Hydroxyapatite(HA) chromatography 已經廣泛的被利用來分離單股DNA和雙股DNA，此技術是利用單股DNA對HA之吸附親和力較其雙股DNA小的原理來做分離，但是之間的交互作用與分離機制目前並無完整的研究討論。因此本研究主要是利用等溫吸附線與恆溫滴定微卡計(ITC)來探討HA與單股DNA和雙股DNA的吸附機制與熱力學分析。在實驗之部分，我們設計不同的條件，包括不同的環境因子(溫度、鹽濃度和pH)以及DNA組成(GC content、length、GC stacking rich & CG stacking rich)等來探討各種不同的效應對吸附行為的影響。 由實驗結果顯示，在等溫吸附線的分析部分，發現對於雙股DNA而言，對HA的交互作用主要是以靜電作用力為主，因此疏水作用力或結構之改變並不會對其造成太大的影響；而對於單股DNA，因為其鹼基是暴露在外，所以與HA之交互作用會受較多因素之影響，例如靜電作用力、疏水作用力與結構之穩定性等，因此在不同的環境因子或是DNA組成的不同，都會造成單股DNA與HA之親和力改變。而且我們也發現單股DNA有多層吸附（multi-layer adsorption）之現象，尤其是在高鹽的環境中，這也說明了單股DNA在吸附過程中受到多重作用力的影響。 而在熱力學分析的部分，發現在不同的鹽濃度下，雙股DNA及單股DNA皆為吸熱的反應，所以此吸附過程為entropy driven，表示去水合在此貢獻中相當重要。並且發現單股DNA之吸附焓皆大於雙股DNA，表示對於單股DNA，疏水作用力之貢獻較大，然而對於雙股DNA，則是靜電作用力貢獻較大。 藉由等溫吸附線與熱力學分析，我們可以清楚了解不同環境因子對於雙股DNA和單股DNA與HA之鍵結行為和機制的影響，此研究結果可提供生物辨識作用方面的基礎資訊。 Hydroxyapatite(HA) chromatography has been used extensively for the separation of single-strand DNA and double-strand DNA. This technique is based on the fact that ssDNA have less affinity for hydroxylapatite than their dsDNA. But the details of the mechanism of the separation of ssDNA and dsDNA by hydroxyapatite are still not clear. In this study, we discussed the effects of environmental factors (i.e. salt concentration, temperature and pH) and different kinds of ssDNA and dsDNA (i.e. GC content, length, secondary structure effects and GC or CG stacking rich…) on the binding behavior. By equilibrium batch analysis, we measure the adsorption isotherm to evaluate the affinity ssDNA and dsDNA with HA, while isothermal titration microcalorimetric was used to measure the adsorption enthalpy. By the kinetics and thermodynamics analysis, we established an interaction mechanism to explain the ssDNA and dsDNA with HA interaction behavior. By isotherm analysis, we realized that dsDNA mainly use electrostatic force to bind with HA. So in high salt concentration, the phosphate of dsDNA interaction was shielded Na+ and the affinity would be decreased. Because the surface of ssDNA molecule contains hydrophobic bases and negative charge phosphate backbone, so both of the hydrophobic and electrostatic interactions affect the affinity of ssDNA for HA. In addition, stability of structure is also important for ssDNA, so increasing in the structural order of ssDNA would increase the binding affinity of ssDNA with HA. However, for oligo ssDNAs (15mer and 60mer) in this study, the hydrophobic interaction is more revealed and multi- layers adsorption were observed. By thermodynamics analysis, we found that the adsorption enthalpies of ssDNA and dsDNA are all endothermic in this study and the dehydration step in the binding process plays a key role. This investigation offers useful knowledge of ssDNA and dsDNA with HA binding behavior and fundamental thermodynamics information in biorecognition system.
