The chemical methods for aptamer grafting on a solid support are similar to those
used for proteinaceous affinity chromatography ligands [115, 116]. Structurally,
oligonucleotides comprise molecular blocks displaying phosphate groups, primary
amines, and hydroxyl groups that could be the starting points for the grafting
reaction on solid supports. However, these groups are not available, since the
functional groups of purine and pyrimidine bases are involved in the formation of
hydrogen bonds, stabilizing the macromolecule, and phosphate groups play the role
of linkers between the bases. It is therefore necessary to chemically modify the
aptamer for grafting compatibility. This modification can be made at the 3
0 or 5
0
terminal end of the chain. In affinity chromatography, it is relatively common to
derivatize molecules with biotin residues and then make complexes with avidin
[4, 117] with interesting results. In the present aptamer ligand context, the oligonucleotide with a biotin molecule can strongly interact with a solid support carrying an
avidin molecule. This molecular complex is stable under current chromatographic
conditions and does not represent a risk of modification of the tertiary structure of the
aptamer. This technology was satisfactorily applied to aptamer ligands in view of the
purification of a recombinant fusion protein from a cell culture supernatant [60]. A
similar approach with biotinylated aptamers anchored on avidin supports has then
been used for other applications [118–122]. In all these cases, the bond is not
covalent but solid enough to perform few affinity separations. Although successful,
these methods are inappropriate for preparative and large-scale applications. They
suffer from several drawbacks. The first is the necessity to derivatize the aptamer
with biotin. The second is to derivatize the solid support with avidin, and the third
limitation is the low binding capacity of the resulting affinity chromatography
construct. All these steps are time-consuming, complex, and expensive.
The introduction of a predetermined chemical group at one of the two terminal
ends (3
0 or 5
0 ) of an aptamer is more appropriate for large-scale chromatography.
Although affinity chromatography technologies involve the immobilization of a
specific ligand after the chemical activation of the solid support [113], the literature
on aptamer ligands describes situations with a reversed approach. As an example, the
cyanuric chloride activation method, very well known in affinity chromatography
[123], has been applied for the activation of the aptamer followed by the solid-phase
grafting [124, 125]. In another example, the same activation was performed to
immobilize an aptamer on a polyethyleneimine-coated surface [126]. Interestingly,
these studies demonstrated that, by varying the molar ratio of aptamer versus
cyanuric chloride (1:0.5; 1:1; 1:5; 1:100), it was possible to evidence an influence
on the coupling yield and on the final affinity properties toward the target protein. In
other examples glutaraldehyde, only marginally used a number of years ago in
affinity chromatography [127, 128], has been described for oligonucleotide grafting.
To this end, amino-derived solid surfaces have been activated with a large excess of
glutaraldehyde. After the elimination of the reagent excess, the activated surface was
exposed to the aptamer, thus forming a sort of a symmetric bond with an aminoterminal linker attached to the aptamer [129, 130]. Although these experiments
allowed obtaining exploitable affinity surfaces, secondary reactions were also engendered. The oligonucleotide displays several possible reaction sites present on
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
113
used for proteinaceous affinity chromatography ligands [115, 116]. Structurally,
oligonucleotides comprise molecular blocks displaying phosphate groups, primary
amines, and hydroxyl groups that could be the starting points for the grafting
reaction on solid supports. However, these groups are not available, since the
functional groups of purine and pyrimidine bases are involved in the formation of
hydrogen bonds, stabilizing the macromolecule, and phosphate groups play the role
of linkers between the bases. It is therefore necessary to chemically modify the
aptamer for grafting compatibility. This modification can be made at the 3
0 or 5
0
terminal end of the chain. In affinity chromatography, it is relatively common to
derivatize molecules with biotin residues and then make complexes with avidin
[4, 117] with interesting results. In the present aptamer ligand context, the oligonucleotide with a biotin molecule can strongly interact with a solid support carrying an
avidin molecule. This molecular complex is stable under current chromatographic
conditions and does not represent a risk of modification of the tertiary structure of the
aptamer. This technology was satisfactorily applied to aptamer ligands in view of the
purification of a recombinant fusion protein from a cell culture supernatant [60]. A
similar approach with biotinylated aptamers anchored on avidin supports has then
been used for other applications [118–122]. In all these cases, the bond is not
covalent but solid enough to perform few affinity separations. Although successful,
these methods are inappropriate for preparative and large-scale applications. They
suffer from several drawbacks. The first is the necessity to derivatize the aptamer
with biotin. The second is to derivatize the solid support with avidin, and the third
limitation is the low binding capacity of the resulting affinity chromatography
construct. All these steps are time-consuming, complex, and expensive.
The introduction of a predetermined chemical group at one of the two terminal
ends (3
0 or 5
0 ) of an aptamer is more appropriate for large-scale chromatography.
Although affinity chromatography technologies involve the immobilization of a
specific ligand after the chemical activation of the solid support [113], the literature
on aptamer ligands describes situations with a reversed approach. As an example, the
cyanuric chloride activation method, very well known in affinity chromatography
[123], has been applied for the activation of the aptamer followed by the solid-phase
grafting [124, 125]. In another example, the same activation was performed to
immobilize an aptamer on a polyethyleneimine-coated surface [126]. Interestingly,
these studies demonstrated that, by varying the molar ratio of aptamer versus
cyanuric chloride (1:0.5; 1:1; 1:5; 1:100), it was possible to evidence an influence
on the coupling yield and on the final affinity properties toward the target protein. In
other examples glutaraldehyde, only marginally used a number of years ago in
affinity chromatography [127, 128], has been described for oligonucleotide grafting.
To this end, amino-derived solid surfaces have been activated with a large excess of
glutaraldehyde. After the elimination of the reagent excess, the activated surface was
exposed to the aptamer, thus forming a sort of a symmetric bond with an aminoterminal linker attached to the aptamer [129, 130]. Although these experiments
allowed obtaining exploitable affinity surfaces, secondary reactions were also engendered. The oligonucleotide displays several possible reaction sites present on
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
113