本實驗主要探討表面核酸探針於微小區域內之雜化反應效率和
對不同標靶分子辨識能力之研究。實驗選用髮夾型鎖核酸(LNA-HP)為探針分子，而所辨識之標靶分子為完全互補(perfect complementary，PC)對照單一鹼基錯配分子(single mismatch，1MM)，以液相原子力顯微鏡(Atomic Force Microscope ， AFM) 之奈米顯影術(nanolithography)成功製備出奈米點狀之LNA-HP 探針分子薄膜，分別與不同標靶分子(PC、1MM)進行雜化反應，雜化過程以原位(insitu)AFM 進行相對高度的測量，得到表面分子的結構資訊。實驗結果指出(1)利用AFM來觀測表面單股探針分子因雜化過程引發的結構
變化是可作為有效的偵測信號、(2)核酸探針區域微小化可有效提升雜化反應之效率並對PC、1MM 之辯識能力有所提升。
另研究不同環境組成是否影響人體端粒DNA 序列之構型。人體端粒由重複(5’-TTAGGG-3’:5’-CCCTAA-3’)n 組成4000~14000 鹼基對(bp)之雙股序列，雙股中的(5’-TTAGGG-3’)於3’端繼續以重複(T2AG3)單元延伸出約100~200 個核苷酸(nt)，此延伸的單股序列稱為G-richDNA，此序列在具有鈉或鉀離子之溶液中，形成特殊的四股結構(G-quadruplexes)。相較之下由(C3TA2)重複單元組成單股序列則稱為C-rich DNA，其序列在低pH 環境中表現出i-motif 摺疊結構。實驗上設計仿人體端粒之21 個核苷酸序列，並於金表面進行分子自組裝。
改變環境溶液條件(鉀離子、pH 值)下，藉由AFM(分析核酸自組裝膜厚、根均方粗糙度分析)和CD(結構定性)光譜分別證實於金表面之人體端粒序列確實因溶液組成改變而有構型上的變化。

The study for surfaced-immobilized nucleic acid probes in
nanometer region in response to hybridization and to discrimination ofdifferent target nuclei acids. The hairpin locked nucleic acid (LNA-HP) isselected to be the probe molecule, and target molecules include perfect
complementary (PC) and single mismatch (1MM). The self-assembledLNA-HP molecular nanospot is successfully prepared by liquid phaseAFM (Atomic Force Microscope)-based nanolithography technique, then
in situ hybridization is carried out by using different targets (PC/1MM).To obtain the information of structure change, we use AFM to analyze therelative heights in the process of hybridization. The experimental results
point out that (1) the structure changes of surface probe molecules maycorrelate with the AFM signal when target sequence hybridizes to the probe, (2) miniaturization of the size of the nucleic acid probe may promote hybridization efficiency and enhance the discrimination between
PC and 1MM.
Studies on whether the different chemical impetus in solution can affect conformation of the human telomeric DNA of sequence is conducted. A human talomeric DNA composed of ( 5’-TTAGGG-3’:5’-CCCTAA-3’ ) repeats, with a 100-200 nt ( T2AG3 ) repetitive unit overhang at 3’ ends is chosen. This extended single-stranded sequence is
called G-rich DNA, which forms the special G-quadruplex structure in solution containing sodium ions or potassium ions. The single-stranded sequence composed of ( C3TA2 ) repetitive units called C-rich DNA displays the i-motif folded structure in the low pH environment. These biomimetic DNA’s are thiol-modified to self-assemble on gold surfaces. Separate measurements with AFM (the molecular thickness and rootmean-
square roughness of the self-assembly monolayer of DNA ) and CD( circular dichroism ) ( structure characterization ) confirm the conformational changes of G-rich and C-rich DNA’s on gold surface are indeed dependent of the presence of cations and protons.

