為探討aptamer與其ligand之交互作用機制，本研究選擇文獻上以L-tyrosinamide為ligand，且利用SELEX程序篩選所得的DNA aptamer（pe35 aptamer）作為研究之目標。本文利用恆溫滴定微卡計（ITC）與圓二色光譜儀（CD），設計不同的實驗條件，包含不同鹽濃度、金屬離子種類（一價（K+、Na+）、二價（Mg2+））、溫度、緩衝液pH、緩衝液種類以及結構相似物(analogues)等。期望藉由熱力學（ITC）與二級結構（CD）分析，來獲得aptamers與ligands辨識機制之相關資訊。
在熱力學分析上，pe35 aptamer與L-tyrNH2結合反應的發生是enthalpy driven，同時結合過程中伴隨著induced-fit程序。在二級結構探討上，pe35 aptamer具有自我形成hairpin之結構而stem區域所形成之helix為B-DNA form。當反應發生後其二級結構將由B-DNA form轉變成具有A-like特性之helix結構。在結合作用力的探討上，反應過程中包含：（1）靜電作用力且L-tyrNH2至少有一個NH３＋參與反應，（2）鹼基堆疊之疏水作用力，（3）質子化耦合效應以及（4）氫鍵等。在構型相似物的比較，研究發現認為L-tyrNH2上的phenolic hydroxyl是pe35 aptamer辨識L-tyrNH2的重要因素之ㄧ，影響因素可能包含提供氫鍵作用力以及參與誘導構型轉變使複合物更加緊密來提高結合常數。在金屬離子對結合行為影響的研究上，不同一價金屬離子（K+、Na+）對於pe35 aptamer與L-tyrNH2結合行為無明顯差異之影響，當提高鹽濃度，均造成結合常數下降。而在二價金屬離子（Mg2+）的研究中，實驗結果發現，Mg2+的存在對於結合行為是不可或缺的，且Mg2+本身即具有誘導pe35 aptamer構型轉變之能力。在熱力學部份，增加Mg2+濃度，則需要提供更多熱量來調整aptamer構型，並由減少entropy cost來驅動反應的進行。
本研究利用ITC所得之結合常數（KA）、熱力學參數（ΔH、ΔS），配合CD結構上的說明，已清楚說明DNA aptamer與其ligand辨識行為之作用力、熱力學以及結構等相關資訊。

Aptamers are macromolecules composed of nucleic acids, such as RNA or DNA, that bind tightly to a specific molecular target. In this study, we used Isothermal Titration Microcalorimetry (ITC) and Circular Dichroism (CD) to study the binding mechanism between a DNA aptamer and L-tyrosinamide. In order to gain further insights into the binding driven force in the recognizing behavior and the thermodynamic discrepancy, binding enthalpy measurements at different system parameters such as salt ion temperature pH value and analogues were carried out. Noteworthily, stabilizing the aptamer structure and enhanced target-aptamer complex formation by magnesium cation was also demonstrated in this study.
ITC results indicate that the binding behavior is an enthalpy driven and entropy cost process. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding behavior is an induced-fit process and the conformation of DNA aptamer changes from B-form to A-from like in the binding process. In addition, binding mechanism analysis suggest that the interaction driven force in the binding process may include electrostatic interactions, hydrophobic interactions, hydrogen bonding and binding-linked protonation process. Furthermore, Mg2+ could not only help the forming of the complex by stable the conformation of the DNA aptamer but also change the structure of DNA aptamer.

【1】 Hud N. V., and Plavec J. “A unified model for the origin of DNA sequence-directed curvature” Biopolymers 2003, 69, 144–158.
【2】 Robertson D. L., and Joyce G. F. “Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA” Nature Struct. Biol. 1990, 344, 467–468.
【3】 Tuerk C., and Gold L. “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA-polymerase” Science 1990, 249, 505–510.
【4】 Ellington A. D., and Szostak J. W. “In vitro selection of RNA molecules that bind specific ligands” Nature 1990, 346, 818–822.
【5】 Breaker R. R. “Natural and engineered nucleic acids as tools to explore biology” Nature 2004, 432, 838–845.
【6】 Batey R. T., Gilbert S. D., and Montange R. K. “Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine” Nature 2004, 432, 411–415.
