Project Description

Project Background

The application of near-infrared II (NIR-II) fluorescent proteins in in vivo imaging presents several challenges, such as scarcity, the requirement for chromophore maturation, and limited emission wavelengths, typically below 800 nm. Despite recent advancements, the development of high-performance NIR-II fluorescent proteins is crucial for enhancing imaging depth and resolution in biological tissues.¹ Traditional NIR-II small-molecule fluorophores often lack the necessary optical and pharmacokinetic properties for clinical applications, highlighting the urgent need for improved methodologies to expand the NIR-II dye library and facilitate early clinical translation of NIR-II bioimaging techniques.²

Recent studies have demonstrated the potential of synthetic protein-seeking NIR-II dyes as chromophores that covalently bind to tag proteins, such as human serum albumin (HSA), through site-specific nucleophilic substitution reactions.³ This approach can be accomplished under gentle physiological conditions without the need for catalysis. For instance, in a study by Xu et al., a chemogenic protein-seeking strategy was employed to create biomimetic NIR-II fluorescent proteins using synthetic dyes like CO- 1080.³ This method significantly enhanced chromophore brightness and photostability, improved biocompatibility, and enabled high-performance NIR-II lymphography and angiography.

Fluorescence imaging has gained extensive attention across various multidisciplinary fields due to its high sensitivity, excellent spatial-temporal resolution, and strong specificity.^4-6 However, intravital deep-tissue imaging remains a significant challenge due to the optical opacity of biological tissues. The extension of fluorescence imaging to the NIR-II window, considered "tissue-transparent," has effectively addressed this issue, creating new opportunities for clinical diagnosis and prognosis.^7-9 With the advent and rapid expansion of NIR fluorescent proteins (FPs), researchers have been inspired to construct more NIR fluorescent probes.^10-11 The internal chromophore of fluorescent proteins is almost non-luminous by itself, but the unique β-canister structure formed through biological self-assembly restricts chromophore rotation and reduces non-radiative transition energy dissipation, resulting in bright luminescence.^12-13

Despite the success of various NIR fluorescent proteins (e.g., iRFP670, iRFP682, iRFP702, iRFP713, iRFP720), these fluorophores are genetically encoded and require slow and oxygen-dependent maturation, making large-scale production challenging.^14-15 Additionally, the lack of NIR-II peak emission fluorophores limits the benefits of NIR-II bioimaging. The development of fluorescent chemogenetic reporters, combining synthetic organic dyes with gene-encoded protein tags, offers improved optical properties and has opened new prospects for on-demand bioimaging and biosensing.^16-17

Inspired by these principles and driven by the urgent need for long-wavelength fluorescent proteins, we aim to design and optimize high-performance NIR-II fluorescent proteins for deep-tissue imaging and medical diagnostics. By mutating key amino acid sites in the DIII region of HSA and optimizing the binding conditions with suitable NIR-II dyes, we intend to overcome the limitations of current technologies and enhance the performance of these fluorescent proteins.

Project Objectives

Overall Objective: The primary goal of this project is to design and optimize high-performance NIR-II fluorescent proteins for deep tissue imaging and medical diagnostics.

Specific Objectives:

1. Mutate Key Amino Acid Sites in the DIII Region of HSA: By targeting specific amino acid sites in the DIII region of human serum albumin (HSA), we aim to enhance the efficiency of dye binding. This involves the strategic mutation of these sites to improve the interaction between the protein and the NIR-II dye.
   
   

   Domain analysis of NIR-II chromophores binding to HSA



2. Achieve Rapid Protein-Dye Binding Under Mild Conditions: One of the critical goals is to enable the rapid binding of the protein and dye under physiological conditions without the need for catalysts. This will involve optimizing the reaction conditions to ensure efficient and stable conjugation of the dye to the protein.
3. Enhance Photostability and Brightness of Fluorescent Proteins: Improving the photostability and brightness of the fluorescent proteins is essential for their application in biological imaging. This will involve testing and optimizing various NIR-II dyes and protein configurations to achieve the best performance in terms of fluorescence intensity and stability.

