two thiol-derived aptamers; this is possible by using dedicated reducing agents such
as tris(2-carboxymethyl)phosphine.
Another important aspect of ligand immobilization is its density distribution on
the surface. It naturally impacts the binding capacity for the target protein to purify.
This parameter is dependent on the efficacy of the grafting reaction and also on the
size of the oligonucleotide [142, 143]. To obtain high grafting density, it is advised
to deal with the smallest size of the oligonucleotide still compatible with the
maintenance of its specificity for the target protein. High-density grafting, however,
may induce interactions between two neighboring oligonucleotides and the reduction of the expected specificity. A compromise between repulsion forces limiting the
grafting yields, the size of nucleotide, and a too high ligand density are case-by-case
issues.
Another point to consider is the availability of the grafted aptamer to dock on the
protein aptatope of the protein to purify. To enhance this probability, it is advised to
use a spacer arm between the solid surface and the aptamer molecule. This is a
classical question largely debated for classical affinity chromatography using polypeptide ligands. The typical adopted solution is the introduction of a spacer arm
[144]. When considering single-chain oligonucleotides under their tertiary structure,
a spacer is already represented by one of the two terminal ends where the functional
chemical group is introduced. However, the main question is related to the length
and the composition of such a spacer.
This question of spacer introduction on aptamer grafting has been discussed by
Balamurugan et al. [115, 143]. First, the introduction of a spacer did not impact the
surface grafting density but significantly increased the effective binding of the target
protein (in this case thrombin). Spacers composed of oligothymidine, associated
with hexaethylene glycol, were very effective to improve the functionality of the
grafted aptamer by a factor of 4. Spacers composed of simple aminohexanoic acid
were also used successfully for the purification of coagulation factors with relatively
large binding capacities [61]. The optimal length of the spacer is not yet a wellknown parameter and may depend not only on the size of the aptamer but also on the
size of the protein to separate. For instance, binding capacities in excess of 20 mg/
mL have been claimed for the separation of human immunoglobulins G (large
protein of about 150 kDa) by using a small aptamer attached on a chromatography
sorbent by means of an 18-carbon spacer [137].
The importance of the spacer and its length, associated with the size of the
aptamer, is also stressed for the preparation of biosensors, where the oligonucleotide
sequence is attached on a gold surface by means of a thiol group [142]. It has been
found that the immobilization of a 5
0 -substituted oligonucleotide with hexanethiol is
better than with an unfunctionalized version of the aptamer, whatever the number of
bases. Moreover, with small oligonucleotides (less than 24 bases), the immobilization reaction yields a well-ordered coating with a relatively high density, whereas
with nucleotides comprising more than 24 bases, the coating coverage tends to be
significantly reduced.
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G. Perret and E. Boschetti
as tris(2-carboxymethyl)phosphine.
Another important aspect of ligand immobilization is its density distribution on
the surface. It naturally impacts the binding capacity for the target protein to purify.
This parameter is dependent on the efficacy of the grafting reaction and also on the
size of the oligonucleotide [142, 143]. To obtain high grafting density, it is advised
to deal with the smallest size of the oligonucleotide still compatible with the
maintenance of its specificity for the target protein. High-density grafting, however,
may induce interactions between two neighboring oligonucleotides and the reduction of the expected specificity. A compromise between repulsion forces limiting the
grafting yields, the size of nucleotide, and a too high ligand density are case-by-case
issues.
Another point to consider is the availability of the grafted aptamer to dock on the
protein aptatope of the protein to purify. To enhance this probability, it is advised to
use a spacer arm between the solid surface and the aptamer molecule. This is a
classical question largely debated for classical affinity chromatography using polypeptide ligands. The typical adopted solution is the introduction of a spacer arm
[144]. When considering single-chain oligonucleotides under their tertiary structure,
a spacer is already represented by one of the two terminal ends where the functional
chemical group is introduced. However, the main question is related to the length
and the composition of such a spacer.
This question of spacer introduction on aptamer grafting has been discussed by
Balamurugan et al. [115, 143]. First, the introduction of a spacer did not impact the
surface grafting density but significantly increased the effective binding of the target
protein (in this case thrombin). Spacers composed of oligothymidine, associated
with hexaethylene glycol, were very effective to improve the functionality of the
grafted aptamer by a factor of 4. Spacers composed of simple aminohexanoic acid
were also used successfully for the purification of coagulation factors with relatively
large binding capacities [61]. The optimal length of the spacer is not yet a wellknown parameter and may depend not only on the size of the aptamer but also on the
size of the protein to separate. For instance, binding capacities in excess of 20 mg/
mL have been claimed for the separation of human immunoglobulins G (large
protein of about 150 kDa) by using a small aptamer attached on a chromatography
sorbent by means of an 18-carbon spacer [137].
The importance of the spacer and its length, associated with the size of the
aptamer, is also stressed for the preparation of biosensors, where the oligonucleotide
sequence is attached on a gold surface by means of a thiol group [142]. It has been
found that the immobilization of a 5
0 -substituted oligonucleotide with hexanethiol is
better than with an unfunctionalized version of the aptamer, whatever the number of
bases. Moreover, with small oligonucleotides (less than 24 bases), the immobilization reaction yields a well-ordered coating with a relatively high density, whereas
with nucleotides comprising more than 24 bases, the coating coverage tends to be
significantly reduced.
116
G. Perret and E. Boschetti