adenine, guanine, and cytosine bases which could react with glutaraldehydeactivated supports [131, 132]. This is also the case with the use of cyanogen bromide
[133, 134], cyanuric chloride [135], and carbodiimides [136] that are very strong
reacting chemical agents. All possible secondary reactions may contribute to
decrease the aptamer affinity for the target protein [137].
A general review on aptamer chemical grafting methods to solid supports has
been published, where several procedures are depicted [115]. Although all the
described methods focused on diagnostic applications, they can be adopted or
adapted for affinity chromatography separations. With hydroxyl-exposed sorbents,
the activation with carbonyldiimidazole is proposed, and then the grafting is operated on a primary amine-derivatized aptamer. This process generates a carbamate
bond, but its stability over time is questionable. Hydroxyl groups from a solid
support can also easily be activated with epichlorohydrin or bisepoxyranes to form
an epoxy exposed group, able to react with either a primary amine-derivatized
aptamer or a thiol derivative. In both cases, the conjugate is particularly stable
under conditions used in affinity chromatography separations, including harsh
cleaning phases. With solid supports presenting primary amine groups, symmetrical
isothiocyanates can be suggested. When in the presence of a sorbent displaying
carboxylic groups, it is proposed to perform condensation reactions with primary
amine-derived aptamers using condensation agents like 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and other analog reagents. Instead of using
condensation agents, carboxylates from the solid support can be activated prior to the
reaction with primary amino-derived aptamers. This is possible with the use of
N-hydroxysuccinimide. These activated supports are commercially available and
can be easily handled for aptamer grafting and constitute one of the effective
methods for aptamer immobilization, as reported in several recently published
reports [61, 110, 137, 138]. A general scheme of main aptamer immobilization
approaches on solid supports is given in Fig. 9.
One of the specific properties of oligonucleotides that is to be considered during
the coupling step is their pronounced acidic character that conflicts with the possible
global negative charge of solid supports. This is particularly evident when using
NHS-activated chromatographic supports, as described for proteins [139]. In practice, upon the contact of a primary-amine aptamer with NHS-activated support, a
grafting reaction takes place releasing a hydroxysuccinimide group. However, at
the same time, some spontaneous hydrolysis reactions are also present with the
NHS-activated carboxylic acids, generating free negative charges that are responsible for the electrostatic repulsion of aptamers in solution. This phenomenon may
reduce the coupling yield very significantly. To prevent or to maintain the repulsion
effect within acceptable limits, an increase of ionic strength of the coupling buffer is
recommended. The presence of sodium chloride at a concentration of 400 mM–1 M
largely attenuates the repulsion effect with the possibility of a higher grafting
density.
Repulsion phenomena can also be attenuated by the modification of the environmental pH [140]. Acidic pH is able to decrease the global positive charge of the
oligonucleotide up to its inversion as it happens for amphoteric molecules (e.g.,
114
G. Perret and E. Boschetti
[133, 134], cyanuric chloride [135], and carbodiimides [136] that are very strong
reacting chemical agents. All possible secondary reactions may contribute to
decrease the aptamer affinity for the target protein [137].
A general review on aptamer chemical grafting methods to solid supports has
been published, where several procedures are depicted [115]. Although all the
described methods focused on diagnostic applications, they can be adopted or
adapted for affinity chromatography separations. With hydroxyl-exposed sorbents,
the activation with carbonyldiimidazole is proposed, and then the grafting is operated on a primary amine-derivatized aptamer. This process generates a carbamate
bond, but its stability over time is questionable. Hydroxyl groups from a solid
support can also easily be activated with epichlorohydrin or bisepoxyranes to form
an epoxy exposed group, able to react with either a primary amine-derivatized
aptamer or a thiol derivative. In both cases, the conjugate is particularly stable
under conditions used in affinity chromatography separations, including harsh
cleaning phases. With solid supports presenting primary amine groups, symmetrical
isothiocyanates can be suggested. When in the presence of a sorbent displaying
carboxylic groups, it is proposed to perform condensation reactions with primary
amine-derived aptamers using condensation agents like 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and other analog reagents. Instead of using
condensation agents, carboxylates from the solid support can be activated prior to the
reaction with primary amino-derived aptamers. This is possible with the use of
N-hydroxysuccinimide. These activated supports are commercially available and
can be easily handled for aptamer grafting and constitute one of the effective
methods for aptamer immobilization, as reported in several recently published
reports [61, 110, 137, 138]. A general scheme of main aptamer immobilization
approaches on solid supports is given in Fig. 9.
One of the specific properties of oligonucleotides that is to be considered during
the coupling step is their pronounced acidic character that conflicts with the possible
global negative charge of solid supports. This is particularly evident when using
NHS-activated chromatographic supports, as described for proteins [139]. In practice, upon the contact of a primary-amine aptamer with NHS-activated support, a
grafting reaction takes place releasing a hydroxysuccinimide group. However, at
the same time, some spontaneous hydrolysis reactions are also present with the
NHS-activated carboxylic acids, generating free negative charges that are responsible for the electrostatic repulsion of aptamers in solution. This phenomenon may
reduce the coupling yield very significantly. To prevent or to maintain the repulsion
effect within acceptable limits, an increase of ionic strength of the coupling buffer is
recommended. The presence of sodium chloride at a concentration of 400 mM–1 M
largely attenuates the repulsion effect with the possibility of a higher grafting
density.
Repulsion phenomena can also be attenuated by the modification of the environmental pH [140]. Acidic pH is able to decrease the global positive charge of the
oligonucleotide up to its inversion as it happens for amphoteric molecules (e.g.,
114
G. Perret and E. Boschetti