High resolution micro-confocal Raman spectrometer-based photo-affinity microarray technology for the investigation of active ingredients - Target protein recognition strategy

Natural products has been used for the prevention and treatment of diseases for a long history. Research on the bioactive components from natural products and their interaction with target proteins are essential for drug discovery. However, studying the binding ability of natural products' active ingredients to target proteins is usually time-consuming and laborious due to their complex and diverse chemical structures. In this study, we have developed a high resolution micro-confocal Raman spectrometer-based photo-affinity microarray (HRMR-PM) technology for the investigation of active ingredients-target protein recognition strategy. The novel photo-affinity microarray was constructed by photo-cross-linking the small molecule with the photo-affinity group (4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzoic acid, TAD) on the photo-affinity linker coated (PALC) slides under 365 nm ultraviolet irradiation. The small molecules on the microarrays with specific binding ability might immobilize target protein, which were characterized by high resolution micro-confocal Raman spectrometer. Using this method, more than a dozen components of Shenqi Jiangtang granules (SJG) were made into small molecule probe (SMP) microarrays. As a result, 8 of them had been identified to have α-glucosidase binding ability according to characteristic Raman shift at about 3060 cm-1. These compounds were further verified by different small molecule-protein interaction analysis methods, including contact angle D-value, surface plasmon resonance (SPR) and molecular docking. The results showed that Ginsenosides Mb, Formononetin and Gomisin D exhibited the strongest binding ability. In conclusion, the HRMR-PM strategy for investigating the interaction between target proteins and small molecules has the advantages such as high throughput, low sample consumption and fast qualitative characterization. This strategy is universal which can be applied in the study of in vitro binding activity of various types of small molecules to target proteins.