臺灣博碩士論文加值系統

在分子生物學與結構生物學領域，試圖去探究具有專一性的分子間作用力是一項相當具有挑戰性的工作。然而，這項工作也是非常有趣的，因為這種類型的作用力支配了所有物質和生物系統的作用。在本次的研究中，我們利用原子力顯微鏡和表面電漿共振技術來探討線形DNA及其標靶DNA（完全互補與單一鹼基突變二種）之間的專一性交互作用。同時，我們也使用了兩種不同濃度的緩衝溶液以便了解在兩條單股DNA雜交時是否會受到離子強度的影響。由原子力顯微鏡所測得的數據，我們以Poisson統計方法去分析出破壞單一分子間交互作用所需的力、線形DNA及其標靶DNA之間所形成鍵的平均數、以及可能存在的非專一性與長距離的交互作用。對單一鹼基突變和完全互補這兩種DNA系統而言，破壞分子間交互作用所需的平均力分別是0.20833 ± 0.07317 nN和0.26296 ± 0.07638 nN。針對完全互補的DNA系統，我們求得破壞兩股DNA之間單一鹼基對的作用力是0.07843 nN。最後，我們利用表面電漿共振技術來研究線形DNA與標靶DNA之間的結合動力學。根據所有的實驗結果，我們成功地利用原子力顯微鏡與表面電漿共振技術來鑑別完全互補與單一鹼基突變兩種DNA系統，並且證實了具有高離子強度的緩衝溶液將有利於DNA的雜交。

Attempting to probe the specific intermolecular forces is a challenging task in molecular and structural biology. However, it is of great interest because such forces dominate the behavior of all materials and biological systems. We used atomic force microscopy (AFM) and surface plasmaon resonance (SPR) to study the specific interaction between the linear DNA and their target DNA (complementary and one-base mutation) in this work. At the same time, we also used the hybridization buffer solutions in two different concentrations to realize whether the ionic strength affects the hybridization between the single-stranded DNA. The Poisson statistical method was used to analyze the rupture force of individual molecule, the mean number of bonds between linear DNA and their target DNA, and the possible non-specific and long-range interactions. The average rupture force for single-base mismatch and complementary system were 0.20833 ± 0.07317 nN and 0.26296 ± 0.07638 nN, respectively. The individual rupture force of double-stranded DNA for the complementary system was also determined to be 0.07843 nN. Finally, we carried out the study of the binding kinetics between linear DNA and their target DNA by SPR. According to our experimental results, AFM and SPR succeeded in distinguishing the difference between totally complementary double-stranded DNA and complement with single-pair mismatch. Besides, it was verified that the hybridization buffer with high ionic strength would be helpful to the DNA hybridization.

Contents
Acknowledgements………………………………………………………….………Ⅰ
Chinese Abstract………………………………………………………………….…Ⅲ
English Abstract…………………………………………………………………..…Ⅳ

Chapter 1 Introduction……………………………………………………………….1

Chapter 2 Experimental Instruments and Principles………………………………….6
2.1 Introduction to Atomic Force Microscopy…………………………….6
2.2 Main Components and General Principle of Microscope Operation…..7
2.2.1 The Atomic Force Microscope Probe……………………………9
2.2.2 Piezoscanner……………………………………………………10
2.2.3 Optical System for Cantilever Deflection Detection…………...11
2.3 General Operating Concepts of AFM………………………………...13
2.3.1 Contact Mode…………………………………………………...15
2.3.2 Non-contact Mode……………………………………………...18
2.3.3 Tapping Mode…………………………………………………..19
2.4 AFM Force Spectroscopy…………………………………………….22
2.4.1 Overview………………………………………………………..22
2.4.2 Typical Forces Curves………………………………………….23
2.4.3 Force Conversion……………………………………………….26
2.5 Poisson Statistical Analysis…………………………………………..28
2.6 Surface Plasmon Resonance………………………………………….30
2.6.1 Total Internal Reflection………………………………………..30
2.6.2 Surface Plasmons……………………………………………….32
2.6.3 Binding Kinetics………………………………………………..32

Chapter 3 Experimental Section…………………………………………………….34
3.1 Materials……………………………………………………………...34
3.2 Modification of AFM Tips and Target DNA Immobilization………..36
3.3 AFM Force Measurements and Data Analysis……………………….37
3.4 SPR Measurements…………………………………………………...38

