Improvement of Aptamer Affinity by Dimerization

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.

Self-bending symmetric cusp beams

“…[3][4][5][6][7][8][9][10][11][12][13] Alternatively, the proximity between two aptamers that bind different thrombin epitopes has been exploited to collect a specific signal of the target. [14][15][16][17] Enhanced sensitivity and reduced reaction time have been achieved with bivalent aptamer structures in which distinct aptamers are conjugated with an optimal linker, 18,19 or co-conjugated to the sensor surface with an optimal density. 20,21 Depending on the length, design, and chemical composition of the linker, 5-to 200-fold enhancement in affinity against thrombin has been reported.…”

Electrochemical Detection of Vascular Endothelial Growth Factor with Aptamer Sandwich

“…The first aptamer (VEap165), with the sequence 5B-ATACCAGTCTATTCAATTGGGCCCGTCCGTATG-GTGGGTGTGCTGGCCAG-3B, binds only to VEGF 165 . 10 The second one (VEap121), with the sequence 5B-TGTGGGGGTGGA-CGGGCCGGGTAGA-3B, binds to both VEGF 165 and VEGF 121 . 11 These aptamers were synthesized by Greiner Bio-One (Tokyo, Japan).…”

“…Some researchers have reported several VEGF-binding aptamers; 8,9 we have described two such aptamers. 10,11 These two aptamers were screened against different isoforms of VEGF-A. The first was screened against VEGF 165 , the most common isoform of VEGF-A, with two domains: a receptorbinding domain and a heparin-binding domain.…”

Electrochemical Detection of Vascular Endothelial Growth Factor by an Aptamer-Based Bound/Free Separation System

“…12 ¦VEap dimer is composed of two of the same truncated mutants of VEap, and each ¦VEap are connected by a double-stranded palindromic sequence. Since VEGF is a homodimeric protein, each domain of ¦VEap dimer will bind to each domain of a VEGF monomer simultaneously.…”

“…Many researchers have reported an improvement in the affinity of aptamer by dimerization, which expands the interaction area between the aptamer and target molecules, 10,11 and we also reported dimeric VEGF aptamers, which improve the affinity of selected aptamers. 7,12 Aptamers can be used as a biosensing element instead of antibodies because aptamers, which can be easily synthesized and are more stable than antibodies, also recognize molecules with a high affinity and specificity. In addition, aptamers have an advantage over antibodies in biosensing in that a structural change in aptamers can be designed using straightforward hybridization rules.…”