the future. Nevertheless, several improvements are expected to turn the applicability
of this technology to full speed, especially at large scale.
A number of aptamer grafting methods have been described, and this represents a
dilemma for any novel user of such an affinity chromatography separation. It appears
that the NHS sorbent activation method represents the most adopted process;
however, no comparative studies have been performed to date. For instance,
epoxy-activated sorbents produce very stable bonds when exposed to primary
amine-derived aptamers, but the grafting reactivity is probably low and needs to
be optimized. This question extends also to thiolated peptides that are also able to
react with epoxy-activated supports. The objective of these possible studies is to
determine a recipe providing the highest binding capacity for protein targets.
The spacers also play an important role to the protein-aptamer docking process. It
is hypothesized that long spacers perform better, but this hypothesis is yet to be
demonstrated in aptamer-based affinity chromatography. Binding capacities of few
mg per mL of sorbent are significantly lower than one can expect, but it could
be increased significantly with optimized conditions of grafting. If capacity of
10 mg/mL of sorbent are reached, the applications on protein separations will go
well beyond the high-value proteins. In all cases, the binding capacity will be much
larger than what is possible with immunosorbents.
Another technical point to elucidate is the aptamer folding process once chemically grafted on a solid surface. To prevent folding problems, it is anticipated that the
use of modified nucleic bases or even configuration freezing with LNA would better
maintain the secondary structures and thus the affinity properties for the target
proteins for an extended period of time [184, 185].
In order to overcome the weak hydrophobic-mediated interaction of aptamers,
progress is ongoing, for instance, post-modifications of nuclear bases or by the use of
PCR, compatible with modified nucleotides [111, 186]. Moreover, these structures
show a high nuclease resistance.
Some of these investigations are in progress in our laboratories.
References
1. Fitzgerald J, Leonard P, Darcy E, Sharma S, O’Kennedy R (2017) Immunoaffinity chromatography: concepts and applications. Methods Mol Biol 1485:27–51
2. Farmaki T (2016) Use of a phosphatidylinositol phosphate affinity chromatography (PIP
chromatography) for the isolation of proteins involved in protein quality control and
proteostasis mechanisms in plants. Methods Mol Biol 1450:223–232
3. Licht P, Pavgi S (1992) Identification and purification of a high-affinity thyroxine binding
protein that is distinct from albumin and prealbumin in the blood of a turtle, Trachemys
scripta. Gen Comp Endocrinol 85:179–192
4. Bansal V, Roychoudhury PK, Mattiasson B, Kumar A (2006) Recovery of urokinase from
integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using
p-aminobenzamidine affinity chromatography. J Mol Recognit 19:332–339
5. Wickerhauser M, Williams C (1984) A single-step method for the isolation of antithrombin III.
Vox Sang 47:397–405
130
G. Perret and E. Boschetti
of this technology to full speed, especially at large scale.
A number of aptamer grafting methods have been described, and this represents a
dilemma for any novel user of such an affinity chromatography separation. It appears
that the NHS sorbent activation method represents the most adopted process;
however, no comparative studies have been performed to date. For instance,
epoxy-activated sorbents produce very stable bonds when exposed to primary
amine-derived aptamers, but the grafting reactivity is probably low and needs to
be optimized. This question extends also to thiolated peptides that are also able to
react with epoxy-activated supports. The objective of these possible studies is to
determine a recipe providing the highest binding capacity for protein targets.
The spacers also play an important role to the protein-aptamer docking process. It
is hypothesized that long spacers perform better, but this hypothesis is yet to be
demonstrated in aptamer-based affinity chromatography. Binding capacities of few
mg per mL of sorbent are significantly lower than one can expect, but it could
be increased significantly with optimized conditions of grafting. If capacity of
10 mg/mL of sorbent are reached, the applications on protein separations will go
well beyond the high-value proteins. In all cases, the binding capacity will be much
larger than what is possible with immunosorbents.
Another technical point to elucidate is the aptamer folding process once chemically grafted on a solid surface. To prevent folding problems, it is anticipated that the
use of modified nucleic bases or even configuration freezing with LNA would better
maintain the secondary structures and thus the affinity properties for the target
proteins for an extended period of time [184, 185].
In order to overcome the weak hydrophobic-mediated interaction of aptamers,
progress is ongoing, for instance, post-modifications of nuclear bases or by the use of
PCR, compatible with modified nucleotides [111, 186]. Moreover, these structures
show a high nuclease resistance.
Some of these investigations are in progress in our laboratories.
References
1. Fitzgerald J, Leonard P, Darcy E, Sharma S, O’Kennedy R (2017) Immunoaffinity chromatography: concepts and applications. Methods Mol Biol 1485:27–51
2. Farmaki T (2016) Use of a phosphatidylinositol phosphate affinity chromatography (PIP
chromatography) for the isolation of proteins involved in protein quality control and
proteostasis mechanisms in plants. Methods Mol Biol 1450:223–232
3. Licht P, Pavgi S (1992) Identification and purification of a high-affinity thyroxine binding
protein that is distinct from albumin and prealbumin in the blood of a turtle, Trachemys
scripta. Gen Comp Endocrinol 85:179–192
4. Bansal V, Roychoudhury PK, Mattiasson B, Kumar A (2006) Recovery of urokinase from
integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using
p-aminobenzamidine affinity chromatography. J Mol Recognit 19:332–339
5. Wickerhauser M, Williams C (1984) A single-step method for the isolation of antithrombin III.
Vox Sang 47:397–405
130
G. Perret and E. Boschetti