Applications of functional dyes in biomedicine and life sciences
Huang, Jingyi
DOI:
10.33612/diss.123828369
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Publication date: 2020
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Huang, J. (2020). Applications of functional dyes in biomedicine and life sciences. University of Groningen. https://doi.org/10.33612/diss.123828369
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145
Chapter 6
Outlook and Future Perspectives
146
Through the work carried out in this thesis, novel functional dyes have been developed for applications in biomedicine and life sciences. Two main areas were explored: self-healing fluorescent probes for super-resolution microscopy described in chapter 2 and chapter 3; photoswitchable biomolecules for photopharmacology and optogenetics described in chapter 4 and chapter 5. This last section summarises some perspectives on how to further improve the photophysics of synthetic organic fluorophores with the intramolecular photostabilization property and explore novel bio-applications for the photoswitchable biomolecules. As for the super-resolution microscopy, intramolecular photostabilization of fluorescent dyes is still less efficient compared to the use of diffusion-based photostabilization. The unnatural amino acids scaffolding as presented in Chapter 2 and Chapter 3 allows for a further discovery and improvement of photostabilizer-dye conjugates by varying the photostabilizing molecules (Figure 6.1). Additionally, for oxazines and perylenes, these types of fluorophores are expected to show different photophysical behaviors after conjugated to nitrophenyl moiety, considering their interaction with antioxidants1-5, amino acids and DNA bases6–9.
Figure 6.1: Triplet state quenchers: ascorbic acid (AA), N,N-methyl viologen (MV),
1,3,5,7-cyclooctatetraene (COT), 4-nitrobenzyl alcohol (NBA), Trolox (TX) and 1,4-diazabicyclo[2.2.2]octane (DABCO).
Furthermore, in this thesis, only redox active compounds containing Trolox (TX) or a nitrophenyl group were tested for the development of a general synthetic strategy of photostabilizer-dye conjugates. It would be interesting to explore how Dexter-type triplet state quenchers, such as cyclooctatetraene (COT),10-12 and diphenylhexatriene (DPHT),13 perform in photostabilizer-dye conjugates. Other antioxidants might be interesting for usage in intramolecular photostabilization, such as MEA, n-propyl gallate (nPG)1 and ascorbic acid (AA)1. Studies on the
147 mechanism from these compounds in solution should be the starting point to explore new compounds for intramolecular photostabilization.
One of the most important goals in chemical and synthetic biology is controlling the expression of defined sets of genes by external stimuli. Such systems allow switching on or off at will the production of proteins of interest, exogenously controlling cell function or constructing increasingly complex gene networks with unprecedented control.14 One of the most attractive stimuli is light. However, current approaches to the photocontrol of biological processes utilize photoresponsive proteins derived from natural photoreceptors or their domains. The photocontrol of gene expression at the RNA level by using aptamers against photoisomerizable small molecules has not been achieved yet.
In chapter 4 and chapter 5, we prepared a photoswitchable ligand. the two isomeric forms of which can be distinguished by a well characterized RNA aptamer, with a 100-fold discrimination and a micromolar binding dissociation constant. The binding can be altered by visiable light. Having performed this research, we are particularly called to design complex synthetic biological devices. Since this is the first step towards the goal of assembling a light-switchable gene control element at the RNA level, future tasks deal with triggering various gene expression systems in a system-independent way.
Figure 6.2: Control of bacterial gene expression by aptamers against photoswitchable small
molecules as genetic control elements. In the present case, the aptamer recognizes only one isomer of the photoswitchable target, represented as the green circle.
148
As a concept shown in Figure 6.1, in order to achieve photocontrol of protein production at the RNA level, a novel artificial photoresponsive gene expression system is designed. In this system, the RNA aptamer is a part of artificial riboswitch which is comprised of a sensor that can selectively bind with only one isomer of the photoswitchable small signaling molecule, an actuator that controls the output signal and a transmitter that channels the signal from the sensor to the actuator. Upon binding with one isomer represented as the green circle, the riboswitch undergoes a structural rearrangement, which can unveil the ribosome binding site and hence activate the downstream gene expression. This process is expected to be reversible.
The possibility of this concept can be of great help in the study of gene expression dynamics as well as to develop photocontrolled biotechnological procedures. Also, different ligands targeted to different aptamers will furthermore provide the opportunity to address several switches in parallel by using light of different wavelengths, and thereby enable complex switching decisions.
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