A general strategy to enhance aptamer affinity by suppressing dissociation through symmetric assembly

TL;DR

This paper introduces a new method to significantly improve the binding strength of aptamers by organizing them symmetrically, leading to better performance in detecting targets like SARS-CoV-2 and VEGF165.

Contribution

A symmetry-guided assembly strategy that enhances aptamer affinity by suppressing dissociation rates.

Findings

Trimeric aptamers showed dissociation constants in the low pM range and koff values in the 10−6 s−1 range.

The trimeric aptamer improved detection sensitivity for VEGF165 by 30-fold in a serum-based assay.

The strategy is modular and applicable to existing aptamers targeting various biomolecules.

Abstract

Aptamers are programmable molecular recognition elements with broad utility in diagnostics, therapeutics, and synthetic biology. However, many aptamers suffer from insufficient affinity due to rapid target dissociation, and no general strategy currently exists to overcome this limitation. Here, we report a symmetry-guided assembly approach that enhances aptamer affinity by suppressing the dissociation rate constant (koff). Three identical aptamer units are spatially organized into a flexible trivalent assembly to enable kinetic cooperativity through rapid rebinding. Applied to aptamers targeting SARS-CoV-2 spike (both trimeric and monomeric S1 subunit), VEGF165 (dimeric), and cardiac troponin I (monomeric), the resulting trimers exhibited dissociation constants (Kd) in the low pM range and koff values in the 10−6 s−1 range, over 100-fold improvements relative to monomers. In a serum-based VEGF165 assay, the trimeric aptamer improved detection sensitivity by 30-fold. This modular, chemistry-based strategy is applicable to existing aptamers and establishes dissociation suppression as a general principle for engineering ultrahigh-affinity aptamers.

Graphical Abstract

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