Enhancing the Affinity of Anti-Human α-Thrombin 15-mer DNA Aptamer and Anti-Immunoglobulin E Aptamer by PolyT Extension

Abstract

Aptamer affinity capillary electrophoresis-laser-induced fluorescence (CE-LIF) for protein detection takes advantage of aptamers for their ease of synthesis and labeling, small size, and having many negative charges. Its success relies on the high binding affinity of aptamers. One 15-mer DNA aptamer (5'-GGT TGG TGT GGT TGG-3', Apt15) shows desirable specificity for human α-thrombin, an important enzyme with multiple functions in blood. However, Apt15 has weak binding affinity, and the use of Apt15 in affinity CE-LIF analysis remains challenging. Here we reported that extension of Apt15 at the 3'-end with a polyT tail having length of 18 T or longer significantly enhanced its affinity and enabled a well-isolated and stable peak for thrombin-aptamer complex in affinity CE. It was likely that the improvement of binding affinity resulted from double binding, an additional interaction of the polyT tail with thrombin in addition to the Apt15 section binding to thrombin. With dye-labeled Apt15 having a T tail, we achieved detection of thrombin at concentrations as low as 0.1 nM by affinity CE-LIF. This aptamer probe specifically bound to human α-thrombin, showing negligible affinity for human β- and γ-thrombin, which are proteolyzed derivatives of human alpha α-thrombin and share similar structure. This strategy of adding a polyT extension also enhanced the binding affinity of anti-immunoglobulin E aptamer in CE-LIF analysis, showing that the affinity enhancement approach is not limited to the thrombin-binding aptamer and has potential for more applications in bioanalysis.

Aptamer-Based Biosensing with a Cationic AIEgen

“…Two model aptamers were chosen for our study: AL40, a 40-nucleotide aptamer with affinity to lysozyme, [34] and ATh, a 15-nucleotide aptamer with affinity to thrombin. [35] To determine the optimal dye concentration for sensing, we measured the fluorescence intensity of TPE-2þ with and without the aptamer. Owing to its amphiphilic nature, the dye at high concentration can aggregate in aqueous solution, which leads to higher fluorescence (Fig.…”

Aptamers against Immunoglobulins: Design, Selection and Bioanalytical Applications

“…Clearly, the complex formation and the stability during separation is influenced by the buffer composition and the best sensitivity in complex detection was observed at the shortest separation time, although the increase of the electric field could cause the dissociation of the aptamer-target complex [166]. To enhance the binding affinity between the aptamer and the target molecule, a polyT extension on the 5 end can be applied to gain well-isolated peak for the aptamer-IgE complex in CE-LIF with higher intensity at the same concentrations compared to the original method [167]. Alternative detection methods include chemiluminescence [168] or PCR [169] involving additional reagents after the separation was performed, thus less convenient than the fluorescence-based detection, but higher sensitivities while still low overall analysis times was offered, e.g., in the case of chemiluminescence detection with an analysis time of 5 min, an LOD in the fM range was achieved [168].…”

Capillary electrophoresis–based immunoassay and aptamer assay: A review

“…The affinity of aptamer to the target molecule can be affected by many factors, including the aptamer length, so it can be increased by adding a poly‐T tail to the end of the nucleotide . For example, Bai and co‐workers used a high‐affinity aptamer with an added poly‐T tail for the selective detection of α‐thrombin without interference from β‐ and γ‐thrombin . Li and co‐workers investigated the affinity of 15‐mer and 29‐mer aptamers to thrombin .…”

“…For example, G‐protein‐BODIPY FL GTPγS, hairpin‐structured DNA‐peptide nucleic acid, concanavalin A‐monosaccharides, dorzolamide‐human carbonicanhydrase II, and tubulin‐anti‐mitotic compounds pairs were examined . Other examples of the aptamer‐based ACE applications are summarized in Table .…”