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Near-infrared Optical Probing of Protein Interactions on Carbon Nanotubes

Applicant Boghossian Ardemis
Number 184822
Funding scheme Project funding (Div. I-III)
Research institution Institut des sciences et ingénierie chimiques EPFL - SB - ISIC
Institution of higher education EPF Lausanne - EPFL
Main discipline Chemical Engineering
Start/End 01.04.2020 - 30.09.2023
Approved amount 798'226.00
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All Disciplines (4)

Discipline
Chemical Engineering
Material Sciences
Biochemistry
Biophysics

Keywords (6)

single-walled carbon nanotube; CRISPR; glucose oxidase; Cas9; near-infrared fluorescence; binding peptide

Lay Summary (French)

Lead
Les protéines jouent un rôle crucial dans le maintien de la vie et le maintien d'une bonne santé. Dans l'exercice de leurs fonctions, ils interagissent souvent avec diverses molécules, ADN et autres protéines. Une compréhension de ces interactions est importante pour créer des technologies biomédicales basées sur les protéines. Dans ce projet, nous visons à développer une nouvelle plateforme qui permettra aux chercheurs d'étudier le comportement et les interactions des protéines à l'aide de la lumière. Nous nous concentrons sur de nouvelles façons biochimiques de relier les protéines aux nanoparticules qui émettent de la lumière à des longueurs d'onde particulières qui ne sont pas absorbées par le matériel biologique, comme le sang, l'eau et la peau. Nous pouvons donc surveiller les interactions protéiques en surveillant les changements dans cette lumière, même lorsque les interactions se produisent à l'intérieur du corps humain.
Lay summary
Objectifs du projet de recherche au début de la recherche et résultats après l'achèvement du projet:
Le principal objectif du projet est de développer des moyens efficaces de relier les protéines aux nanoparticules émettrices de lumière appelées nanotubes de carbone à paroi simple. Plus précisément, nous souhaitons étudier (i) la liaison directe des protéines à la surface du nanotube, (ii) la liaison des protéines à l'ADN qui adhère à la surface du nanotube, et (iii) la liaison indirecte des protéines à la surface du nanotube en utilisant l'ADN comme lien de connexion . Ces approches seront utilisées comme base pour de nouvelles avancées biomédicales, y compris des technologies pour surveiller les niveaux de glucose dans le sang et pour surveiller les interactions protéiques pour l'édition du génome, en utilisant la lumière.

Contexte scientifique et sociétal du projet de recherche:
Ce projet permettra de réaliser de nouvelles technologies de détection optique pouvant être utilisées en présence de fluides biologiques ou même dans le corps humain. Ces technologies peuvent être utilisées non seulement pour diagnostiquer et traiter des maladies, mais aussi pour concevoir des protéines thérapeutiques.
Direct link to Lay Summary Last update: 13.02.2020

Responsible applicant and co-applicants

Employees

Abstract

The fluorescence emissions of single-walled carbon nanotubes (SWCNTs) share a unique combination of desirable properties that make them ideal for optical sensing applications. They benefit from near-infrared (NIR) emissions that are distinct from the absorption and autofluorescence of biological tissue and proteins, enabling their use for in vivo sensing as well as characterization of autofluorescent cells. Their indefinite photostability allows continuous, long-term optical monitoring. Furthermore, their nanometer size coincides with the length scale of typical protein interactions. Combined with their single-molecule sensitivity limits, SWCNTs represent a formidable material for sensitive optical monitoring applications in the life sciences. Despite their growing use for small molecule detection, their use in monitoring more complex and specific biological interactions, such as protein-protein and protein-DNA, remains limited. The lack of effective bioconjugative techniques and fundamental understanding of protein-SWCNT interactions prevents this nanomaterial from optically characterizing foundational protein-based interactions.The goal of this proposal is to develop, compare, and improve bioconjugative protein-SWCNT approaches and to apply these approaches to new biosensing and protein monitoring applications. The proposed study consists of three objectives that focus on three distinct modes of protein immobilization onto the SWCNT surface: 1. Characterization of nonspecific protein adsorption onto the SWCNT surface; 2. Study of specific protein binding to immobilized double-stranded DNA (dsDNA) on the SWCNT surface; and 3. Orientated immobilization of proteins onto the SWCNT surface using a DNA hybridization method recently developed in our lab. In the first objective, we screen the binding affinity of a library of computationally designed peptides towards SWCNTs. We also screen the non-specific adsorption of a selection of common proteins onto SWCNTs and identify binding motifs through structure and sequence alignment. These results, combined with the results of the peptide affinity screen, will be used in designing amino acid-based protein anchors that will facilitate protein adsorption. These anchors will be specifically applied to enhance immobilization of glucose oxidase (GOx), a model protein with applications in optical NIR SWCNT-based glucose sensing. In the second objective, we focus on using SWCNT fluorescence to monitor protein-dsDNA interactions. We will verify enzyme binding and DNA-cutting on dsDNA that is immobilized onto the SWCNT surface using gel electrophoresis. The NIR SWCNT fluorescence will be used to monitor enzyme activity, and single-molecule measurements will be used to elucidate enzyme mechanism. This platform will be applied to restriction enzymes, which serve as model enzymes with known mechanisms of DNA restriction, and Cas9, a CRISPR-based enzyme that lacks a detailed mechanistic understanding. Finally, in the third objective, we will develop and apply a new bioconjugative approach to immobilizing proteins onto the SWCNT surface based on dsDNA hybridization. Cysteine-mutated proteins will be covalently conjugated to single-stranded DNA (ssDNA). Protein immobilization will occur through hybridization of the ssDNA with an adsorbed ssDNA strand on the SWCNT surface. This strategy will allow us to immobilize proteins while keeping the sp2 bonds of the SWCNT intact, a necessary requirement for preserving the NIR fluorescence. This strategy will be compared to existing bioconjugative strategies and applied to several proteins, including GOx, to demonstrate its versatility and applicability to NIR glucose sensing. By exploring three distinct immobilization strategies, non-specific protein adsorption, specific protein-DNA binding, and anchoring through dsDNA hybridization, we allow ourselves previously unfounded ways for NIR optical monitoring of protein interactions. Although this proposal specifically focuses on applications related to NIR glucose sensing and monitoring single-molecule Cas9 activity, this platform can be expanded to general NIR monitoring of protein interactions.
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