Raman Scattering Reveals Ion-Dependent G-Quadruplex Formation in the 15-mer Thrombin-Binding Aptamer upon Association with α-Thrombin

Abstract

The discovery of DNA aptamers that bind biomolecular targets has enabled significant innovations in biosensing. Aptamers form secondary structures that exhibit selective high-affinity interactions with their binding partners. The binding of its target by an aptamer is often accompanied by conformational changes, and sensing by aptamers often relies on these changes to provide readout signals from extrinsic labels to detect target association. Many biosensing applications involve aptamers immobilized to surfaces, but methods to characterize conformations of immobilized aptamers and their in situ response have been lacking. To address this challenge, we have developed a structurally informative Raman spectroscopy method to determine conformations of the 15-mer thrombinbinding aptamer (TBA) immobilized on porous silica surfaces. The TBA is of interest because its binding of α-thrombin depends on the aptamer forming an antiparallel G-quadruplex, which is thought to drive signal changes that allow thrombin-binding to be detected. However, specific metal cations also stabilize the G-quadruplex conformation of the aptamer, even in the absence of its protein target. To develop a deeper understanding of the conformational response of the TBA, we utilize Raman spectroscopy to quantify the effects of the metal cations, K + (stabilizing) and Li + (nonstabilizing), on G-quadruplex versus unfolded populations of the TBA. In K + or Li + solutions, we then detect the association of α-thrombin with the immobilized aptamer, which can be observed in Raman scattering from the bound protein. The results show that the association of α-thrombin in K + solutions produces no detectable change in aptamer conformation, which is found in the Gquadruplex form both before and after binding its target. In Li + solutions, however, where the TBA is unfolded prior to α-thrombin association, protein binding occurs with the formation of a G-quadruplex by the aptamer.

Surface-Area Enhanced Raman Spectroscopy of DNA in Porous Silica: A Quantitative and Reproducible Alternative to Plasmonic-Based SERS

“…Silica particles with pore diameters as large as 100 nm are commercially available and have been employed in similar experiments to investigate protein binding to ligands at supported lipid bilayers. , Porous silica has a high surface energy that can lead to nonspecific adsorption, especially of proteins. Fortunately, there are numerous polyethylene glycol (PEG) reagents that can be used to backfill the areas between active DNA sites, greatly reducing nonspecific adsorption of protein targets to these surfaces . A final limitation of this methodology for investigating interfacial biorecognition is the long time required for low concentrations of tight binders to achieve equilibrium with DNA sites in the particles; these slow kinetics reflect the time required for molecules to diffuse over large distances and accumulate within the particles.…”

“…Fortunately, there are numerous polyethylene glycol (PEG) reagents that can be used to backfill the areas between active DNA sites, greatly reducing nonspecific adsorption of protein targets to these surfaces. 42 A final limitation of this methodology for investigating interfacial biorecognition is the long time required for low concentrations of tight binders to achieve equilibrium with DNA sites in the particles; these slow kinetics reflect the time required for molecules to diffuse over large distances and accumulate within the particles. Planar supports for biosensing are able to achieve fast response times because the small population of binding sites can be equilibrated with a small volume of flowing solution, greatly increasing the transport of molecules to the surface.…”

“…39 Immobilizing DNA onto the interior surfaces of a porous silica particle generates internal (within-particle) concentrations of DNA as high as ∼30 mM, 40 well above the detection limits for unenhanced Raman scattering. We have recently applied this methodology to monitor the reaction steps that immobilize DNA onto porous silica surfaces, 40 to observe hybridization of ssDNA with fully and partially complementary strands, 39 and to detect the association of immobilized DNA with small molecules 41 and proteins 42 from solution.…”

Programmable DNA Templates for Silver Nanoclusters Synthesis To Develop On-Demand FRET Aptasensor

“…Compared to antibodies, aptamers offer several advantages, including chemical stability, structural feasibility, and ease of chemical synthesis and modification . Consequently, aptamers have a wide range of applications such as medical diagnosis, , environmental analysis, , and food safety monitoring. − Currently, aptamer-modified AgNCs have garnered increasing attention due to the synergistic benefits offered by the combination of AgNCs and aptamers. By appending C-rich sequences to the aptamer, the DNA strands within these aptasensors are engineered to possess a dual functionality.…”