, during the incubation process, some of
the sequences might bind to the immobilization matrixes, resulting in nonspecific
aptamers. In order to ensure the specificity of the aptamer recognition with target
analytes solely, negative SELEX was frequently applied to eliminate such possibility.
To our knowledge, the first negative SELEX practice was reported by Ellington
and Szostak [47], when they successfully developed DNA aptamers against small
organic dye molecules. After three selection cycles, they incubated the library with