| Author: | Chow, Chiu Lok Joshua |
| Title: | Femtosecond time-resolved spectroscopic study on excited state dynamics of DNA and RNA guanine quadruplexes and their binding interaction with Thioflavin T |
| Advisors: | Kwok, Wai Ming (ABCT) |
| Degree: | Ph.D. |
| Year: | 2026 |
| Subject: | Quadruplex nucleic acids Fluorescent probes Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xlii, 265 pages : color illustrations |
| Language: | English |
| Abstract: | Guanine-quadruplexes (GQs) are non-canonical, four-stranded nucleic acid structures with diverse topologies. They are essential for genome stability and play a crucial role in biotechnology. Consequently, probing GQs has become a significant research focus. However, the factors governing the intrinsic fluorescence dynamics and deactivation pathways of GQs, as well as those of the fluorescent ligand Thioflavin T (ThT) when probing GQs, remain insufficiently understood. This thesis addresses this gap by employing steady-state and femtosecond broadband time-resolved spectroscopic techniques to systematically investigate the excited-state dynamics of GQs and ThT binding with GQs. First, intrinsic fluorescence dynamics of human telomeric and promoter region DNA GQs with varying structures were characterized. Human telomeric GQs exhibit a deactivation mechanism involving two long-lived charge transfer (CT) G-G excimers coupled with proton transfer (PT) in the core region. The energy relaxation and lifetimes of these CT excimers are governed by the G-tetrad stacking mode and planarity. For instance, a larger overlapping region between neighboring G-tetrads promotes greater CT character, resulting in lower-energy radiative relaxation. In a more planar G-tetrad, PT is further facilitated, which extends the CT excimer lifetime. In contrast, promoter region GQs lack obvious excimers with CT behavior and instead display long-lived neutral excimers and previously unreported CT exciplex states. The CT exciplex involves PT between guanine bases in the G-tetrad and thymidine or 2′-deoxyadenosine in the loop region, resulting in markedly red-shifted fluorescence. Second, human telomeric RNA GQs reveal multichannel deactivation pathways, including ultrafast (<1 ps) CT formation, which is not observed in DNA GQs, and a high-energy CT excimer deactivated rapidly via PT in the tetrad core. The emission energy of these CT excimers is similar to that of neutral excimers (~330 nm) in DNA GQs. The different nature of these excimers can be attributed to the greater structural rigidity of RNA GQs compared to DNA GQs. On the other hand, there is a unique exciplex without PT involvement, exhibiting strongly red-shifted fluorescence and originating from CT between adenosine and uridine bases in the loop region. Third, the fluorescence dynamics of ThT were elucidated. ThT's fluorescence originates from a locally excited state and is quenched after transition to a twisted intramolecular charge transfer (TICT) state. The binding of ThT to GQs inhibits the formation of the TICT state. The degree of inhibition is dependent on GQ topology, resulting in various degrees of fluorescence enhancement. Tighter binding sites more strongly restrict the twisting of ThT, leading to greater fluorescence enhancement. Furthermore, an ultrafast FRET process (<3 ps) occurs in GQ-ThT complexes after GQs excitation, with GQs acting as the donors and ThT as the acceptor. The efficiency of FRET is based on the GQ structure and the ThT binding mode. These insights into the excitation dynamics of GQs and bound ThT in GQ advance the understanding of GQ and ThT deactivation mechanisms and facilitate the development of GQ-based probing techniques and nanobiotechnological applications. |
| Rights: | All rights reserved |
| Access: | open access |
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