RNA binding motifs 扮演多功能角色，如參與RNA splicing, 解旋RNA hairpins和抑制dsDNA轉錄等，對於細胞內的平衡與轉譯的控制扮演重要的角色。然而，他們彼此間的關係為何?是否這些功能有著共同的物理性質以致於一個功能可以經透過演化和稍許的化學修飾被修改成另一個? 在本文中，我們演示了兩個結構上一樣但序列間不同的RNA Recognition Motifs (RRM1和RRM2)，兩者對於結合RNA和ssDNA具有不同的親和力 (差了約百倍左右)。RRM1對RNA有更強的結合力且被發現在沒有ATPs的情況下能解旋ds(TG)6，而對RNA有較弱結合力的RRM2則無法解旋dsDNA。經由一系列的實驗與模擬分析，包含fluorescence anisotropy、F-EMSA、FRET、生物資訊 (保守程度)、蛋白質-蛋白質對接、Gaussian-accelerated (Ga-) MD simulations及X-ray與NMR的證據支持，我們認為對於RNA骨幹和鳥嘌呤 (Guanine)有專一結合力RNA結合蛋白較強的結合者可以演化成不需要ATPs的dsDNA解旋酶。在這過程中，部分ssDNA的結合位點也可作為dsDNA的結合位點，結合住dsDNA的major grooves，並在dsDNA間歇性地打開時快速地抓著一股ssDNA以幫助解旋。因為實驗和計算兩者都發現一個RRM1結合ssDNA後，兩個RRM1將更容易結合ssDNA，所以RRM1解旋dsDNA被認為有偕同性。因此，我們主張與ssDNA/dsDNA相關的蛋白質的功能可以從RNA chaperone藉由修改極少數量的氨基酸組成而得到，這支持了一個從只有RNA跟蛋白質的世界衍生到有RNA、蛋白質與DNA共存的世界。本研究也提供了對於在未來設計基因編輯相關的解旋酶和及設計抑制專一dsDNA序列的轉錄抑制劑的一個重要參考。

RNA binding motifs serve multiple functions, including RNA splicing, unwinding of RNA hairpins and suppression of (ds)DNA transcription, and play important roles in intracellular homeostasis and translational control. However, it is not clear whether these functions share a physical origin and are related evolutionarily? In this study, we showcase two RNA Recognition Motifs (RRM1 and RRM2) that share a similar structural fold but differ in primary sequences (~25% sequence identity). The two RRMs differ in their RNA and ssDNA binding affinity by >100 fold. RRM1 that has a stronger RNA binding affinity is found to be able to unwind ds(TG)6 in the absence of ATPs while RRM2 cannot. After a series of experiments and molecular dynamics simulations analyses including fluorescence anisotropy, FRET, F-EMSA, residue conservation, protein-protein docking, Gaussian- accelerated molecular dynamic (GaMD) simulations and structural evidence from x-ray/NMR, we propose that RRMs that can favorably bind backbone of RNA and have specificity in guanine can be evolved into energy-independent dsDNA helicase. In this process, part of ssDNA binding site in RRM1 can also serve as part of the dsDNA binding site that anchors the major grooves of dsDNA. Multiple RRM1s could cooperatively bind transiently opened dsDNA to unwind the double strands, which is supported by both experi-mental and computational evidence demonstrating when one RRM1 binds ssDNA, the second one can bind the same ssDNA more favorably. Our analyses suggest a RNA chaperone could be evolved to bind relevant ssDNA and/or unwind specific dsDNA by modifying less than a handful residues. This argument supports the hypothesis that life is evolved from a RNA+ protein world to a RNA+ protein + DNA world. This study also offers an important reference for the design of gene editing-associated helicases and transcriptional inhibitors that bind specific sequences of dsDNA.

摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vi
表目錄 vii
1. 緒論 1
2. 方法 4
2.1 分子動力學模擬 4
2.1.1 創建分子動力模擬的起始結構 5
2.1.2 加水與離子 9
2.1.3 分子動力模擬的相關參數設置: 9
2.2 Gaussian Accelerated Molecular Dynamics simulations (GaMD) 12
2.3 用FTDock來進行Protein-dsDNA Docking 13
2.4 高斯網絡模型 (Gaussian Network Model; GNM) 15
2.5 用Intrinsic Dynamics Domains (IDDs)來過濾FTDOCK結果，並且將docking 複合體結構叢集化 (clustering)，再照叢集大小來排序docking複合體 16
2.6 方均根差 (Root-Mean Square Deviation; RMSD) 16
2.7 接觸分析 (Contact Analysis) 17
2.8 分子力學泊松-玻爾茲曼表面積 (Molecular Mechanics Poisson–Boltzmann Surface Area; MM-PBSA) 18
2.9 保守分數 (Conservation score) 19
2.10 Flexible Structure Alignment by Chaining Aligned fragment pairs allowing Twists (FATCAT) Pairwise Alignment 19
2.11 用螢光非等向性 (Fluorescence Anisotropy)來量測Kd 20
2.12 用螢光電泳遷移率分析化學當量 (stoichiometry) 21
3.結果 22
3.1 蛋白-核酸結合力的實驗與模擬結果之對照 22
3.2 RRM1/RRM2結合(UG)6的能量的相同處以及相異處比較 23
3.3 RRM1/RRM2結合ss(TG)6的能量的相同處以及相異處比較 31
3.4 RRM1與ss(TG)6/(UG)6結合時在殘基結合上的相同處以及相異處比較 39
3.5 RRM2與ss(TG)6/(UG)6結合時在殘基結合上的相同處以及相異處比較 43
3.6 蛋白質殘基與核酸鹼基的親和力解析 47
3.6.1 RRM1結合核糖核酸比去氧核醣核酸更好的原因主要是因為核糖上的二號碳中的氧比胸腺嘧啶的甲基團更重要 47
3.6.2 鳥嘌呤比胸腺嘧啶/尿嘧啶更重要 49
3.6.3 RRM2上的點突變E200W與I253R佐證殘基能量分析的正確性: 50
3.7 以FRET實驗證明RRM1可解旋ds(TG)6 51
3.8 殘基接觸核酸的機率分析 52
3.9 用Gaussian Accelerated (Ga-) MD解旋dsDNA的結果 55
3.10 RRMs與核甘酸的化學計量數 (stoichiometry) 58
3.11 模擬2:1與1:1的能量比較 (蛋白質:核甘酸) 59
3.12 RRM1可彎曲ss(TG)6，但 RBD不能彎曲ss(TG)6 60
4. 討論 62
4.1 各模擬系統中前20名殘基二級結構分布: 62
4.2 RRM1/2 結合ssDNA/RNA能量結果與點突變實驗對照 63
4.3 Thymine /Uracil 與Guanine的重要性比較 65
4.4 RRM1的演化推論 67
5. 結論 69
參考文獻 71

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