Research Methods

Protein Engineering: Utilizing site-directed mutagenesis techniques, we will mutate key amino acid sites in the DIII region of human serum albumin (HSA) to cysteine. This strategic modification is intended to enhance the efficiency and specificity of dye binding

Dye Selection and Optimization: We will select appropriate NIR-II dyes and optimize their binding conditions with the mutated HSA. This process involves screening various dyes and adjusting the reaction parameters to achieve optimal binding efficiency and stability.

Binding Experiments: Protein-dye binding experiments will be conducted under mild physiological conditions to ensure biocompatibility and practical applicability. Techniques such as spectroscopic analysis and gel electrophoresis will be employed to verify the binding efficacy and stability of the conjugates.

Performance Testing: The synthesized fluorescent proteins will be rigorously tested for photostability, brightness, and biocompatibility. These tests will include assessing fluorescence intensity under various conditions, long-term stability, and compatibility with biological systems.

Expected Results and Future Prospects

Theoretical Expectations:

Through the strategic mutation of HSA's DIII region and the optimization of binding conditions with NIR-II dyes, we anticipate the development of highly efficient NIR-II fluorescent proteins. These proteins are expected to exhibit superior brightness, photostability, and binding efficiency, making them suitable for advanced imaging applications. The theoretical foundation suggests that the combination of site-directed mutagenesis and dye selection will result in fluorescent proteins that outperform existing alternatives in terms of emission intensity and stability under physiological conditions.

Practical Applications:

The optimized NIR-II fluorescent proteins will have significant practical applications in high-resolution deep tissue imaging. These proteins are anticipated to provide unprecedented clarity and resolution in visualizing biological processes, thus facilitating accurate monitoring of complex biological events and medical diagnostics. The ability to achieve high-contrast images in deep tissue will be invaluable for various medical applications, including the precise detection of pathological conditions and the guidance of surgical procedures.

Technological Applications:

Looking ahead, the application of this technology in broader fields of biological imaging holds great promise. For instance, in oncology, these fluorescent proteins could enable the early detection and accurate localization of tumors, significantly improving treatment outcomes. In vascular imaging, the enhanced resolution and depth penetration could facilitate the detailed visualization of blood vessels, aiding in the diagnosis and management of vascular diseases.

Further Research:

Future research will explore the potential of combining different proteins and dyes to achieve a wider spectrum of fluorescent colors for multispectral imaging. This approach aims to create a versatile imaging platform capable of simultaneously visualizing multiple biological targets with high specificity and minimal interference. The development of such a platform could revolutionize the field of bioimaging, providing comprehensive insights into the dynamic interactions within biological systems.

Reference

1. Tian, R., Feng, X., Wei, L. et al. A genetic engineering strategy for editing near-infrared-II fluorophores. Nat Commun 13, 2853 (2022).
2. Zhu, S. et al. Repurposing Cyanine NIR-I dyes accelerates clinical translation of near-infrared-II (NIR-II) bioimaging. Adv. Mater. 30, e1802546 (2018).
3. Xu, J., Zhu, N., Du, Y. et al. Biomimetic NIR-II fluorescent proteins created from chemogenic protein-seeking dyes for multicolor deep-tissue bioimaging. Nat Commun 15, 2845 (2024).
4. Wu, X. et al. Tether-free photothermal deep-brain stimulation in freely behaving mice via wide-field illumination in the near-infrared-II window. Nat. Biomed. Eng. 6, 754-770 (2022).
5. Wang, S. et al. Fluorescence imaging of pathophysiological microenvironments. Chem. Soc. Rev. 50, 8887-8902 (2021).
6. Song, L. et al. Fluorescence microsphere probe for rapid qualitative and quantitative detection of trypsin activity. Nanoscale Adv. 1, 162-167 (2019).
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8. Li, C., Chen, G., Zhang, Y., Wu, F. & Wang, Q. Advanced fluorescence imaging technology in the near-infrared-II window for biomedical applications. J. Am. Chem. Soc. 142, 14789-14804 (2020).
9. Liu, M., Zhang, Z., Yang, Y. & Chan, Y. Polymethine-based semi-conducting polymer dots with narrow-band emission and absorption/emission maxima at NIR-II for bioimaging. Angew. Chem. Int. Ed. 60, 983-989 (2021).
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