Publication
DNA-protein interactions lie at the crux of life's essential processes. As such, various technologies have been developed to characterize these interactions. The distinct advantages of these technologies can be leveraged to study different facets of these interactions. In this thesis, we aim to establish a ternary system for optical monitoring of DNA-protein interactions. Single-walled carbon nanotubes (SWCNTs) are especially good for optical detection because of their photostable near-infrared (NIR) fluorescence, but so far it has been limited for use as binary systems. These systems are used to detect biomolecule interaction with only DNA or only protein, but unable to examine more complex systems that include DNA-protein interactions. This thesis overcomes this limitation by developing a ternary system consisting of immobilized DNA and protein on nanotubes. We demonstrate the applicability of this system as a new platform for studying protein binding and activity on DNA. Additionally, our biocompatible system serves as a novel bioconjugative approach for protein immobilization on SWCNTs, where we aim to combine the reactivity and orthogonality of DNA, the target selectivity of proteins, and the sensitive NIR fluorescence of SWCNTs, which is not achievable in any state-of-the-art biosensors. In this context, this thesis was divided into three stages: (1) resolving the DNA-SWCNT structure, (2) applications of DNA-protein-SWCNT and (3) improving the ternary hybrid for fluorescence detection. First, we elucidated the structure-function relationship of double-stranded DNA (dsDNA)-SWCNT. Previously, the duplex structure of DNA on SWCNTs was debatable. We performed a restriction enzyme (RE)-based electrophoretic assay on DNA-SWCNT hybrids with various DNA sequences. We suggested a model with B-DNA lying on the SWCNT along the long axis of the SWCNT. Additionally, we developed an analytical assay based on DNA-specific fluorescence dyes and single-stranded DNA (ssDNA)-specific nuclease, enabling the analysis at higher throughput and providing a universal method for routine analysis. Overall, we successfully validated the Watson-Crick base pairing of dsDNA on SWCNTs for the first time. Secondly, we showcased the sensing applications of DNA-protein-SWCNT in two different configurations: (1) DNA-protein conjugates and (2) DNA-protein interactions. In the first case, we bridged proteins to SWCNTs via dsDNA bridges, which was verified by fluorescence microscopy. We developed a facile approach to construct new materials for specific analyte detection, as we showed in an example of DNA-horseradish peroxidase (HRP)-SWCNT, which detects hydrogen peroxide. In the second case, we found that both RE and Cas9 remain enzymatically active and are able to digest specific sequences on SWCNTs. Correspondingly, we suggested a potential application of DNA-protein interactions within sensors based on SWCNT NIR fluorescence. Finally, we demonstrated that by protein library