5 An Update on Aptamer-Based Affinity Chromatography
Advancements
In this section, the state of the art of the most important steps for the preparation of an
aptamer-based affinity sorbent is reviewed.
5.1 RNA Versus DNA Aptamer Ligands
Since RNA- and DNA-based aptamers have both been described as affinity chromatography ligands, a legitimate question would be to understand the preference of
the ones versus the others.
Although they have an equivalent ability to specifically recognize a protein target,
DNA aptamers appear particularly attractive to preparative chromatographic applications. This is related to the better chemical stability of DNA, compared to RNA.
Structurally, the main difference is the pentose that is ribose for RNA and deoxyribose for DNA. The 2
0 -hydroxyl groups in the ribose of RNA aptamers are susceptible to deprotonation, thus acting as a nucleophile center, capable to induce the
hydrolysis of the phosphodiester bond. In fact, the OH group is adjacent to phosphorus and allows a transesterification with the hydrolysis of the phosphodiester
bridge, between two nucleotides. This reaction mechanism is accelerated, particularly in alkaline conditions, and depends on the degree of protonation of the oxygen
atom in position 2
0 . The reaction starts with the deprotonation of the hydroxyl group
to generate a highly reactive O
À anion that breaks the link of phosphate with the OH
of position 5
0 of the next ribose and forms a cyclic phosphodiester between the 3
0 and
2
0 positions of the same ribose [105]. The deprotonation occurs not only under
alkaline conditions but also to a more limited extent in acidic conditions. The
deprotonation can be equally easily induced by the presence of deprotonationpromoting molecules (e.g., alkaline cations, such as potassium and magnesium,
and even cationic amino acids, such as histidine); briefly, the process is initiated
in the presence of a sort of “proton pump” whatever its nature and origin. This
mechanism is dependent on temperature considering that from 4
C and 50
C, the
hydrolysis rate increases of about three orders of magnitude. The nucleotide base
composition is also a factor influencing the transesterification rate of RNA. Conversely, DNA aptamers with a deoxyribose do not expose hydroxyl groups, thus
rendering the structure more stable. Another additional minor advantage is the
presence of a more hydrophobic thymine nucleobase, compared to uracil rendering
the constructs more rigid, compared to RNA molecules. Although DNA aptamers
appear more adapted as affinity chromatography ligands for protein preparative
applications, stabilized RNA structures are adopted in a number of cases. An elegant
way to reduce the hydrolysis propensity of RNA aptamers is to use UTP and CTP
modified with a fluorine atom, which replaces the 2
0 hydroxyl groups of the ribose
[106]. Another interesting post-SELEX chemical modification is the introduction of
an O-methyl group (20-OMe). However, in practice, since post-modifications may
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
111
Advancements
In this section, the state of the art of the most important steps for the preparation of an
aptamer-based affinity sorbent is reviewed.
5.1 RNA Versus DNA Aptamer Ligands
Since RNA- and DNA-based aptamers have both been described as affinity chromatography ligands, a legitimate question would be to understand the preference of
the ones versus the others.
Although they have an equivalent ability to specifically recognize a protein target,
DNA aptamers appear particularly attractive to preparative chromatographic applications. This is related to the better chemical stability of DNA, compared to RNA.
Structurally, the main difference is the pentose that is ribose for RNA and deoxyribose for DNA. The 2
0 -hydroxyl groups in the ribose of RNA aptamers are susceptible to deprotonation, thus acting as a nucleophile center, capable to induce the
hydrolysis of the phosphodiester bond. In fact, the OH group is adjacent to phosphorus and allows a transesterification with the hydrolysis of the phosphodiester
bridge, between two nucleotides. This reaction mechanism is accelerated, particularly in alkaline conditions, and depends on the degree of protonation of the oxygen
atom in position 2
0 . The reaction starts with the deprotonation of the hydroxyl group
to generate a highly reactive O
À anion that breaks the link of phosphate with the OH
of position 5
0 of the next ribose and forms a cyclic phosphodiester between the 3
0 and
2
0 positions of the same ribose [105]. The deprotonation occurs not only under
alkaline conditions but also to a more limited extent in acidic conditions. The
deprotonation can be equally easily induced by the presence of deprotonationpromoting molecules (e.g., alkaline cations, such as potassium and magnesium,
and even cationic amino acids, such as histidine); briefly, the process is initiated
in the presence of a sort of “proton pump” whatever its nature and origin. This
mechanism is dependent on temperature considering that from 4
C and 50
C, the
hydrolysis rate increases of about three orders of magnitude. The nucleotide base
composition is also a factor influencing the transesterification rate of RNA. Conversely, DNA aptamers with a deoxyribose do not expose hydroxyl groups, thus
rendering the structure more stable. Another additional minor advantage is the
presence of a more hydrophobic thymine nucleobase, compared to uracil rendering
the constructs more rigid, compared to RNA molecules. Although DNA aptamers
appear more adapted as affinity chromatography ligands for protein preparative
applications, stabilized RNA structures are adopted in a number of cases. An elegant
way to reduce the hydrolysis propensity of RNA aptamers is to use UTP and CTP
modified with a fluorine atom, which replaces the 2
0 hydroxyl groups of the ribose
[106]. Another interesting post-SELEX chemical modification is the introduction of
an O-methyl group (20-OMe). However, in practice, since post-modifications may
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
111