3.1 Oligonucleotide Libraries as Ligand Banks
Aptamers are sequences of nucleotides that can dock on small protein sub-parts
(called aptatopes) and are capable to reach a very high degree of specificity.
Oligonucleotide libraries can comprise up to 10
15 individual sequences. Having
such a large number of structures in a given volume means that the concentration
of each of them is extremely low and, thus, it would be very hard to isolate the
binding structure and to identify it for further use. What allowed aptamers to succeed
in the development of affinity ligands was the concept of in vitro evolution,
associated with polymerase chain reaction (PCR). In fact, this technology allows
amplifying individual oligonucleotide molecules up to the concentration needed for
further analysis. This mechanism of separation and amplification was elegantly
developed at the end of the 1990s under the name SELEX [57, 63]. Furthermore,
with the advances of chemical synthesis, it is relatively easy today to produce
oligonucleotides in large quantities, as required for their chemical immobilization
on chromatography sorbents. From this collection of molecules, it is possible to
extract the right structure by a sort of reversed affinity adsorption, where the
immobilized molecule (grafted on solid particles or captured on a filter) is the target
protein [64]. After this step, the affinity oligonucleotide is desorbed, amplified,
sequenced, and finally synthesized prior to chemical grafting.
3.2 Single-Chain Oligonucleotide Ligands
An aptamer ligand for affinity chromatography is a single-chain DNA or RNA
oligonucleotide of a given length, constituted of a determined sequence of nucleic
bases. While the sequence of bases is a major characteristic for their functionality,
the construct is not linear but rather folded by the aggregation of the planar structures
of the nucleic bases. The structuration starts with the association of bases (base
pairing), as a consequence of hydrogen bond formation between two complementary
structures, independently on their position all along the sequence string. This base
pairing naturally distorts the entire molecule with the formation of short doublehelical structure portions with paired nucleotides and other single-stranded regions
with unpaired bases (see Fig. 2).
For instance, small RNA molecules show structures with multiple internal
and side loops. Actually, to comply with atomic distances, the nucleic acid string
generates structures with variable configurations, such as stemloops [65], hairpins
[66], tetraloops [67], pseudoknots [65], and G-quartets [68] (Fig. 3). These peculiar
structures confer unique three-dimensional shapes, creating thus the conditions of
high affinity and specificity for a given target. These structures that have been
naturally observed for RNA can occur in the same manner for single-stranded DNA.
It is interesting to note here that the nucleic acid folding contrasts largely with
what happens during the formation of tridimensional structures in proteins. This is
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
99
Aptamers are sequences of nucleotides that can dock on small protein sub-parts
(called aptatopes) and are capable to reach a very high degree of specificity.
Oligonucleotide libraries can comprise up to 10
15 individual sequences. Having
such a large number of structures in a given volume means that the concentration
of each of them is extremely low and, thus, it would be very hard to isolate the
binding structure and to identify it for further use. What allowed aptamers to succeed
in the development of affinity ligands was the concept of in vitro evolution,
associated with polymerase chain reaction (PCR). In fact, this technology allows
amplifying individual oligonucleotide molecules up to the concentration needed for
further analysis. This mechanism of separation and amplification was elegantly
developed at the end of the 1990s under the name SELEX [57, 63]. Furthermore,
with the advances of chemical synthesis, it is relatively easy today to produce
oligonucleotides in large quantities, as required for their chemical immobilization
on chromatography sorbents. From this collection of molecules, it is possible to
extract the right structure by a sort of reversed affinity adsorption, where the
immobilized molecule (grafted on solid particles or captured on a filter) is the target
protein [64]. After this step, the affinity oligonucleotide is desorbed, amplified,
sequenced, and finally synthesized prior to chemical grafting.
3.2 Single-Chain Oligonucleotide Ligands
An aptamer ligand for affinity chromatography is a single-chain DNA or RNA
oligonucleotide of a given length, constituted of a determined sequence of nucleic
bases. While the sequence of bases is a major characteristic for their functionality,
the construct is not linear but rather folded by the aggregation of the planar structures
of the nucleic bases. The structuration starts with the association of bases (base
pairing), as a consequence of hydrogen bond formation between two complementary
structures, independently on their position all along the sequence string. This base
pairing naturally distorts the entire molecule with the formation of short doublehelical structure portions with paired nucleotides and other single-stranded regions
with unpaired bases (see Fig. 2).
For instance, small RNA molecules show structures with multiple internal
and side loops. Actually, to comply with atomic distances, the nucleic acid string
generates structures with variable configurations, such as stemloops [65], hairpins
[66], tetraloops [67], pseudoknots [65], and G-quartets [68] (Fig. 3). These peculiar
structures confer unique three-dimensional shapes, creating thus the conditions of
high affinity and specificity for a given target. These structures that have been
naturally observed for RNA can occur in the same manner for single-stranded DNA.
It is interesting to note here that the nucleic acid folding contrasts largely with
what happens during the formation of tridimensional structures in proteins. This is
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
99