目 錄
中文摘要…………………………………………………………………i
英文摘要………………………………………………………………...iii
目錄……………………………………………………………………....v
圖目錄…………………………………………………………………...ix
表目錄………………………………………………………………….xiii
第壹章 緒論…………………………………………………………....1
1-1 前言………………………………………………………...1
1-2 核酸摺疊結構(DNA folding structures)………………......3
1-2.1 髮夾型核酸……………………………………....3
1-2.2 G-quadruplexes…………………………………..6
1-2.3 i-motif DNA……………………………………...9
1-3 研究動機……………………………………………….....11
第貳章 實驗儀器及材料……………………………………………..13
2-1 原子力顯微鏡(Atomic Force Microscope;AFM)……......13
2-1.1 AFM原理……………………………………….13
2-1.2 實驗用AFM 模式………………………………15
2-1.3 AFM奈米顯影術操作步驟…………………….15
2-1.4 AFM數據分析………………………………….16
2-2 圓二色光譜儀(Circular Dichroism ; CD)………………19
2-2.1 CD原理…………………………………………19
2-2.2 CD參數設定……………………………………20
2-3 實驗材料………………………………………………...21
2-4 局部區域雜化實驗方法………………………………...23
2-4.1 清洗容器器皿…………………………………..23
2-4.2 製備gold substrate /Au (111)…………………...24
2-4.3 生物藥品配置…………………………………..25
2-4.4 製備基質薄膜 (matrix)………………………...27
2-4.5 奈米尺度內的自組裝薄膜(SAMs)…………….28
2-4.6 核酸雜化反應…………………………………..29
2-5 鉀離子驅動G-rich DNA quadruplex形成………………30
2-6 不同環境pH 造成C-rich DNA 構型差異……………..31
第參章 結果與討論…………………………………………………..32
3-1 Gold substrate 液相下AFM 高度影像……………….....32
3-2 Matrix Layer 液相下AFM 高度影像…………………...33
3-3 區域微小化對雜化辨識反應之影響…………………...34
3-3.1 LNA-HP 與完全互補標靶分子(PC)的雜化反
應..........................................................................34
3-3.2 LNA-HP 與單一鹼基錯配標靶分子(1MM)的雜
化反應…………………………………………..36
3-3.3 各類探針分子雜化速率及辨識能力的討論…..38
3-4 鉀離子對G-rich DNA quadruplex 結構之影響……....40
3-4.1 極微量離子環境中，利用AFM 對金表面之
G-rich DNA 自組裝單層膜(SAMs)進行平均粗
糙度及膜厚分析………………………………..40
3-4.2 鉀離子溶液浸泡後，利用AFM 對金表面之
G-rich DNA SAMs 進行平均粗糙度及膜厚分
析………………………………………………..42
3-4.3 鉀離子影響G-rich DNA 的quadruplex 結構之
結果討論………………………………………..44
3-5 溶液pH 對C-rich DNA i-motif 構型之影響…………45
3-5.1 利用AFM 對不同pH 溶液所製備SAMs 進行膜
厚分析…………………………………………..45
3-5.2 在不同pH 值下利用CD 對C-rich 做結構定
性………………………………………………..47
3-5.3 環境pH 影響C-rich DNA 的i-motif 構型之結
果討論…………………………………………..48
第肆章 結論…………………………………………………………. 50
參考文獻………………………………………………………………..53

(1) You, Y.; Moreira, B. G.; Behlke, M. A.; Owczarzy, R. Nucleic Acids Res.
2006, 34, 60: Design of LNA probes that improve mismatch
discrimination
(2) Deaton, R.; Garzon, M.; Murphy R. C.; Rose, J. A.; Franceschetti, D.
R.; Stevens, S. E. Phys. Rev. Lett. 1998, 80, 417: Reliability and
efficiency of a DNA-based computation
(3) Gao, Y.; Wolf, L. K.; Georgiadis, R. M. Nucleic Acids Res. 2006, 34,
3370: Secondary structure effects on DNA hybridization kinetics: a
solution versus surface comparison
(4) Dendy, D. S.; Wu, P.; Grainger, D. W. Proc. Natl. Acad. Sci. 2007. 104.
8223: Array feature size influence nucleic acids surface capture in
DNA microarrays
(5) Mao, Y.; Liu, D.; Wang, S.; Luo, S.; Wang, W.; Yang, Y.; Ouyang, Q.;
Jiang, L. Nucleic Acids Res. 2007, 35, 33: Alternating–electric–field
-enhanced reversible switching of nanocontainers with pH
(6) Bonnet, G.; Tyagi, S.; Libchaber, A.; Kramer, F. R. Proc. Natl. Acad.
Sci. 1999. 96. 6171: Themodynamic basis of the enhanced
specificity of structured DNA probes
(7) Wei, F,; Chen, C.; Zhang, N.; Zhao, X. S. J. Am. Chem. Soc. 2005, 127,
5306: Recognition of single nucleotide polymorphisms using
scanning potential hairpin denaturation
(8) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J. -i.; Morio, K. –i.; Doi, T.;
Imanishi, T. Tetrahedron Lett. 1998, 39, 5401: Stability and structural
features of the duplexs containing nucleoside analogues with a fixed N-type conformation, 2’-O,4’-C-methyleneribonucleosides
(9) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar,R.;
Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607:
LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine,
guanine, 5-ethylcytosine, thymine and uracil bicyclonucleoside
monomers, oligomerisation, and unprecedented nucleic acid
recognition
(10) Natsume, T.; Ishikawa, Y.; Dedachi, K.; Tsukamoto, T.; Kurita, N.
Chem. Phys. Lett. 2007, 446, 151: Effect of base mismatch on the
electronic properties of DNA-DNA and LNA-DNA double strands:
density -functional theoretical calculations
(11) Wang, L.; Yang, C. J.; Medley, C. D.; Benner, S. A.; Tan, W. J. Am.
Chem. Soc. 2005, 127, 15664: Locked nucleic acid molecular
beacons
(12) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916:
Characterization of DNA probes immobilized on gold surfaces
(13) Peeters, S.; Stakenborg, T.; Reekmans, G.; Laureyn, W.; Lagae, L.;
Aerschot, A. V.; Ranst, M. V. Biosens. Bioelectron. 2008, 24, 72:
Impact of spacers on the hybridization efficiency of mixed
self-assembled DNA/ alkanethiol films
(14) Wright, W. E. et al. Genes Dev. 1997. 11, 2801: Normal human
chromosomes have long G-rich telomeric overhangs at one end
(15) Gellert, M.; Lipsett, M. N.; Davies, D. R. Proc. Natl Acad. Sci. U.S.A.
