Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance

Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR. 1. Introduction DNA-protein interactions are involved in several biological processes like transcription, replication, DNA repair, or recombination. The specificity of such recognition processes originates from direct and indirect readout mechanisms. The variety of these mechanisms involves variations of the electrostatic potential due to groove narrowing or specific hydrogen bond donors and acceptors of the DNA helix that are recognized by a complementary set of amino acids [1]. Several methods have been developed to analyze DNA-protein interactions. Generally, they can be divided into two groups. Label-based methods require the ligation of the analyte and/or ligand with reporters like enzymes, fluorescent dyes, or radioisotopes. These labels possess the disadvantage that they can adulterate the results by interfering with the molecular interaction. Blocking the active binding site or affecting the conformation of the analyte can lead to false negatives. Moreover, unspecific background binding leading to false positives is another issue in these assays [2, 3]. In label-free approaches like atomic force microscopy-dynamic force spectroscopy experiments [4, 5], acoustic biosensors based on quartz crystal resonators [6], calorimetric biosensors [7], and surface plasmon resonance (SPR) inherently properties (e.g., mass) of the interacting molecules are measured. Therefore, these techniques avoid labeling steps and the disadvantages mentioned above. This article will focus on the most widely used label-free detection method: