Parallel synthesis and screening of optical probes for G-quadruplex DNA
File(s)
Author(s)
Majid, Aatikah
Type
Thesis
Abstract
In addition to the well-known double helix, DNA has the ability to form other non-canonical structures. One of the most stable alternative secondary structures is the guanine-quadruplex (G4) DNA. Sequences with the potential to form G4s in the human genome are prevalent in telomeres, transcriptional and translational regions. Therefore, G4s have been proposed to play an important role in DNA replication, regulation of gene expression, RNA translation and telomere maintenance. However, the detection and visualisation of these structures in live cells still proves challenging.
Unlike fluorescent intensity probes, which are concentration dependent, fluorescence lifetime probes can more easily visualise G4s even with a large excess of duplex DNA present. Previous work in our group has shown that polyaromatic triangulenium probes can be used to differentiate between different DNA topologies based on changes in lifetime, which presents a superior detection method as compared to fluorescence intensity. However, predicting optical properties of probes is not trivial task and the synthesis and screening of individual probes is a time-consuming and labour-intensive process.
This thesis presents the use of a semi-automated platform for the synthesis and screening of fluorescence lifetime probes for G4s based on the triangulenium and Thiazole Orange scaffolds. Two libraries based on these scaffolds were synthesised and screened in parallel to evaluate their ability to act as fluorescence lifetime-based probes for G4s. This led to the successful discovery of two new fluorescence lifetime probes, one from each series.
Interestingly, the library based on the triangulenium scaffold showed a novel switch-on/switch-off effect in the presence of G4s and duplex DNA respectively, allowing these probes to behave as both fluorescent intensity and fluorescence lifetime probes. The cause of this interesting photo-physical effect was investigated, showing the binding conformation of these probes greatly affects the fluorescence lifetime.
Unlike fluorescent intensity probes, which are concentration dependent, fluorescence lifetime probes can more easily visualise G4s even with a large excess of duplex DNA present. Previous work in our group has shown that polyaromatic triangulenium probes can be used to differentiate between different DNA topologies based on changes in lifetime, which presents a superior detection method as compared to fluorescence intensity. However, predicting optical properties of probes is not trivial task and the synthesis and screening of individual probes is a time-consuming and labour-intensive process.
This thesis presents the use of a semi-automated platform for the synthesis and screening of fluorescence lifetime probes for G4s based on the triangulenium and Thiazole Orange scaffolds. Two libraries based on these scaffolds were synthesised and screened in parallel to evaluate their ability to act as fluorescence lifetime-based probes for G4s. This led to the successful discovery of two new fluorescence lifetime probes, one from each series.
Interestingly, the library based on the triangulenium scaffold showed a novel switch-on/switch-off effect in the presence of G4s and duplex DNA respectively, allowing these probes to behave as both fluorescent intensity and fluorescence lifetime probes. The cause of this interesting photo-physical effect was investigated, showing the binding conformation of these probes greatly affects the fluorescence lifetime.
Version
Open Access
Date Issued
2024-01-05
Date Awarded
2024-04-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Kuimova, Marina
Vilar, Ramon
Thompson, Alex
Sponsor
Engineering and Physical Sciences Research Council
Grant Number
CHSA-G98193
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)