1962, 48, 2013: Helix formation by guanylic acid
(16) Alberti, P.; Bourdoncle, A.; Sacc`a, B.; Lacroix, L.; Mergny, J. L. Org.
Biomol. Chem. 2006, 4, 3383: DNA nanomachines and nanostructures involving quadruplexes
(17) Pilch, D. S.; Plum, G. E.; Breslauer, K. J. Curr. Opin. Struct. Biol. 1995,
5, 334: The thermodynamics of DNA structures that contain lesions
of guanine tetrads
(18) Patel, D. J. Nature 2002, 417, 807: A molecular propeller
(19) Parkinson G. N.; Lee, M. P.; Neidle, S. Nature 2002, 417, 876: Crystal
structure of parallel quadruplexes from human telomeric DNA
(20) Hardin, C. C.; Watson, T.; Corregan, M.; Bailey, C. Biochemistry 1992,
31, 833: Cation-dependent transition between the quadruplex and
Watson -Crick hairpin forms of d(CGCG3GCG)
(21) Wang, Y.; Patel, D. J. Structure 1993, 1, 263: Solution structure of the
Tetrahymena telomeric repeat d(T2G4)4 G-tetraplex
(22) Kang, C. et al. Nature 1992, 356, 126: Crystal structure of four -
stranded Oxytricha telomeric DNA
(23) Phillips, K. et al. J. Mol. Biol. 1997, 273, 171: The crystal structure of
a parallel-stranded guanine tetraplex at 0.95Å resolution
(24) Breslauer, K. J. et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7694
(25) Liu, D.; Bruckbauer, A.; Abell, C.; Balasubramanian, S.; Kang, D. J.;
Klenerman, D. and Zhou, D. J. Am. Chem. Soc. 2006, 128, 2067: A
reversible pH-Driven DNA nanoswitch array
(26) Li, X.; Peng, Y.; Ren, J.; Qu, X. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 19658: Carboxyl-modified single-walled carbon nanotubes
selectively induce human telomeric i-motif formation
(27) MultimodeTM SPM Instruction Manual. 1996, USA, Digital Instrument
Inc.
(28) 國立中央大學化學工程與材料工程所:利用表面電漿共振生物
感測器及單分子模型槽研究蛋白質在固/液及氣/液界面上之結構
吳旻珈撰
(29) http://arrandee.com/products/How_to_use/body_how_to_use.html
(30) Peeters, S.; Stakenborg, T.; Reekmans, G.; Laureyn, W.; Lagae, L.;
Aerschot, A. V.; Ranst, M. V. Biosens. Bioelectron. 2008, 24, 72:
Impact of spacers on the hybridization efficiency of mixed
self-assembled DNA/ alkanethiol films
(31) Sikorav, J.-L.; Orland, H.; Braslau, A. J. Chem. Phys. 2009, 113, 3715:
Mechanism of thermal renaturation and hybridization of nucleic
acids: Kramers’ process and universality in Watson-Crick base
pairing
(32) Peterson, A. W.; Wolf, L.K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002,
124, 14601: Hybridization of mismatched or partially matched
DNA at surfaces
(33) Han, W. –H.; Liao, J. –M.; Chen, K. –L.; Wu, S. –M.; Chiang, Y. –W.;
Lo, S. –T.; Chen C. –L.; Chiang C. –M. Anal. Chem. 2010, 82, 2395:
Enhanced Recognition of Single-Base Mismatch Using Locked
Nucleic Acid-Integrated Hairpin DNA Probes Revealed by Atomic
Force Microscopy Nanolithography
(34) Cederquist, K. B.; Golightly, R. S.; Keating, C. D. Langmuir 2008,
24, 9162: Molecular Beacon−Metal Nanowire Interface: Effect of
Probe Sequence and Surface Coverage on Sensor Performance
(35) Yang,Y.; Liu, G.; Liu, H.; Li, D.; Fan, C.; Liu, D. Nano Lett., 2010,
57
10 , 1393: An Electrochemically Actuated Reversible DNA
Switch
(36) Hou, X.; Guo, W.; Wang, Y.; Liu, D.; Jiang, L. et al. J. Am. Chem. Soc.
2009, 131, 7800: A Biomimetic Potassium Responsive
Nanochannel :G-Quadruplex DNA Conformational Switching in
a Synthetic Nanopore
(37) Zhou, X. –H.; Kong D. –M.; Shen H. –X. Anal. Chem. 2010, 83, 789:
Ag+ and Cysteine Quantitation Based on G-Quadruplex−Hemin
DNAzymes Disruption by Ag+
(38) Peng, Y.; Wang, X.; Xiao, Y.; Feng, L.; Zhao, C.; Ren, J.; Qu, X. J.
Am. Chem. Soc. 2009, 131, 13813: i-Motif Quadruplex
DNA-Based Biosensor for Distinguishing Single- and
Multiwalled Carbon Nanotubes