Investigation of Replication protein A – Werner syndrome helicase interactions and application of Replication protein A - aptamer interactions to biosensor

Abstract

RPA70 (replication protein A) is an ssDNA binding proteinRPA70 plays a very important role in genetic stability through interactions with various proteins. This study can be divided into three chapters. The first is the study of the interaction between RPA70 and WRN (Werner's syndrome protein), and the second is the study of biosensors using RPA70. The third is the study about improvement of RPA70A performance in biosensors. Chapter Ⅰ describes a study of the interaction between RPA70 and WRN. Werner syndrome protein (WRN) is human DNA helicases that contribute to genome maintenance. They interact with replication protein A (RPA), and these interactions dramatically enhance the unwinding activities of both helicases. Even though the interplay between these helicases and RPA is particularly important in the chemo resistance pathway of cancer cells, the precise binding regions, interfaces, and properties have not yet been characterized. Here we present systematic NMR analyses and fluorescence polarization anisotropy assays of both helicase- RPA interactions for defining core binding regions and binding affinities. Our results showed that two acidic repeats of human WRN bind to RPA70N and RPA70A. Our results suggest that each helicase interaction has unique features, although they both fit an acidic peptide into abasic cleft for RPA binding. Our findings shed light on the protein interactions involved in overcoming the DNA-damaging agents employed in the treatment of cancer and thus potentially provide insight into enhancing the efficacy of cancer therapy. Chapter Ⅱ describes a development of biosensor using RPA70. Most prevalent infectious diseases worldwide are caused by mediators such as insects and characterized by high mortality and morbidity, thereby creating a global public health concern. Therefore, a sensitive, selective detection platform for diagnosing diseases in the early stages of infection is needed to prevent disease spread and protect public health. Here, we use DNA aptamers specific to the nucleocapsid protein (NP) of the severe fever with thrombocytopenia syndrome (SFTS) virus and synthesized ssDNA-binding protein-conjugated liposomes encapsulated with horseradish peroxidase (HRP) for application in a simple and universal platform. This platform achieved highly sensitive detection of the NP by measuring the colorimetric signal following lysis of the HRP encapsulated liposomes, mediated by a mixture of 3,3’,5,5’- tetramethylbenzidine and H2O2 solution. The limit of detection was 0.009 ngꞏmL-1, and NP was successfully detected in diluted human serum with a high recovery rate. Moreover, this method was specific and did not exhibit cross-reactivity among NPs of other virus types. These results demonstrated the efficacy of the proposed method as a highly sensitive, specific, and universal diagnostic tool for potential application in monitoring of the early stages of infectious diseases. Chapter Ⅲ shows the improvement of RPA70A performance in biosensors through protein engineering. This study aimed to develop a simple, universal, and sensitive colorimetric biosensor to detect various biomarkers, using target-specific aptamer as recognition element and streptavidin-fusion replication protein A (binding domain A of human Replication Protein A of 70 kDa) linked to biotin-horseradish peroxidase (i.e., STARPA70A@ biotin-HRP) with a colorimetric assay. The proposed assay is based on a colorchange from white to blue owing to interactions between the aptamer and RPA70A in the presence of the target, observed at visible light or a UV-vis spectrometer. Consequently, lowconcentration targets were effectively detected owing to interactions between aptamers with RPA70A. Furthermore, the linear range of the proposed sensing platform was from 1 pgꞏmL-1 to 100 ngꞏmL-1; detection limit, 0.2 pgꞏmL-1. This universal colorimetric assay is a suitable alternative to screen disease biomarkers aimed at sensitive detection, owing to its simplicity, sensitivity, and specific.