Applications of Surface Plasmon Resonance for Advanced Studies Involving Nucleic Acids

Abstract

Surface plasmon resonance (SPR) is increasingly recognized as one of the most widely used techniques for studying nucleic acid interactions. The main advantage of SPR is its ability to measure the binding affinities and association/dissociation kinetics of complexes in real-time, in a label-free environment, and using relatively small quantities of materials. The method is based on the immobilization of one of the binding partners, ligand, on a dedicated sensor surface. Immobilization is followed by the injection of the other partner, analyte, over the surface containing the ligand. The binding is monitored by subsequent changes in the refractive index of the medium close to the sensor surface upon injection of the analyte. In the field of Nucleic Acid, SPR has been intensively used in the study of various artificial and naturally occurring RNA/DNA molecules interaction with large molecular weight mass proteins and small organic molecules because of its ability to detect highly dynamic complexes that are difficult to investigate using other techniques. This mini review aims to provide a short guideline for setting up SPR experiments to identify nucleic acid complexes and assess their binding affinity or kinetics. It covers protocols for (i) nucleic acid immobilization methods, including biotin-streptavidin, metal ion-based affinity, and amine coupling, (ii) analyte-binding analysis, (iii) affinity and kinetic measurements, and (iv) data interpretation. Determining the affinity and kinetics of nucleic acid interactions through SPR is essential for gaining insights into molecular-level binding mechanisms, thus supporting advancements in nucleic acid nanotechnology. The review also highlights the various sections of SPR applications in nucleic acid research, including nucleic acid-probe immobilization, interactions with biomolecules, aptamer studies, and small molecule binding, concluding with perspectives on future developments in the field.

Keywords: Bioconjugation; Biophysics; Dissociation Constant; Nucleic Acid Nanoparticles.

Fig. 1.. Working Principle of Surface Plasmon Resonance for Biomolecular Interaction Analysis.

Polarized light is directed at a metal-dielectric interface (e.g., a gold sensor chip surface). When light is incident at a specific angle (θ°), it excites surface plasmons, generating a resonance effect that decreases the intensity of reflected light. The resonance angle shifts in response to refractive index changes at the sensor surface, which occur when an analyte binds to an immobilized ligand. The resulting sensorgram (inset) graphically represents the interaction in real time, with the response signal (y-axis) indicating the binding event and dissociation phase as the analyte binds to and dissociates from the ligand on the sensor surface.

Fig. 2.. Common SPR conjugation strategies illustrating various SPR conjugation strategies used to immobilize ligands onto the sensor surface.

Panel 2A shows an NH2 sensor coupled to a carboxy-functionalized ligand using EDC/NHS chemistry, a common method for covalent bonding. Panel 2B demonstrates the use of a maleimide sensor with a thiol ligand, where the maleimide group covalently binds to the thiol group of the ligand. In Panel 2C, a biotin sensor is conjugated to a streptavidin ligand, exploiting the strong biotin-streptavidin affinity for stable immobilization. Panel 2D presents a thiol sensor coupled with a thiolated ligand, forming a covalent bond between the thiol group on the ligand and the sensor surface. Panel 2E depicts hydrophobic sensors used to immobilize hydrophobic ligands through hydrophobic interactions. Panel 2F illustrates the use of liposome sensors to immobilize vesicles, a strategy particularly useful in membrane protein studies. In Panel 2G, an NTA sensor is activated with Ni ions, which subsequently bind His-tagged proteins via affinity interactions. Panel 2H shows a carboxy sensor functionalized with aminofunctionalized protein A, which then binds IgG antibodies with high affinity, allowing the attachment of specific ligands. Finally, Panel 2I illustrates a carboxy sensor functionalized with amine-functionalized antibodies using EDC/NHS chemistry, enabling the attachment of ligands with high affinity.

Fig. 3.. Examples of SPR sensors used for the investigation of nucleic acids interaction.

A) Target hybridization kinetics as a function of probe density were analyzed, with probe density, measured by SPR, ranging from 2 × 10¹² to 12 × 10¹² molecules/cm²; figure adapted with permission from Ref # Copyright © 2001 Oxford University Press. B) Hybridization and dissociation data obtained using SPFS (represented by circles) are fitted with curves derived from the extended Langmuir adsorption model. Figure adapted from Ref # Copyright © 2001 Oxford University Press. C) Analysis of ribosomal protein S3 binding to human 8-oxoguanine DNA N-glycosylase 1; figure adapted from Ref # Copyright © 2004, American Chemical Society. D) SPR analysis of the pRNA-3WJ. Figure adapted from Ref #99; copyright © 2024, published by Cold Spring Harbor Laboratory Press for the RNA Society.

Fig. 4.

A. Schematic illustration of protein microarray synthesis from a DNA microarray via surface transcription and translation. Herein, dsDNA templates encode His6-tagged proteins captured by Cu(II)-NTA on detector elements. Figure adapted from Ref # Copyright © 2004, American Chemical Society. B. Example of sensitive SPR biosensor using ZnO@Au nanomaterial and a classical sandwich strategy with biotin-streptavidin for secondary signal amplification to detect human IgG (hIgG); figure adapted with permission from Ref # Copyright © 2022 Elsevier B.V.

Fig. 5.

Overview of the dual SPR-based assay for studying RNase H activity. (A) Initial baseline in buffer, where no interactions occur with immobilized oligonucleotides. (B) Injection of tested antisense oligonucleotides (AONs). The formation of the cON-R:AON duplex is detected in the upstream area, with no response in the downstream area. (C) RNase H mixed with AONs is injected. A reduction in the upstream sensor response indicates AON-mediated RNase H cleavage of cON-R, while cleaved fragments are recaptured in the downstream area, causing an increase in its sensor response. (D) Final baseline in the buffer. The change between the initial and final baselines in both sensing areas (a decrease upstream and an increase downstream) reflects the amount of cleaved probes. Figure adapted with permission from Ref # Copyright © 2010 Elsevier B.V.