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Development of Novel Far-Red/Near-In...

Sheng, Wei.

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Development of Novel Far-Red/Near-Infrared Dye-hCRBPII Based Imaging Tags for Background-Free Live Cell Imaging.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Development of Novel Far-Red/Near-Infrared Dye-hCRBPII Based Imaging Tags for Background-Free Live Cell Imaging./
作者:	Sheng, Wei.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
面頁冊數:	325 p.
附註:	Source: Dissertations Abstracts International, Volume: 80-11, Section: B.
Contained By:	Dissertations Abstracts International80-11B.
標題:	Chemistry. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13865509
ISBN:	9781392144671

Development of Novel Far-Red/Near-Infrared Dye-hCRBPII Based Imaging Tags for Background-Free Live Cell Imaging.
Sheng, Wei.

Development of Novel Far-Red/Near-Infrared Dye-hCRBPII Based Imaging Tags for Background-Free Live Cell Imaging. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 325 p.

Source: Dissertations Abstracts International, Volume: 80-11, Section: B.

Thesis (Ph.D.)--Michigan State University, 2019.

This item must not be sold to any third party vendors.

Modern fluorescence imaging technologies, including deep-tissue imaging and super-resolution microscopies, require novel fluorescent labeling tags possessing non-conventional optical features, among which most desired ones are high brightness in the far-red/near-infrared (NIR) region and turn-on/off control in a spatiotemporal manner. Previously, we demonstrated the ability of fine tuning the absorption spectra of a protein-bound natural chromophore over an unprecedented range (474 ~ 664 nm). The goal of this PhD research is to exploit protein-ligand interactions for the development of protein-based pigments as NIR fluorescent tags for background-free live cell imaging.In the past half century, tremendous efforts have been invested in the optimization and derivatization of GFP-like fluorescent proteins (FPs). More recently, growing attention on phytochrome-based FPs has even upsized the repertoire of available FPs with many enhanced optical features. Giving this advancement, certain pitfalls are still limiting their uses in modern fluorescence imaging. In this context, synthetic dyes provide a broader chemical space for tailoring desired optic features including spectral wavelengths, brightness, stability, and many more photophysical and/or photochemical functionalities.To achieve high contrast imaging with minimal background interference, three different strategies have been applied here. 1) NIR emission is approached by utilizing a dye capable of specific complexation with a target protein via imine bond formation. Upon protonation of the imine, the complex experiences a large bathochromic shift as a result of a strong intramolecular charge transfer (ICT) process. A light-triggered imine isomerization is further incorporated to furnish a photoswitchable tag and negate the routine wash steps in live cell experiments. Rational protein engineering affords a faster variant that allows unprecedented spatiotemporal control of this no-wash bright NIR imaging. (2) A rare organic super photobase is identified, exhibiting a 14-unit change in pKa upon light excitation. Steady-state and ultrafast spectroscopic measurements ascribe this event to an excited-state proton transfer (ESPT) process. This ESPT feature is recapitulated in a protein-ligand micro-environment, yielding protein-dye complexes with extremely high fluorescence quantum yields (up to 92%) and large pseudo-Stokes shifts (> 200 nm). Our optimal mutant bound to the dye boasts millisecond binding rate and enables live cell imaging with negligible background. (3) A general approach to fluorogenicity, i.e., the ability to turn on fluorescence, is designed by coupling a quenching moiety capable of photoinduced electron transfer (PeT) to our dyes. The fluorescence is negligible before the Michael addition of engineered cysteine residue (the trigger) with the quencher moiety. A 30-fold fluorescence enhancement is achieved in vitro with an electronically tuned quencher group. Currently, further modifications are in progress to optimize the quenched system for in vivo applications.

ISBN: 9781392144671Subjects--Topical Terms:

516420
Chemistry.
Subjects--Index Terms:

Fluorescent dye

Development of Novel Far-Red/Near-Infrared Dye-hCRBPII Based Imaging Tags for Background-Free Live Cell Imaging.
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506 $a This item must not be sold to any third party vendors.
520 $a Modern fluorescence imaging technologies, including deep-tissue imaging and super-resolution microscopies, require novel fluorescent labeling tags possessing non-conventional optical features, among which most desired ones are high brightness in the far-red/near-infrared (NIR) region and turn-on/off control in a spatiotemporal manner. Previously, we demonstrated the ability of fine tuning the absorption spectra of a protein-bound natural chromophore over an unprecedented range (474 ~ 664 nm). The goal of this PhD research is to exploit protein-ligand interactions for the development of protein-based pigments as NIR fluorescent tags for background-free live cell imaging.In the past half century, tremendous efforts have been invested in the optimization and derivatization of GFP-like fluorescent proteins (FPs). More recently, growing attention on phytochrome-based FPs has even upsized the repertoire of available FPs with many enhanced optical features. Giving this advancement, certain pitfalls are still limiting their uses in modern fluorescence imaging. In this context, synthetic dyes provide a broader chemical space for tailoring desired optic features including spectral wavelengths, brightness, stability, and many more photophysical and/or photochemical functionalities.To achieve high contrast imaging with minimal background interference, three different strategies have been applied here. 1) NIR emission is approached by utilizing a dye capable of specific complexation with a target protein via imine bond formation. Upon protonation of the imine, the complex experiences a large bathochromic shift as a result of a strong intramolecular charge transfer (ICT) process. A light-triggered imine isomerization is further incorporated to furnish a photoswitchable tag and negate the routine wash steps in live cell experiments. Rational protein engineering affords a faster variant that allows unprecedented spatiotemporal control of this no-wash bright NIR imaging. (2) A rare organic super photobase is identified, exhibiting a 14-unit change in pKa upon light excitation. Steady-state and ultrafast spectroscopic measurements ascribe this event to an excited-state proton transfer (ESPT) process. This ESPT feature is recapitulated in a protein-ligand micro-environment, yielding protein-dye complexes with extremely high fluorescence quantum yields (up to 92%) and large pseudo-Stokes shifts (> 200 nm). Our optimal mutant bound to the dye boasts millisecond binding rate and enables live cell imaging with negligible background. (3) A general approach to fluorogenicity, i.e., the ability to turn on fluorescence, is designed by coupling a quenching moiety capable of photoinduced electron transfer (PeT) to our dyes. The fluorescence is negligible before the Michael addition of engineered cysteine residue (the trigger) with the quencher moiety. A 30-fold fluorescence enhancement is achieved in vitro with an electronically tuned quencher group. Currently, further modifications are in progress to optimize the quenched system for in vivo applications.
590 $a School code: 0128.
650 4 $a Chemistry. $3 516420
650 4 $a Organic chemistry. $3 523952
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653 $a Imaging probe
653 $a Near-infrared cell imaging
653 $a Organic chemistry
653 $a Photochemistry
653 $a Protein engineering
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