【7】 Mandal M., Lee M., Barrick J. E., Weinberg Z., Emilsson G. M., Ruzzo W. L., and Breaker R. R. “A glycine-dependent riboswitch that uses cooperative binding to control gene expression” Science 2004, 306, 275–279.
【8】 Gilbert S. D., Stoddard C. D., Wise S. J., and Batey R. T. “Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain” J. Mol. Biol. 2006, 359, 754–768.
【9】 Bunka D. H. J., and Stockley P. G. “Aptamers come of age – at last” Nature Reviews Microbiology 2006, 4, 588–596.
【10】 Cox J. C., and Ellington A. D. “Automated Selection of Anti-Protein Aptamers” Bioorganic and Medicinal Chemistry 2001, 9 (10), 2525–2531.
【11】 Bock C., Coleman M., Collins B., Davis J., Foulds G., Gold L., Greef C., Heil J., Heilig J. S., Hicke B., Hurst M. N., Husar G. M., Miller D., Ostroff R., Petach H., Schneider D., Vant-Hull B., Waugh S., Weiss A., Wilcox S. K., and Zichi D. “Photoaptamer arrays applied to multiplexed proteomic analysis” Proteomics 2004, 4 (3), 609–618.
【12】 Eulberg D., Buchner K., Maasch C., and Klussmann S. “Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist” Nucleic Acids Res. 2005, 33(4), e45.
【13】 Gronewold T. M., Glass S., Quandt E., and Famulok M. “Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors” Biosens. Bioelectron. 2005, 20, 2044–2052.
【14】 Savran C. A., Knudsen S. M., Ellington A. D., and Manalis S. R. “Micromechanical detection of proteins using aptamer-based receptor molecules” Anal. Chem. 2004, 76, 3194–3198.
【15】 Bang G. S., Cho S., and Kim B. G. “A novel electrochemical detection method for aptamer biosensors” Biosens. Bioelectron. 2005, 21, 863–870.
【16】 Nutiu R., and Li Y. “Aptamers with fluorescence-signaling properties” Methods 2005, 37, 16–25.
【17】 Nutiu R., and Li Y. “Structure-switching signaling aptamers” J. Am. Chem. Soc. 2003, 125, 4771–4778.
【18】 Yang C. J., Jockusch S., Vicens M., Turro N. J., and Tan W. “Light switching excimer probes for rapid protein monitoring in complex biological fluids” Proc. Natl. Acad. Sci. 2005, USA 102, 17278–17283.
【19】 Navani N. K., and Li Y. “Nucleic acid aptamers and enzymes as sensors” Curr. Opin. Chem. Biol. 2006, 10, 272–281.
【20】 Lu Y., and Liu J. W. “Functional DNA nanotechnology: emerging applications of DNAzymes and aptamers” Current Opinion in Biotechnology 2006, 17, 580–588.
【21】 Ravelet C., Grosset C., and Peyrin E. “Liquid chromatography, electrochromatography and capillary electrophoresis applications of DNA and RNA aptamers” J. Chromatrogr. A 2006, 1117, 1–10.
【22】 Romig T. S., Bell C., and Drolet D. W. “Aptamer affinity chromatography: combinatorial chemistry applied to protein purification” J. Chromatogr. B 1999, 731, 275–284.
【23】 Connor A. C., and McGown L. B. “Aptamer stationary phase for protein capture in affinity capillary chromatography” J. Chromatogr. A 2006, 1111, 115–119.
【24】 Deng Q., German I., Buchanan D., and Kennedy R. T. “Retention and Separation of Adenosine and Analogues by Affinity Chromatography with an Aptamer Stationary Phase” Anal. Chem. 2001, 73, 5415–5421.
【25】 Vo T. U., and McGown L. B., “Effects of G-quartet DNA stationary phase destabilization on fibrinogen peptide resolution in capillary electrochromatography” Electrophoresis 2006, 27, 749–756.
【26】 Michaud M., Jourdan E., Villet A., Ravel A., Grosset C., and Peyrin E. “A DNA Aptamer as a New Target-Specific Chiral Selector for HPLC” J. Am. Chem. Soc. 2003, 125, 8672–8679.
