專一性的蛋白質-DNA/RNA結合在基因調控中扮演著關鍵的角色。在胚胎發育中，TDP-43是個必要的保守DNA/RNA結合蛋白。在2014年，Saldi等人以生物資訊及實驗發現並證實TDP-43是 一個RNA伴護蛋白並具有解開雙股螺旋的RNA轉譯產物之能力。因此，我們嘗試著去切分TDP-43的區域功能並利用螢光非等向性(Fluorescence Anisotropy)、螢光電泳移動位切試驗(F-EMSA)、分子動態電腦模擬(MD simulations)以及螢光恢復試驗(Fluorescence Recovery Assay)等來量測與驗證其與DNA/RNA的結合特性，如結合常數(Kd)、希爾方程(hill coefficient)、結合當量及解旋活性。TDP-43的RNA結合相關區域(包含著RRM1及RRM2及RRM1加成RRM2的RBD區域)其對應於DNA/RNA的結合常數顯示TDP-43偏好與RNA結合更甚於與DNA結合，而分子動態電腦模擬也呼應這一點結論。TDP-43與(TG)6的結合當量結果也印證其結合時協同作用的可能性。在量測TDP-43的結合當量的過程中，我們發現了螢光電泳移動位切試驗在分析方法上可能的偏差，因為蛋白質與核酸的結合過程中可能會影響到螢光標定物的量子產率(Quantum Yield)，而此現象已被歸類為蛋白誘導螢光增強(PIFE)現象。透過分析電泳膠體上非結合的螢光標定核酸區塊，我們得到相似且更好的分析結果對比於傳統的蛋白結合區塊分析方法。此外，分子動態電腦模擬的結果亦符合相對應的實驗結論，其能量的計算以及生物資訊學上保守分數的計算顯示出RRM1對應RRM2中相同或相異的殘基提供了一些演化學上的可能觀點。從實驗結果得知RRM1對比於RRM2有較高的(TG)6核酸結能力同時透過專一的結合能力RRM1具有較強的雙股解旋能力。另外，透過分子電腦模擬的結果分析揭示TDP-43是藉由與鳥苷(Guanosine，一個於RNA折疊的關鍵核苷酸)的結合達成與(TG)6專一性的結合。沿此線索，我們亦發現TDP-43的RRM1可能具有解開形成人類端粒DNA結構:G-四聯體(G-quadruplex)的能力，而相關的研究仍繼續進行中。

Sequence specific protein-DNA/RNA recognition plays a key role in genetic regulations. A conserved DNA/RNA binding protein, TDP-43, is essentially involved in embryonic development and neurodegenerative disease formation. In 2014, Saldi et al. discovered that TDP-43 possesses RNA chaperone activity and has the ability to resolve the double stranded structure of RNA transcripts. Therefore, we try to understand the DNA/RNA binding properties and RNA chaperone activities of TDP-43 by dissecting it into domain functions through determining constant (Kd), hill coefficient (nH), stoichiometry, and unwinding activity by fluorescence anisotropy (FA), fluorescence based electrophoretic mobility shift assay (F-EMSA), fluorescence recovery assay, and molecular dynamic (MD) simulations. The Kd of TDP-43’s RNA binding domains (RBD, which is composed of RRM1 and RRM2) demonstrates the binding preference of RNA to DNA. The stoichiometry confirms the cooperative binding mode of RRM1 and RBD to ss(TG)6. During determining the stoichiometry of protein-nucleic acids interactions by quantifying the fluorescence intensity in F-EMSA, we have found a potential bias of bound fraction analysis since the protein induced fluorescence enhancement (PIFE) phenomenon could influence the quantum yield of the fluorophore on nucleic acids. Through analyzing the free-fraction nucleic acids in F-EMSA, we get similar and better result of bound-fraction analysis. Furthermore, our MD simulations also agree with the binding properties and stoichiometry of RNA binding domains. The energy calculation of MD simulations and conservation scores of RRM1 and RRM2 reveal the similar and different binding sites of RRM1 versus RRM2 in evolutionary point of view. RRM1 possessing higher binding affinity and conservation than RRM2 can facilitate the unwinding of double stranded DNA through specific interaction with its target, ss(TG)6. In addition, the analysis of MD simulations upon nucleic acids illustrates the important interaction of Guanosine – a key player in RNA folding/misfolding mechanism. To follow the clue, we discover a human telomeric G-quadruplex can also be resolved by the RRM1 domain of TDP-43, and the related project is on going.

