The applications of ImidAPol remain to be explored. One could consider, for instance, using it
for selectively extracting MPs from a crude supernatant that also contains soluble proteins or for
immobilizing a purified MP onto Ni:NTA beads with the view of screening drugs or identifying or
purifying biological molecules that bind to the immobilized MP. As mentioned above, the presence of
many imidazole moieties implies that, at acidic pH, such as exists in the lumen of endosomes,
ImidAPol particles will become zwitterionic. As discussed in Chap. 15 (§ 15.2.3), this could be of
interest for the cytosolic release of drugs or in the formulation of vaccines.
13.3.3 Oligonucleotide-Tagged Amphipol
In Study 13.9, Christel Le Bon and colleagues examined the possibility of functionalizing A8-35 with
oligodeoxynucleotides (ODNs) (Le Bon et al. 2014a). The reversible immobilization of MPs via an
ODN tag presents major assets for the regeneration and reconfiguration of biosensors and biochip
surfaces. In addition, ODNs offer rich possibilities of modulating the stability and specificity of the
attachment, by playing on their length and sequence, and they lend themselves particularly well to
multiplexing. Indeed, they provide a chemically mild method for the site-selective immobilization of
multiple MPs onto solid supports: traditional, well-established deoxyribonucleic acid (DNA)
microarrays could be easily adapted as immobilization platforms. Previous studies have demonstrated
sequence-specific and site-selective immobilization of semisynthetic protein-ODN conjugates onto
surfaces modified with complementary nucleotide sequences (Zdyrko et al. 2003; Bano et al. 2009).
However, these methods require protein engineering or chemical modification and cannot be easily
adapted to MPs, which tend to be fragile and hard to handle. An alternative strategy is to first
reconstitute the target MP into liposomes containing a cholesterol-modified ODN and then bind the
proteoliposomes onto surfaces modified with the complementary ODN (cODN) (Bailey et al. 2009).
Reconstitution of MPs into artificial vesicles, however, is a delicate, protein-specific process, and its
optimization is time-consuming, whereas trapping MPs with APols is straightforward.
In Study 13.9 is described a route for synthesizing and purifying ODN-grafted A8-35, as well as
the solution properties of the well-behaved particles into which the resulting “OligAPol” assembles
(see Chap. 4, § 4.4.2, Fig. 4.31). The ability of OligAPol to trap and anchor MPs onto cODN-carrying
surfaces was demonstrated using two different types of solid supports, magnetic beads and gold NPs.
In both cases, adsorption was shown to be specifically mediated by the formation of the DNA duplex.
Magnetic beads gave access to measuring adsorption yields, gold NPs to the homogeneity and
reversibility of the adsorption. Anti-BR antibodies were shown to bind specifically to MP/OligAPol
complexes immobilized onto gold NPs via the cODN (Le Bon et al. 2014a).
Figure 13.17 illustrates some of these experiments. Gold NPs immobilized onto poly-L-lysinemodified glass slides were used to (i) explore the possibility of using OligAPol for anchoring MPs onto
gold surfaces and (ii) directly observe, by fluorescence microscopy, the hybridization of MP/OligAPol
complexes and cODN onto solid supports and the binding of antibodies to immobilized MPs. The
strong electrostatic interaction between the positively charged polylysine and the negatively charged
surface of cODN-coated gold NPs ensured a stable immobilization. Differential interference contrast
images showed well-dispersed particles (Fig. 13.17A). BR was trapped in a 1:1 mixture of OligAPol
and Alexa Fluor 647-labeled A8-35 (FAPol AF647 ; Fernandez et al. 2014; see Chap. 4, Table 4.5). After
incubation with these complexes, cODN-functionalized gold NPs exhibited a strong and homogeneous
fluorescence signal (Fig. 13.17B). Single-particle analysis of the fluorescence images showed a ratio
between the average signal recorded on gold NPs and the background of $10 (Fig. 13.17G), indicative
of a low level of non-specific binding of the BR/OligAPol/FAPol AF647 complexes and free OligAPol/
FAPol AF647 particles to the polylysine-coated glass. Similar results were obtained upon incubation of
13.3 Alternative Tags
613
for selectively extracting MPs from a crude supernatant that also contains soluble proteins or for
immobilizing a purified MP onto Ni:NTA beads with the view of screening drugs or identifying or
purifying biological molecules that bind to the immobilized MP. As mentioned above, the presence of
many imidazole moieties implies that, at acidic pH, such as exists in the lumen of endosomes,
ImidAPol particles will become zwitterionic. As discussed in Chap. 15 (§ 15.2.3), this could be of
interest for the cytosolic release of drugs or in the formulation of vaccines.
