induce conformational changes with reduced specificity for the targets, it is desirable
to perform the SELEX process directly with the previously modified nucleotides.
This is possible using mutant T7 polymerases [107]; nevertheless, the efficiencies of
reverse transcription are generally low. Other artificial means have been described to
obtain more stable RNA aptamers [108, 109], but they need post-SELEX chemical
modifications. Alternatively, an interesting successful RNA aptamer ligand, made
using 2
0 -fluoro-modified ribonucleotides, is described for the purification of immunoglobulins G from human blood [110].
The last important consideration to compare the two chemistries is the synthesis
cost. The synthesis of DNA aptamers is cheaper than RNA, and the difference
increases with the length of the oligonucleotide chain.
Although interesting the use of unnatural nucleotides [111] appears to be currently limited because of the lack of a large-scale synthesis method.
In our laboratories, we have focused our applications on the selection of
DNA-based aptamers. They have demonstrated their stability under intensive use
in the purification of several proteins from the human blood coagulation cascade
with repeated alkaline cleaning cycles, involving the use of sodium hydroxide
[61, 112].
5.2 Aptamer Grafting Methods for Chromatography
Exploitations
The preparation of an affinity chromatography sorbent for protein purification
comprises a critical phase: the ligand grafting on the solid support. After years of
work devoted to the definition of the most effective chemical immobilization for
protein immobilization, a number of methods have practically been abandoned,
while others are still in use. This natural selection has been driven by the availability
of reagents, the minimum complications, the stability of the linkage, and a reasonable cost. A large diversity of chemical reactions has been developed around the
activation of the solid support to render it ready to react with selected chemical
groups of the affinity ligand. The most representative chemical groups present on
ligands are primary amines, carboxylic acids, hydroxyls, and thiol groups. Throughout the years, scientists proposed numerous reactions with such chemical groups, but
little by little, the attention was focused on primary amines. Specific attention
has been paid to the creation of either a stable peptide bond or a stable
alkylamine. They are obtained by using epoxy-activated supports, divinyl-sulfone,
carbonyldiimidazole-derived chromatography sorbents, and N-hydroxysuccinimideactivated media [113]. While in most cases the protein immobilization on reactive
solid supports is made directly, in certain cases, the modification of the ligand is
necessary to maintain the best performance as described for antibodies [114]. The
latter are modified by partial oxidation of the glycan to protect the polypeptidic
affinity sites.
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G. Perret and E. Boschetti
to perform the SELEX process directly with the previously modified nucleotides.
This is possible using mutant T7 polymerases [107]; nevertheless, the efficiencies of
reverse transcription are generally low. Other artificial means have been described to
obtain more stable RNA aptamers [108, 109], but they need post-SELEX chemical
modifications. Alternatively, an interesting successful RNA aptamer ligand, made
using 2
0 -fluoro-modified ribonucleotides, is described for the purification of immunoglobulins G from human blood [110].
The last important consideration to compare the two chemistries is the synthesis
cost. The synthesis of DNA aptamers is cheaper than RNA, and the difference
increases with the length of the oligonucleotide chain.
Although interesting the use of unnatural nucleotides [111] appears to be currently limited because of the lack of a large-scale synthesis method.
In our laboratories, we have focused our applications on the selection of
DNA-based aptamers. They have demonstrated their stability under intensive use
in the purification of several proteins from the human blood coagulation cascade
with repeated alkaline cleaning cycles, involving the use of sodium hydroxide
[61, 112].
5.2 Aptamer Grafting Methods for Chromatography
Exploitations
The preparation of an affinity chromatography sorbent for protein purification
comprises a critical phase: the ligand grafting on the solid support. After years of
work devoted to the definition of the most effective chemical immobilization for
protein immobilization, a number of methods have practically been abandoned,
while others are still in use. This natural selection has been driven by the availability
of reagents, the minimum complications, the stability of the linkage, and a reasonable cost. A large diversity of chemical reactions has been developed around the
activation of the solid support to render it ready to react with selected chemical
groups of the affinity ligand. The most representative chemical groups present on
ligands are primary amines, carboxylic acids, hydroxyls, and thiol groups. Throughout the years, scientists proposed numerous reactions with such chemical groups, but
little by little, the attention was focused on primary amines. Specific attention
has been paid to the creation of either a stable peptide bond or a stable
alkylamine. They are obtained by using epoxy-activated supports, divinyl-sulfone,
carbonyldiimidazole-derived chromatography sorbents, and N-hydroxysuccinimideactivated media [113]. While in most cases the protein immobilization on reactive
solid supports is made directly, in certain cases, the modification of the ligand is
necessary to maintain the best performance as described for antibodies [114]. The
latter are modified by partial oxidation of the glycan to protect the polypeptidic
affinity sites.
112
G. Perret and E. Boschetti