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Improvement of Aptamer Affinity by Dimerization

View Article: PubMed Central - PubMed

ABSTRACT

To increase the affinities of aptamers for their targets, we designed an aptamer dimer for thrombin and VEGF. This design is based on the avidity of the antibody, which enables the aptamer to connect easily since it is a single-strand nucleic acid. In this study, we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer. Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimer has a Kd value which is 1/10 of that of the monomers from which it is composed. Also, the designed aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers against vascular endothelial growth factor (VEGF165), which is a homodimeric protein. As in the case of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower Kd value than that of the monomer. This study demonstrated that the dimerization of aptamers effectively improves the affinities of those aptamers for their targets.

No MeSH data available.


The equilibrium responses are plotted as a function of the aptamer concentration. VEGF165 was immobilized on a sensor chip and aptamers were injected for the SPR measurement. (A) VEa5, (B) del5-1, (C) VEa5-VEa5, (D) del5-1-del5-1 were assayed.
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f4-sensors-08-01090: The equilibrium responses are plotted as a function of the aptamer concentration. VEGF165 was immobilized on a sensor chip and aptamers were injected for the SPR measurement. (A) VEa5, (B) del5-1, (C) VEa5-VEa5, (D) del5-1-del5-1 were assayed.

Mentions: We have already confirmed by SPR measurement that VEa5 binds VEGF165 with a Kd value of 120 nM (Fig. 4A) [11]. The affinities of del5-1 and the aptamer dimers against VEGF165 were also evaluated by SPR measurement. Aptamer solutions of different concentrations were assayed on VEGF165-immobilized sensor chips and the corresponding signals were obtained. Figure 4 shows the signal at equilibrium plotted as a function of the aptamer concentration. The Scatchard plot analysis showed that del5-1 had a Kd value of 500 nM (Table 3). In both VEa5 and del5-1, the constants of dissociation with VEGF165 of the aptamer dimers without linkers were less than 1/10 of those of the monomer aptamers (Table 3). Meanwhile, when dimer aptamers with dT linkers were injected, the equilibrium response values were not even partially dependent on the aptamer concentrations, and the Scatchard plots were not linear (data not shown). This might be caused by the low stabilities of aptamer dimers with dT linkers.


Improvement of Aptamer Affinity by Dimerization
The equilibrium responses are plotted as a function of the aptamer concentration. VEGF165 was immobilized on a sensor chip and aptamers were injected for the SPR measurement. (A) VEa5, (B) del5-1, (C) VEa5-VEa5, (D) del5-1-del5-1 were assayed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927496&req=5

f4-sensors-08-01090: The equilibrium responses are plotted as a function of the aptamer concentration. VEGF165 was immobilized on a sensor chip and aptamers were injected for the SPR measurement. (A) VEa5, (B) del5-1, (C) VEa5-VEa5, (D) del5-1-del5-1 were assayed.
Mentions: We have already confirmed by SPR measurement that VEa5 binds VEGF165 with a Kd value of 120 nM (Fig. 4A) [11]. The affinities of del5-1 and the aptamer dimers against VEGF165 were also evaluated by SPR measurement. Aptamer solutions of different concentrations were assayed on VEGF165-immobilized sensor chips and the corresponding signals were obtained. Figure 4 shows the signal at equilibrium plotted as a function of the aptamer concentration. The Scatchard plot analysis showed that del5-1 had a Kd value of 500 nM (Table 3). In both VEa5 and del5-1, the constants of dissociation with VEGF165 of the aptamer dimers without linkers were less than 1/10 of those of the monomer aptamers (Table 3). Meanwhile, when dimer aptamers with dT linkers were injected, the equilibrium response values were not even partially dependent on the aptamer concentrations, and the Scatchard plots were not linear (data not shown). This might be caused by the low stabilities of aptamer dimers with dT linkers.

View Article: PubMed Central - PubMed

ABSTRACT

To increase the affinities of aptamers for their targets, we designed an aptamer dimer for thrombin and VEGF. This design is based on the avidity of the antibody, which enables the aptamer to connect easily since it is a single-strand nucleic acid. In this study, we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer. Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimer has a Kd value which is 1/10 of that of the monomers from which it is composed. Also, the designed aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers against vascular endothelial growth factor (VEGF165), which is a homodimeric protein. As in the case of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower Kd value than that of the monomer. This study demonstrated that the dimerization of aptamers effectively improves the affinities of those aptamers for their targets.

No MeSH data available.