Chapter 4 Results and Discussion…………………………………………………...39
4.1 Surface Modification…………………………………………………39
4.2 Specific Interaction between Thiolated Linear DNA Probe and Target DNA…………………………………………………………………..42
4.3 Application of the Poisson Statistical Method……………………….45
4.4 Rupture Force Measured under Different Salt Concentration………..48
4.5 Kinetics of DNA Hybridization………………………………………50
4.6 Discussion…………………………………………………………….55
4.7 Conclusion……………………………………………………………57
Chapter 5 References………………………………………………………………..59




















Figure of Contents
Figure 2-1 AFM probe schematic picture…………………………………………….6
Figure 2-2 Schematic diagram of the AFM equipment………………………………8
Figure 2-3 Quad Photodetector Arrangement……………………………………….11
Figure 2-4 Operating Concepts of AFM (Contact mode is instanced)……………...15
Figure 2-5 AFM image acquisition at constant force……………………………….17
Figure 2-6 AFM image acquisition at constant height……………………………...17
Figure 2-7 Working point selection during tapping mode…………………………..20
Figure 2-8 A schematic drawing of three AFM operating modes…………………..21
Figure 2-9 Schematic diagram of the vertical tip movement during the approach and retract parts of a force spectroscopy experiment………………………..23
Figure 2-10 Plot of approach (red) and retract (blue) curves for a contact mode cantilever and clean mica in air………………………………………...24
Figure 2-11 Plot of approach (red) and retract (blue) curves in water……………...26
Figure 2-12 A: Light passing from a denser medium to a less dense one is refracted towards the plane of the interface. B: Over a critical angle of incidence, total internal reflection occurs and no light passes into the less dense medium…………………………………………………………………31
Figure 2-13 A representative sensorgram of Biacore 3000 system…………………33
Figure 4-1 Response signal of the self-assembly process of thiolated DNA on the gold substrates measured by SPR. Self-assembly of 250 nM thiolated DNA on the gold substrate in 0.4 M phosphate buffer, pH 7.3. After 3 h, the SPR response has reached a plateau…………………………………41
Figure 4-2 Typical force–distance curves of thiolated linear DNA probe–cDNA1 (solid line) and thiolated linear DNA probe–cDNA2 (dotted line) in hybridization buffer at pH 8.0…………………………………………...43
Figure 4-3 Rupture force distribution histograms for thiolated linear DNA probe with the target DNA. (A) Rupture force histogram corresponding to the force distribution of 5’-thiol-modified tip and cDNA2-coated surface (average rupture force 0.20833 ± 0.07317 nN). (B) Rupture force histograms corresponding to the force distribution of 5’-thiol-modified tip and cDNA1-immobilized surface (average rupture force 0.26296 ± 0.07638 nN). Each of the histograms was derived from 100–200 force measurements of adhesion events measured at different spots acquired with a single tip………………………………………………………….44
Figure 4-4 Force variance vs. mean force for the complementary double-stranded DNA system acquired in 0.1 M hybridization buffer at pH 8.0. Each point represents a data set taken with a different sample location, as shown in Table 4-1………………………………………………………………...46
Figure 4-5 Rupture force distribution histogram for thiolated linear DNA probe with the complementary target DNA acquired in 0.5 M hybridization buffer at pH 8.0 and the average rupture force is 0.27186 ± 0.05897 nN. The histogram was derived from 131 force measurements of adhesion events measured at different spots acquired with a single tip…………………..49
Figure 4-6 Sensorgram of single-pair mismatch system in 0.5 M hybridization buffer at pH 8.0…………………………………………………………………51
Figure 4-7 Sensorgram of complementary system in 0.5 M hybridization buffer at pH 8.0………………………………………………………………………..52
Figure 4-8 Sensorgram of complementary system in 0.1 M hybridization buffer at pH 8.0………………………………………………………………………..53













Table of Contents
Table 4-1 Results of the Rupture Forces between Thiolated Linear DNA Probe and Target DNA (complementary) in 0.1M Hybridization Buffer……………47
Table 4-2 Results of the Rupture Forces between Thiolated Linear DNA Probe and Target DNA (one-base mutation) in 0.1M Hybridization Buffer………...47
Table 4-3 Kinetic Constants in Single-pair Mutation and Complementary System…………………………………………………………………...54
Table 4-4 Kinetic Constants of Complementary System in Different Ionic Strength………………………………………………………………….54

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