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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 linked thrombin-binding aptamer.
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f1-sensors-08-01090: The linked thrombin-binding aptamer.

Mentions: The sequences of the designed thrombin-binding aptamers are shown in Table 1. The 15-mer thrombin-binding aptamer having a G-quartet structure recognizes the fibrinogen-binding exosite of thrombin via a T-loop and inhibits the thrombin activity (Fig. 1) [14]. The 29-mer thrombin-binding aptamer recognizes the heparin-binding site of thrombin and has no thrombin-inhibitory activity (Fig. 1) [15]. We connected the 15-mer and the 29-mer aptamers using poly(dT) linkers of various lengths. The distance between the fibrinogen-binding exosite and the heparin-binding site is about 3.4 nm, and the total length of five sequential thymine bases is about 5 nm. Considering this, we varied the number of dTs inserted between the 15-mer and the 29-mer (0, 5, 10, 15 or 20 dTs).


Improvement of Aptamer Affinity by Dimerization
The linked thrombin-binding aptamer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3927496&req=5

f1-sensors-08-01090: The linked thrombin-binding aptamer.
Mentions: The sequences of the designed thrombin-binding aptamers are shown in Table 1. The 15-mer thrombin-binding aptamer having a G-quartet structure recognizes the fibrinogen-binding exosite of thrombin via a T-loop and inhibits the thrombin activity (Fig. 1) [14]. The 29-mer thrombin-binding aptamer recognizes the heparin-binding site of thrombin and has no thrombin-inhibitory activity (Fig. 1) [15]. We connected the 15-mer and the 29-mer aptamers using poly(dT) linkers of various lengths. The distance between the fibrinogen-binding exosite and the heparin-binding site is about 3.4 nm, and the total length of five sequential thymine bases is about 5 nm. Considering this, we varied the number of dTs inserted between the 15-mer and the 29-mer (0, 5, 10, 15 or 20 dTs).

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.