DNA Aptamer掌性異構物辨識之熱力學與機制探討

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唐紹祖(Shao-tzu Tang) 查詢紙本館藏

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系統生物與生物資訊研究所

論文名稱

DNA Aptamer掌性異構物辨識之熱力學與機制探討
(Thermodynamic Basis of Chiral Recognition in a DNA Aptamer)

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摘要(中)

核酸適體(Aptamer)為對目標分具特異性及強親合性的寡聚核苷酸(Oligonucleotides)，因此可以被用來作為掌性異構物分離用的選擇劑，但對核酸適體對掌性異構的辨識(Chiral recognition)之機制探討並不多，故本研究選擇L-Argininamide(L-Arm)對應的DNA核酸適體（DNA aptamer），對L-Argininamide(L-Arm)及其鏡相異構物D-Argininamide (D-Arm)的掌性辨識為研究之目標。本實驗設計不同的實驗條件，包含不同鹽濃度、金屬離子種類（K+、Na+）、溫度、反應溶液pH值及緩衝液種類等，利用恆溫滴定微卡計（ITC）與圓二色光譜儀（CD），期望藉由熱力學（ITC）與二級結構（CD）分析，來獲得核酸適體掌性辨識性機制之相關資訊。
經由熱力學分析得知，L-Arm的核酸適體分別與L-Arm和D-Arm結合反應均為是Enthalpy driven及Entropy cost的程序，且結合過程中均伴隨著較鬆散的Apt構形的有序化(Conformational ordering)。在結合作用力的探討上，反應過程中均包含：（1）L-Arm和D-Arm帶正電的胺基 (NH３＋)和核酸間的靜電作用力造成核酸Counterion的釋放；（2）去質子化耦合效應以及（3）氫鍵等。不同金屬離子（K+、Na+）對於Apt與L-Arm及D-Arm和Apt的結合行為之影響均無明顯差異，當提高鹽濃度，均造成結合常數下降。比較L-Arm與Apt的結合反應和D-Arm與Apt的結合反應，由反應熱容量變化(?Cp)差異得到L-Arm和Apt結合所造成Apt構形的轉變(非極性表面的包埋)會完全相異於D-Arm結合所造成構形的轉變(極性表面的包埋)；而L-Arm與D-Arm鄰近掌中心位置質子化的Amino group和Apt間靜電作用力的不同是造成Apt對L,D-Arm具掌性辨識的主因之一。
本研究利用ITC所得之結合常數（KA）、熱力學參數（ΔH、ΔS），配合CD所得之構形變化，說明核酸適體掌性辨識之作用力、熱力學以及結構等相關資訊，此研究結果可提供以核酸適體作為掌性選擇劑設計及鏡相異構物分離應用之理論基礎。

摘要(英)

Chiral separations have become an area of increasing interest in pharmaceutical or biological fields, since most of biological molecules, many drugs and food additives are chiral compounds. Their physiological behaviors are related their chirality. DNA Aptamers (oligonucleotides) are a new group of chiral selectors and they can bind with a wide rang of target molecules with high affinity and specificity. As a new group of chiral selectors, there still exists a deficiency in the understanding of the molecular basis of chiral recognition in a DNA aptamer. Herein a comparative study of the L-argininamide-binding DNA aptamer complexation with L-Argininamide (L-Arm) and its enantiomer is investigated by spectroscopic and calorimetric methods. The influence of various experimental conditions (such as temperature, pH and salt concentration) on the L-Arm and D-Arm binding properties was studied in order to provide information about the chiral recognition mechanism of the DNA aptamer.
Isothermal titration calorimetry studies revealed that both L-Arm and D-Arm binding with the aptamer are an enthalpy driven and entropy cost process. Ionic strength experiments, along with pH experiments, indicate the protonated amino group of argininamide (both of enantiomers) participates in electrostatic interaction and a stronger electrostatic interaction of D-Arm binding with the aptamer than L-Arm binding with the aptamer. Both the larger change in ellipticity and more entropy cost suggest the larger conformational change of the aptamer upon the D-Arm binding than L-Arm binding. The stronger electrostatic interaction and the larger conformational change of the aptamer upon D-Arm binding also cause a contrary direction of the heat capacity change (?Cp) of L-Arm binding. This work indicates that the electrostatic interaction provided by protonated amino group with the aptamer and the conformational change of the nucleic acid upon the binding reactions are the sources of chiral recognition in the DNA aptamer.

關鍵字(中)

★ 熱力學
★ 掌性異構物
★ 核酸適體
★ 恆溫滴定微卡計

關鍵字(英)

★ ITC
★ thermodynamics
★ Chiral recognition
★ Aptamer

論文目次

中文摘要 I
Abstract III
目錄 V
圖目錄 VIII
表目錄 XI
第一章緒論 1
第二章文獻回顧 3
2.1鏡相異構物的介紹 3
2.1.1 鏡相異構物簡介 3
2.1.2 鏡相異構物不同的生化效應 5
2.1.3 鏡相異構的分離 7
2.1.4 掌性辨識理論之探討 12
2.1.5 D型胜肽(D-Peptide)及L型核酸(L-Nucleic acid) 14
2.2適體(Aptamers)介紹 16
2.2.1核酸適體及其篩選 16
2.2.2胜肽適體、核酸開關及核酸酶 19
2.2.3核酸適體相關應用 21
2.3核酸適合體與配體結合行為之探討 26
2.3.1 結合熱容量變化(ΔCp)分析之研究 26
2.3.2 L-Arginine及L-Argnininamide和核酸適體的交互作用 29
2.4 恆溫滴定微卡計及圓二色光譜儀 31
2.4.1恆溫滴定微卡計之簡介 31
2.4.2結合常數與速率常數分析 34
2.4.3圓二色光譜及在蛋白質、核酸結構分析上的應用 36
第三章實驗藥品與儀器設備 40
3.1 實驗藥品 40
3.2 儀器設備 41
3.3 實驗步驟 42
3.3.1 恆溫滴定微卡計實驗（MicroCal VP-ITC） 42
3.4 ITC實驗參數設定與數據分析 45
3.4.1 VP-ITC實驗參數設定 45
第四章結果與討論 47
4.1結合反應對核酸適體構形影響差異的分析 47
4.2探討結合反應熱力學的異同 53
4.3 L-Arm與D-Arm分別和Apt結合機制之探討 60
4.3.1結合反應之熱容量變化分析 60
4.3.2鹽類效應與金屬離子效應 67
4.3.3 溶液pH值影響與質子化耦合效應 79
第五章結論 85
第六章參考文獻 88

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2008-7-22

推文