【27】 Ruta J., Ravelet C., Grosset C., Fize J., Ravel A., Villet A., and Peyrin E. “Enantiomeric Separation Using an L-RNA Aptamer as Chiral Additive in Partial-Filling Capillary Electrophoresis” Anal. Chem. 2006, 78(9), 3032–3039.
【28】 Blank M., and Blind M. “Aptamers as tools for target validation” Curr. Opin. Chem. Biol. 2005, 9 (4), 336–342.
【29】 Lee J. F., Stovall G. M., and Ellington A. D. “Aptamer therapeutics advance” Curr. Opin. Chem. Biol. 2006, 10, 282–289.
【30】 Rusconi C. P., Roberts J. D., Pitoc G. A., Nimjee S. M., White R. R., Quick G., Scardino E., Fay W. P., and Sullenger B. A. “Antidote-mediated control of an anticoagulant aptamer in vivo” Nat. Biotechnol. 2004, 22, 1423–1428.
【31】 Convery M. A., Rowsell S., Stonehouse N. J., Ellington A. D., Hirao I., Murray J. B., Peabody D. S., Phillips S. E. V., and Stockley P. G. “The crystal structure of an RNA aptamer protein complex at 2.8Å resolution” Nature Struct. Biol. 1998, 5, 133–139.
【32】 Rowsell S., Stonehouse N. J., Convery M. A., Adams C. J., Ellington A. D., Hirao I., Peabody D. S., Stockley P. G., and Phillips S. E. V.. “Crystal structures of a series of RNA aptamers complexed to the same protein target” Nature Struct. Biol. 1998, 970–975.
【33】 Hermann T., and Patel D. J. “Biochemistry - Adaptive recognition by nucleic acid aptamers” Science 2000, 287, 820–825.
【34】 Noeske J., Buck J., Furtig B., Nasiri H. R., Schwalbe H., and Wohnert J. “Interplay of ''induced fit'' and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch” Nucleic Acids Research 2007, 35 (2), 572–583.
【35】 Carothers J. M., Oestreich S. C., and Szostak J. W. “Aptamers selected for higher-affinity binding are not more specific for the target ligand” J. Am. Chem. Soc. 2006, 128, 7929–7937.
【36】 Bishop G. R., Ren J. S., Polander B. C., Jeanfreau B. D., Trent J. O., and Chaires J. B. “Energetic basis of molecular recognition in a DNA aptamer” Biophysical Chemistry 2007, 126, 165–175.
【37】 Famulok M. “Molecular Recognition of Amino Acids by RNA-Aptamers: An L-Citrulline Binding RNA Motif and Its Evolution into an L-Arginine Binder” J. Am. Chem. Soc. 1994, 116, 1698–1706.
【38】 Huang Z., and Szostak J. W. “Evolution of aptamers with a new specificity and new secondary structures from an ATP aptamer” RNA 2003, 9, 1456–1463.
【39】 Mannironi C., Scerch C., Fruscoloni P., and Tocchini-Valentini G. P. “Molecular recognition of amino acids by RNA aptamers: The evolution into an L-tyrosine binder of a dopamine-binding RNA motif” RNA 2000, 6, 520–527.
【40】 Sayer N. M., Cubin M., Rhie A., Bullock M., Tahiri-Alaoui A., and James W. “Structural determinants of conformationally selective, prion-binding aptamers” Joural of Biological Chemistry 2004, 279 (13), 13102–13109.
【41】 Dey A. K., Griffiths C., Lea S. M., and James W. “Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1” RNA 2005, 11, 873–884.
【42】 Muller M., Weigand J. E., Weichenrieder O., and Suess B. “Thermodynamic characterization of an engineered tetracycline-binding riboswitch” Nucleic Acids Research 2006, 34, 2607–2617.
【43】 Ladbury J. E. “Application of Isothermal Titration Calorimetry in the Biological Sciences: Things Are Heating Up! ” Biotechniques 2004, 37, 885–887.
【44】 Leavitt S., and Freire E. “Direct measurement of protein binding energetics by isothermal titration calorimetry” Curr. Opin. Struct. Biol. 2001, 11, 560–566.
【45】 Saboury A. A. “A review on the ligand binding studies by isothermal titration calorimetry” Journal of the Iranian Chemical Society 2006, 3 (1), 1–21.
