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.

The structure and function of biological macromolecules change due to intermolecular deuterium bond formation or deuterium substitution with environmental D2O. In this study, surface-enhanced Raman spectroscopy (SERS) was used to detect interaction sites between D2O and ssDNA and their action mechanisms. SERS peaks of ssDNA changed with increasing D2O proportions, and the site of action mainly involved A and G bases, whose number strengthened the interaction between sequences and D2O and hence the SERS peak intensities. Fixing the number of A and G bases prevented changes in their positions from significantly altering the map. We also identified the interaction between ssDNA sequences that easily formed a G-quadruplex structure and D2O. The amplitude of the SERS peak intensity change reflected the ssDNA structural stability and number of active sites. These findings are highly significant for exploring genetic exchanges and mutations and could be used to determine the stability and structural changes of biological macromolecules.

This study aimed to optimize the detection conditions for surface-enhanced Raman spectroscopy (SERS) of single-stranded DNA (ssDNA) in four different buffers and explore the interaction between gonyautoxin (GTX1/4) and its aptamer, GO18. The influence of the silver colloid solution and MgSO4 concentration (0.01 M) added under four different buffered conditions on DNA SERS detection was studied to determine the optimum detection conditions. We explored the interaction between GTX1/4 and GO18 under the same conditions as those in the systematic evolution of ligands by exponential enrichment technique, using Tris-HCl as the buffer. The characteristic peaks of GO18 and its G-quadruplex were detected in four different buffer solutions. The change in peak intensity at 1656 cm−1 confirmed that the binding site between GTX1/4 and GO18 was in the G-quadruplex plane. The relative intensity of the peak at 1656 cm−1 was selected for the GTX1/4–GO18 complex (I1656/I1099) to plot the ratio of GTX1/4 in the Tris-HCl buffer condition (including 30 μL of silver colloid solution and 2 μL of MgSO4), and a linear relationship was obtained as follows: Y = 0.1867X + 1.2205 (R2 = 0.9239). This study provides a basis for subsequent application of SERS in the detection of ssDNA, as well as the binding of small toxins and aptamers.