聚电解质（DNA）表面吸附和相互作用及Toehold协助下的DNA链替换反应
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摘要
双链DNA分子是电负性很强的生物大分子,同时也是刚性很强的聚电解质。它能够与多价阳离子和带正电荷的聚电解质如聚乙烯亚胺(PEI)作用并形成带电量不一样的复合物。在本论文中,我们采用双偏振干涉测量技术研究了聚乙烯亚胺与双链DNA在表面的相互作用过程同时研究了不同带电量的DNA/阳离子复合物在硅羟基和氨基表面的吸附行为。研究发现,当高浓度PEI流经PEI-DNA复合层表面时,PEI能够与DNA发生相互作用并使复合层发生溶胀进而使得DNA分子以DNA/PEI复合物的形式从芯片表面剥离下来。同时,我们发现,增加体系盐浓度可以使得这种剥离现象消失。
通过对DNA/阳离子复合物在两种芯片表面的吸附行为研究,我们发现,相同复合物在氨基表面比在硅羟基表面吸附得更加紧密。当复合物间存在有强电荷排斥时,后续吸附的复合物能够使先前吸附的复合物发生构象调整同时由于复合物间的排斥作用使得后续吸附的复合物只能以部分接触的方式吸附到表面。当复合物间电荷排斥很弱时,并不能引起构象的调整,但是由于芯片表面很拥挤,后续吸附的复合物同样只能以部分接触的方式堆积到表面。
通过碱基间严格可预测的互补配对并在toehold协助下,DNA链间能发生链替换反应,从而实现了DNA链引发的杂交链增长反应并作为一种新型的信号放大技术被广泛运用于目标小分子,DNA及其它特定物质的检测。本论文第五章中,我们通过将引发链DNA固定到芯片表面,利用双偏振干涉测量技术研究了芯片表面DNA杂交链增长反应的详细过程并计算得到了芯片表面不同盐浓度下和不同固定方式下杂交链增长反应效率。研究发现随着杂交链增长反应的进行,形成的长的双链DNA倾向于倒向芯片表面而非伸向溶液里面使得释放出来的单链DNA引发后续杂交链替换反应的效率降低从而使得整个芯片表面杂交链增长反应的效率比溶液中低。
最后一章中,我们提出了一种基于2-硝基苄基紫外光切断的新型光控toehold的可控形成方法。利用该方法通过控制光照强度(光照时间)可以控制toehold的形成量并生成纯的1:1比例的具有活性toehold的双链DNA结构。该结构可用于引发DNA分支迁移反应从而实现了光控的DNA分支迁移反应。与先前报道的活性toehold体系不同的是,我们利用光将隐藏在发卡DNA环部的toehold释放出来,该过程不用加化学试剂同时不会产生DNA副产物。此外,通过调节光照强度,可以简单方便地控制toehold的产生量进而调节toehold协助下的DNA分支迁移反应的速率。该方法具有构建光响应的DNA纳米结构以及DNA循环系统的潜质。
Double-stranded DNA (dsDNA) is a kind of highly negatively charged biopolymer. It is also a kind of stiff polyelectrolyte. dsDNA can interact with multivalent cations and other polyelectrolyte with positive charges such as polyethyleneimine (PEI), forming complexes with different charge conditions. In the present work, the interaction between PEI and DNA on silica surface and the adsorption behaviors of DNA/cation complexes with different charge conditions on silica and amino chip surfaces were studied using dual polarization interferometry (DPI). We determined that when PEI is injected over the PEI-DNA complex bilayer, DNA is stripped off under high PEI concentrations. Our results prove that stripping results from the formation of overcharged DNA/PEI complexes and from the strong electrostatic repulsion between the first PEI layer and the overcharged DNA/PEI complex. Therefore, stripping can be avoided under specific salt conditions because of the charge shielding effect.
The adsorption of DNA/Ca2+, DNA/Cu2+, and DNA/Co(NH3)63+complexes on amino and silica chip surface were investigated using dual polarization interferometry. A more compact DNA/cation complex layer formed on the amino chip surface compared with the silica chip surface at the same cation condition. The real-time mass, thickness, and density changes were monitored during the adsorption process. The overall results show that the approaching complexes can cause the conformation rearrangement of pre-adsorbed complexes and the adsorbed complexes affect the deposition pattern of the approaching complexes during the adsorption of DNA/Ca2+and DNA/Cu2+complexes on both chip surfaces. The relatively strong electrostatic repulsion between the approaching and adsorbed complexes results in multiple mass loading rate changes and loose attachment of the following complexes. The weak repulsion between the DNA/Co(NH3)63+complexes cannot induce this kind of rearrangement. Thus, no multiple mass loading rate changes were observed. Meanwhile, the adsorbed DNA/Co(NH3)63+can also affect the deposition pattern of the following complexes because of the geometric resistance.
The specificity and predictability of Watson-Crick base pairing make DNA a powerful and versatile material for engineering at the nanoscale. A kind of DNA hybridization chain reaction (HCR) has recently been designed based on toehold-mediated DNA strand-displacement reactions, which has been used as a new signal amplification technique for DNA and other molecules detection. In the fifth chapter, DNA HCR on a solid-liquid interface was investigated using DPI. The effects of salt concentration and different immobilization positions on HCR efficiency and DNA conformation were also investigated using DPI. With the HCR going on, the formed long dsDNA structure lay on the chip surface, which suppressed the activity of the released single-strand part on DNA hairpin.
