Fluorescent nucleic acid probes for DNA sensing and imaging


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Leck, Georgina Helen (2021). Fluorescent nucleic acid probes for DNA sensing and imaging. University of Birmingham. Ph.D.

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Fluorescent probes are widely used to study biological samples. In recent years, fluorescently modified nucleic acid probes have been investigated for the detection of DNA single point variations. Single point variations have been linked to certain diseases with a genetic component and are viewed as biomarkers. Despite recent advances in the detection of such biomarkers, commercially available detection assays of this type are often time-consuming, complex in experimental design and tend to produce only qualitative data (i.e. simply denoting whether a mutation is present or not). As complex diseases such as cancer are becoming better understood, and in particular the way in which these diseases develop and mutate, there is a drive for the development of versatile probes that can detect several mutations at once, while also providing quantitative data (e.g. the proportion of a cancerous mutation present within a given sample). The work herein describes the development of such probes which aim to overcome the challenges that are currently faced.

The fluorescent probes developed in this thesis are simple in design, comprising a short nucleic acid sequence, which is complementary to the region of interest, and a fluorescent molecule incorporated directly into the sugar-phosphate backbone as a reporter group.

1,8- Naphthalimides were the choice of fluorophore for the majority of this work, owing to their longer wavelength absorption and emission profiles in comparison to many nucleic acid probes in the literature, their tuneable photophysical properties, large Stokes shift and photostability. They are also solvatochromic, giving emission profiles that are highly sensitive to their local environment. This effect has been explored as a method for discriminating between single nucleobases within a target sequence of DNA. In addition, their synthetic versatility has allowed the development of four different classes of fluorescent tag for probe incorporation, each with varying photophysical properties and DNA sensing capabilities.

Owing to their longer wavelength absorption and emission profiles, the naphthalimide probes are also suitable for use in imaging techniques; this aspect has been explored using fluorescence in-situ hybridisation (FISH) and DNA-PAINT.

Finally, building on work previously studied in the group, anthracene-based nucleic acid probes have also been developed. These probes were effective at sensing and quantifying the BRAF V600E mutation, a single point variation in genomic DNA associated with many types of cancer such as melanoma and colorectal cancer. Work in this thesis builds on the development of these probes into assays for use on patient samples.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Engineering & Physical Sciences
School or Department: School of Chemistry
Funders: Engineering and Physical Sciences Research Council
Subjects: Q Science > QD Chemistry
URI: http://etheses.bham.ac.uk/id/eprint/12026


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