【46】 Egawa T., Tsuneshige A., Suematsu M., and Yonetani T. “Method for determination of association and dissociation rate constants of reversible bimolecular reactions by isothermal titration calorimeters” Analytical Chemistry 2007, 79 (7), 2972–2978.
【47】 Baker B. M., and Murphy K. P. “Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry” Biophys. J. 1996, 71, 2049–2055.
【48】 Fukada H., and Takahashi K. “Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride” Proteins-Structure Function and Genetics 1998, 33 (2), 159–166.
【49】 Fukada H., and Takahashi K. “Differential Scanning Calorimetric Study of the Thermal Unfolding of Taka-amylase A from Aspergillus oryzae” Biochemistry 1987, 26, 4063–4068.
【50】 Parker M. H., Lunney E. A., Ortwine D. F., Pavlovsky A. G., Humblet C., and Brouillette C. G. “Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1(matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry” Biochemistry 1999, 38, 13592–13601.
【51】 Kaul M., Barbieri C. M., and Pilch D. S. “Coupling of Drug Protonation to the Specific Binding of Aminoglycosides to the A Site of 16 S rRNA: Elucidation of the Number of Drug Amino Groups Involved and their Identities” J. Mol. Biol. 2003, 326, 1373–1387.
【52】 Pilch D. S., Kaul M., Barbieri C. M., and Kerrigan J. E. “Thermodynamics of aminoglycoside-rRNA recognition” Biopolymers 2003, 70 (1), 58–79.
【53】 Barbieri C. M., and Pilch D. S. “Complete Thermodynamic Characterization of the Multiple Protonation Equilibria of the Aminoglycoside Antibiotic Paromomycin: A Calorimetric and Natural Abundance 15N NMR Study” Biophysical Journal 2006, 90, 1338–1349.
【54】 Nguyen B., Stanek J., and Wilson W. D. “Binding-linked protonation of a DNA minor-groove agent” Biophys. J. 2006, 90, 1319–1328.
【55】 Petrosian S. A., and Makhatadze G. I. “Contribution of proton linkage to the thermodynamic stability of the major cold-shock protein of Escherichia coli CspA” Protein Science 2000, 9, 387–394.
【56】 Spolar R. S., and Record M.T. “Coupling of local folding to site-specific binding of proteins to DNA” Science 1994, 263, 777–784.
【57】 Ren J. S., Jenkins T. C., and Chaires J. B. “Energetics of DNA intercalation reactions” Biochemistry 2000, 39, 8439–8447.
【58】 Sharp K. A. “In Thermodynamics in Biology” Di Cera, E., Ed.; Oxford University Press: New York, 2000, pp 113–130.
【59】 Bergqvist S., Williams M. A., O''Brien R., and Ladbury J. E. “Heat capacity effects of water molecules and ions at a protein-DNA interface” J. Mol. Biol. 2004, 336, 829–842.
【60】 Barbieri C. M., Srinivasan A. R., and Pilch D. S. “Deciphering the origins of observed heat capacity changes for aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA a-sites: a calorimetric, computational, and osmotic stress study” J. Am. Chem. Soc. 2004, 126, 14380–14388.
【61】 Famulok M., and Mayer G. “Chemical biology - Aptamers in　nanoland” Nature 2006, 439 (7077), 666–669.
【62】 Liu J. W., and Lu Y. “Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles” Angew. Chem. Int. Ed. 2006, 45, 90 –94.
【63】 Liu J. W., Mazumdar D., and Lu Y. “A simple and sensitive "dipstick" test in serum based on lateral flow separation of aptamer-linked nanostructures” Angew. Chem. Int. Ed. 2006, 45, 7955 –7959.
【64】 Record M. T. J., Anderson C. F., and Lohman T. M. “Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity” Q. Rev. Biophys. 1978, 11, 103–178.
【65】 Andre C., Xicluna A., and Guillaume Y. C.　“Aptamer-oligonucleotide binding studied by capillary electrophoresis: Cation effect and separation efficiency” Electrophoresis 2005, 26, 3247–3255.
【66】 Vianini E., Palumbo M., and Barbara G. “In vitro selection of DNA aptamers that bind L-Tyrosinamide” Bioorganic & Medicinal Chemistry 2001, 9, 2543–2548.
