Optimization of Gonyautoxin1/4-Binding G-Quadruplex Aptamers by Label-Free Surface-Enhanced Raman Spectroscopy

Abstract

Nucleic acids with G-quadruplex (G4) structures play an important role in physiological function, analysis and detection, clinical diagnosis and treatment, and new drug research and development. Aptamers obtained using systematic evolution of ligands via exponential enrichment (SELEX) screening technology do not always have the best affinity or binding specificity to ligands. Therefore, the establishment of a structure-oriented experimental method is of great significance. To study the potential of surface-enhanced Raman spectroscopy (SERS) in aptamer optimization, marine biotoxin gonyautoxin (GTX)1/4 and its G4 aptamer obtained using SELEX were selected. The binding site and the induced fit of the aptamer to GTX1/4 were confirmed using SERS combined with two-dimensional correlation spectroscopy. The intensity of interaction between GTX1/4 and G4 was also quantified by measuring the relative intensity of SERS bands corresponding to intramolecular hydrogen bonds. Furthermore, the interaction between GTX1/4 and optimized aptamers was analyzed. The order of intensity change in the characteristic bands of G4 aptamers was consistent with the order of affinity calculated using microscale thermophoresis and molecular dynamics simulations. SERS provides a rapid, sensitive, and economical post-SELEX optimization of aptamers. It is also a reference for future research on other nucleic acid sequences containing G4 structures.

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer

“…Thanks to an intensive investigation of the systematic evolution of ligands through the exponential enrichment (SELEX) technique over last 20 years [14], new variations of SELEX such as bead- [15], capillary electrophoresis- [16], and cell-based SELEX [17] have emerged, markedly enhancing the affinity and specificity of the screened aptamers. Up to now, thousands of protein-binding aptamers [18] and several hundred small molecule-binding aptamers [19] have been reported, and their affinities to the targets can reach sub-nM [20]. With their high specificity, affinity, and accessibility, aptamers are very well-suited for the detection and analysis of biomarkers [21,22] and have proved to be powerful alternatives to antibodies.…”