Publication

Establishing a Ternary System for Optical Monitoring of DNA-Protein Interactions with Single-Walled Carbon Nanotubes

Shang-Jung Wu
2020
Thèse EPFL
Résumé

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 screening and protein engineering we were able to fine-tune the interaction between proteins and ssDNA-SWCNTs. In particular, we were able to identify the electrostatic interactions and solvent-exposed Asp to be the deterministic factor of non-specific adsorption. With proper amplifier designing by incorporating sp3-defects on SWCNTs, we envision that the detection of DNA-protein interactions via SWCNT NIR fluorescence can be accomplished in vitro and in vivo.

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Concepts associés (39)
Nanotube de carbone
thumb|Représentation d'un nanotube de carbone. (cliquer pour voir l'animation tridimensionnelle). thumb|Un nanotube de carbone monofeuillet. thumb|Extrémité d'un nanotube, vue au microscope électronique. Les nanotubes de carbone (en anglais, carbon nanotube ou CNT) sont une forme allotropique du carbone appartenant à la famille des fullerènes. Ils sont composés d'un ou plusieurs feuillets d'atomes de carbone enroulés sur eux-mêmes formant un tube. Le tube peut être fermé ou non à ses extrémités par une demi-sphère.
Interaction protéine-protéine
thumb|upright=1.2|L'inhibiteur de la ribonucléase en forme de fer à cheval (en représentation « fil de fer ») forme une interaction protéine–protéine avec la protéine de la ribonucléase. Les contacts entre les deux protéines sont représentés sous forme de taches colorées. Une Interaction protéine–protéine apparait lorsque deux ou plusieurs protéines se lient entre elles, le plus souvent pour mener à bien leur fonction biologique.
Acide désoxyribonucléique
vignette|Structure de la double hélice d'ADN. vignette|Structure chimique de l'ADN illustrant les quatre configurations des paires AT et GC entre les deux armatures de la double hélice, constituées d'une alternance de phosphate et de désoxyribose. L'acide désoxyribonucléique, ou ADN, est une macromolécule biologique présente dans presque toutes les cellules ainsi que chez de nombreux virus. L'ADN contient toute l'information génétique, appelée génome, permettant le développement, le fonctionnement et la reproduction des êtres vivants.
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