【67】 Merino E. J., and Weeks K. M. “Facile conversion of aptamers into sensors using a 2 ''-ribose-linked fluorophore” J. Am. Chem. Soc. 2005, 127 (37), 12766–12767.
【68】 Michaud M., Jourdan E., Ravelet C., Villet A., Ravel A., Grosset C., and Peyrin E. “Immobilized DNA aptamers as target-specific chiral stationary phases for resolution of nucleoside and amino acid derivative enantiomers” Anal. Chem. 2004, 76, 1015–1020.
【69】 Leulliot N., and Varani G. “Current topics in RNA-protein recognition: control of specificity and biological function through induced fit and conformational capture” Biochemistry 2001, 40, 7947–7956.
【70】 Williamson J. R. “Induced fit in RNA–protein recognition” Nature Struct. Biol. 2000, 7, 834–837.
【71】 Andrews T. J., Lorimer G. H., and Tolbert N. E. “Ribulose diphosphate oxygenase” I. Synthesis of phosphoglycolate by fraction-1 protein of leaves. Biochemistry 1973, 12(1), 11–18.
【72】 Kaushik M., Kukreti R., Grover D., Brahmachari S. K., and Kukreti S. “Hairpin-duplex equilibrium reflected in the A→B transition in an undecamer quasipalindrome present in the locus control region of the human ß-globin gene cluster” Nucleic Acids Res. 2003, 31, 6904–6915.
【73】 Hoshika S., Minakawa N., and Matsuda A. “Synthesis and physical and physiological properties of 4 ''-thioRNA: application to post-modification of RNA aptamer toward NF-kappa B” Nucleic Acids Res. 2004, 32 (13), 3815–3825.
【74】 Bozza M., Sheardy R. D., Dilone E., Scypinski S., and Galazka M. “Characterization of the secondary structure and stability of an RNA aptamer that binds vascular endothelial growth factor” Biochemistry 2006, 45, 7639–7643.
【75】 Yamauchi T., Miyoshi D., Kubodera T., Nishimura A., Nakai S., and Sugimoto N. “Roles of Mg2+ in TPP-dependent riboswitch” FEBS Letters 2005, 579, 2583–25.
【76】 Johnson W. C. “CD of nucleic acids” In Nakanishi,K., Berova,N. and Woody,R.W. (eds), Circular Dichroism: Principles and Applications. VCH, New York, NY, 1994, pp. 523–540.
【77】 Cowan J. A., Ohyama T., Wang D. Q., and Natarajan K. “Recognition of a cognate RNA aptamer by neomycin B: quantitative evaluation of hydrogen bonding and electrostatic interactions” Nucleic Acids Res. 2000, 28 (15), 2935–2942.
【78】 Thomas J. R., Liu X. J., and Hergenrother P. J. “Biochemical and thermodynamic characterization of compounds that bind to RNA hairpin loops: Toward an understanding of selectivity” Biochemistry 2006, 45 (36), 10928–10938.
【79】 Gold B. “Effect of Cationic Charge Localization on DNA Structure” Biopolymers (Nucleic Acid Sci) 2002, 65,173–179.
【80】 Manning G. S. “Comments on selected aspects of nucleic acid electrostatics. Biopolymers” 2003, 69, 137–143.
【81】 McDonald R. J., Dragan A. I., Kirk W. R., Neff K. L., Privalov P. L., and Maher L. J. “DNA Bending by Charged Peptides: Electrophoretic and Spectroscopic Analyses” Biochemistry 2007, 46, 2306–2316.
【82】 Baumann C., Smith S., Bloomfield V., and Bustamante C. “Ionic effects on the elasticity of single DNA molecules” Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6185–6190.
【83】 Hud N. V., and Polak M. “DNA-cation interactions: the major and minor grooves are flexible ionophores” Current Opinion in Structural Biology 2001, 11, 293–301.
【84】 Zaug A. J., and Cech T. R. “The intervening sequence RNA of Tetrahymena is an enzyme” Science 1986, 231, 470-475.
【85】 Santoro S. W., and Joyce G. F. “A general purpose RNA-cleaving DNA enzyme” Proc. Natl. Acad. Sci. 1997, 94, 4262-4266.