Abstract (6)
摘要 (8)
Acknowledgement (9)
Chapter 1: Introduction (10)
1.1: The importance of TDP-43 (11)
1.2: The domain function of TDP-43 (11)
1.2.1: The N-terminus of TDP-43 promotes TDP-43 oligomerization (12)
1.2.2: The biophysical properties of TDP-43 RRM1 and RRM2 (12)
1.2.3: The functional role of TDP-43 C-terminus (13)
1.3: The importance of RNA chaperone activity and TDP-43 (13)
1.4: Neurodegenerative diseases and telomere shortening (14)
1.4.1: The structure and function of human telomere (14)
1.4.2: The regulatory role of Guanine-rich sequence, G-quadruplex (15)
1.4.3: TDP-43 is a G-quadruplex binding protein involved in cellular transportation (15)
1.5: Specific Aims (16)
Chapter 2: Material and Methods (18)
2.1: Protein purification (19)
2.2: Quantification of nucleic acid oligos (20)
2.3: Fluorescence based electrophoretic mobility shift assay (F-EMSA) (21)
2.4: Image analysis for protein-ssDNA stoichiometry determination (21)
2.5: Fluorescence anisotropy assay for binding constant and hill coefficient measurement (22)
2.6: F-EMSA for dsDNA/dsRNA unwinding (23)
2.7: Fluorescence recovery for time-course unwinding assay (23)
2.8: MD simulations (24)
2.8.1: Preparation of the initial 3D structures (24)
2.8.2: Creation of input files for MD Simulations (25)
2.8.3: Energy minimization (25)
2.8.4: Equilibration and production run (25)
2.8.5: Binding Gibbs free energy change calculations using MM/PBSA (26)
2.8.6: ABMD Simulations Method (26)
2.8.7: Protein-nucleotide docking (27)
2.8.8: Conservation score (28)
2.9: Steady-state Förster resonance energy transfer measurement (28)
2.10: Relative mobility (RM) analysis (28)
2.11: Quantum Yield determination (29)
Chapter 3: Results (30)
3.1: The binding properties of RRM1, RRM2, and RBD to TG/rUrG repeats measured by fluorescence anisotropy (31)
3.2: The stoichiometry of RRM1 and RBD binding to ss(TG)6 determined by F-EMSA (32)
3.3 The protein-nucleic acids binding affinity comparison of experiments to MD simulations (33)
3.4 The difference of binding energy between RRM1-(rUrG)6 and RRM2-(rUrG)6 (34)
3.5 The binding-energy difference between RRM1-ss(TG)6 and RRM2-ss(TG)6 (36)
3.6: RRM domains prefer Guanine rather than Thymine upon binding to either ss(TG)6 or (rUrG)6 (38)
3.7: RRM1 domain unwinds double-stranded structure of TG-repeat sequence (39)
3.7.1: F-EMSA for unwinding ability of RRM1, RRM2, and RBD. (39)
3.7.2: Fluorescence Recovery Assay for time-course measuring of unwinding process. (40)
3.8: Adaptively Biased MD (ABMD) simulations reveal that RRM1 can better facilitates the opening of ds(TG)6 than RRM2 (41)
3.9: RRM1 can resolve intermolecular anti-parallel and parallel G-quadruplexes (41)
Chapter 4: Discussion (44)
4.1: The importance of unwinding activities of RRM1 in TDP-43 (45)
4.2: The importance of Guanine binding by RRM1 domain (46)
4.3: Multiple RRM1s and RBDs binding to (rUrG)6 may trigger oligomerization of TDP-43. (46)
4.4: The potential role of resolving G-quadruplexes by TDP-43 RRM1 (48)
4.5: F-EMSA for biochemical assays of protein-nucleic acids interactions (48)
Tables (51)
Figures (71)
Chapter 5: References (107)
Chapter 6: Appendix (118)

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