13.3.3 Oligonucleotide-Tagged Amphipol
In Study 13.9, Christel Le Bon and colleagues examined the possibility of functionalizing A8-35 with
oligodeoxynucleotides (ODNs) (Le Bon et al. 2014a). The reversible immobilization of MPs via an
ODN tag presents major assets for the regeneration and reconfiguration of biosensors and biochip
surfaces. In addition, ODNs offer rich possibilities of modulating the stability and specificity of the
attachment, by playing on their length and sequence, and they lend themselves particularly well to
multiplexing. Indeed, they provide a chemically mild method for the site-selective immobilization of
multiple MPs onto solid supports: traditional, well-established deoxyribonucleic acid (DNA)
microarrays could be easily adapted as immobilization platforms. Previous studies have demonstrated
sequence-specific and site-selective immobilization of semisynthetic protein-ODN conjugates onto
surfaces modified with complementary nucleotide sequences (Zdyrko et al. 2003; Bano et al. 2009).
However, these methods require protein engineering or chemical modification and cannot be easily
adapted to MPs, which tend to be fragile and hard to handle. An alternative strategy is to first
reconstitute the target MP into liposomes containing a cholesterol-modified ODN and then bind the
proteoliposomes onto surfaces modified with the complementary ODN (cODN) (Bailey et al. 2009).
Reconstitution of MPs into artificial vesicles, however, is a delicate, protein-specific process, and its
optimization is time-consuming, whereas trapping MPs with APols is straightforward.
In Study 13.9 is described a route for synthesizing and purifying ODN-grafted A8-35, as well as
the solution properties of the well-behaved particles into which the resulting “OligAPol” assembles
(see Chap. 4, § 4.4.2, Fig. 4.31). The ability of OligAPol to trap and anchor MPs onto cODN-carrying
surfaces was demonstrated using two different types of solid supports, magnetic beads and gold NPs.
In both cases, adsorption was shown to be specifically mediated by the formation of the DNA duplex.
Magnetic beads gave access to measuring adsorption yields, gold NPs to the homogeneity and
reversibility of the adsorption. Anti-BR antibodies were shown to bind specifically to MP/OligAPol
complexes immobilized onto gold NPs via the cODN (Le Bon et al. 2014a).
Figure 13.17 illustrates some of these experiments. Gold NPs immobilized onto poly-L-lysinemodified glass slides were used to (i) explore the possibility of using OligAPol for anchoring MPs onto
gold surfaces and (ii) directly observe, by fluorescence microscopy, the hybridization of MP/OligAPol
complexes and cODN onto solid supports and the binding of antibodies to immobilized MPs. The
strong electrostatic interaction between the positively charged polylysine and the negatively charged
surface of cODN-coated gold NPs ensured a stable immobilization. Differential interference contrast
images showed well-dispersed particles (Fig. 13.17A). BR was trapped in a 1:1 mixture of OligAPol
and Alexa Fluor 647-labeled A8-35 (FAPol AF647 ; Fernandez et al. 2014; see Chap. 4, Table 4.5). After
incubation with these complexes, cODN-functionalized gold NPs exhibited a strong and homogeneous
fluorescence signal (Fig. 13.17B). Single-particle analysis of the fluorescence images showed a ratio
between the average signal recorded on gold NPs and the background of $10 (Fig. 13.17G), indicative
of a low level of non-specific binding of the BR/OligAPol/FAPol AF647 complexes and free OligAPol/
FAPol AF647 particles to the polylysine-coated glass. Similar results were obtained upon incubation of
13.3 Alternative Tags
613