In the last chapter, we present for the first time a new photocontrolled toehold formation method to generate1:1ratio DNA duplexes for toehold-mediated branch migration reactions. This method is based on the photocleavage of2-nitrobenzyl linker-embedded DNA hairpin precursor structures. Different from previously reported overhanging-toehold systems, light is employed to activate the hidden toehold without addition of any chemicals or formation of waste DNA molecules. More importantly, the amount of released toehold can be easily controlled by fine-tuning the irradiation dose, allowing the rate of the toehold-mediated branch migration reaction to be regulated by changing the initial UV irradiation time. Our system shows potential for the construction of light-responsive dynamic DNA nanostructures and DNA circuits.
通过对DNA/阳离子复合物在两种芯片表面的吸附行为研究,我们发现,相同复合物在氨基表面比在硅羟基表面吸附得更加紧密。当复合物间存在有强电荷排斥时,后续吸附的复合物能够使先前吸附的复合物发生构象调整同时由于复合物间的排斥作用使得后续吸附的复合物只能以部分接触的方式吸附到表面。当复合物间电荷排斥很弱时,并不能引起构象的调整,但是由于芯片表面很拥挤,后续吸附的复合物同样只能以部分接触的方式堆积到表面。
通过碱基间严格可预测的互补配对并在toehold协助下,DNA链间能发生链替换反应,从而实现了DNA链引发的杂交链增长反应并作为一种新型的信号放大技术被广泛运用于目标小分子,DNA及其它特定物质的检测。本论文第五章中,我们通过将引发链DNA固定到芯片表面,利用双偏振干涉测量技术研究了芯片表面DNA杂交链增长反应的详细过程并计算得到了芯片表面不同盐浓度下和不同固定方式下杂交链增长反应效率。研究发现随着杂交链增长反应的进行,形成的长的双链DNA倾向于倒向芯片表面而非伸向溶液里面使得释放出来的单链DNA引发后续杂交链替换反应的效率降低从而使得整个芯片表面杂交链增长反应的效率比溶液中低。
最后一章中,我们提出了一种基于2-硝基苄基紫外光切断的新型光控toehold的可控形成方法。利用该方法通过控制光照强度(光照时间)可以控制toehold的形成量并生成纯的1:1比例的具有活性toehold的双链DNA结构。该结构可用于引发DNA分支迁移反应从而实现了光控的DNA分支迁移反应。与先前报道的活性toehold体系不同的是,我们利用光将隐藏在发卡DNA环部的toehold释放出来,该过程不用加化学试剂同时不会产生DNA副产物。此外,通过调节光照强度,可以简单方便地控制toehold的产生量进而调节toehold协助下的DNA分支迁移反应的速率。该方法具有构建光响应的DNA纳米结构以及DNA循环系统的潜质。
Double-stranded DNA (dsDNA) is a kind of highly negatively charged biopolymer. It is also a kind of stiff polyelectrolyte. dsDNA can interact with multivalent cations and other polyelectrolyte with positive charges such as polyethyleneimine (PEI), forming complexes with different charge conditions. In the present work, the interaction between PEI and DNA on silica surface and the adsorption behaviors of DNA/cation complexes with different charge conditions on silica and amino chip surfaces were studied using dual polarization interferometry (DPI). We determined that when PEI is injected over the PEI-DNA complex bilayer, DNA is stripped off under high PEI concentrations. Our results prove that stripping results from the formation of overcharged DNA/PEI complexes and from the strong electrostatic repulsion between the first PEI layer and the overcharged DNA/PEI complex. Therefore, stripping can be avoided under specific salt conditions because of the charge shielding effect.
The adsorption of DNA/Ca2+, DNA/Cu2+, and DNA/Co(NH3)63+complexes on amino and silica chip surface were investigated using dual polarization interferometry. A more compact DNA/cation complex layer formed on the amino chip surface compared with the silica chip surface at the same cation condition. The real-time mass, thickness, and density changes were monitored during the adsorption process. The overall results show that the approaching complexes can cause the conformation rearrangement of pre-adsorbed complexes and the adsorbed complexes affect the deposition pattern of the approaching complexes during the adsorption of DNA/Ca2+and DNA/Cu2+complexes on both chip surfaces. The relatively strong electrostatic repulsion between the approaching and adsorbed complexes results in multiple mass loading rate changes and loose attachment of the following complexes. The weak repulsion between the DNA/Co(NH3)63+complexes cannot induce this kind of rearrangement. Thus, no multiple mass loading rate changes were observed. Meanwhile, the adsorbed DNA/Co(NH3)63+can also affect the deposition pattern of the following complexes because of the geometric resistance.
The specificity and predictability of Watson-Crick base pairing make DNA a powerful and versatile material for engineering at the nanoscale. A kind of DNA hybridization chain reaction (HCR) has recently been designed based on toehold-mediated DNA strand-displacement reactions, which has been used as a new signal amplification technique for DNA and other molecules detection. In the fifth chapter, DNA HCR on a solid-liquid interface was investigated using DPI. The effects of salt concentration and different immobilization positions on HCR efficiency and DNA conformation were also investigated using DPI. With the HCR going on, the formed long dsDNA structure lay on the chip surface, which suppressed the activity of the released single-strand part on DNA hairpin.
In the last chapter, we present for the first time a new photocontrolled toehold formation method to generate1:1ratio DNA duplexes for toehold-mediated branch migration reactions. This method is based on the photocleavage of2-nitrobenzyl linker-embedded DNA hairpin precursor structures. Different from previously reported overhanging-toehold systems, light is employed to activate the hidden toehold without addition of any chemicals or formation of waste DNA molecules. More importantly, the amount of released toehold can be easily controlled by fine-tuning the irradiation dose, allowing the rate of the toehold-mediated branch migration reaction to be regulated by changing the initial UV irradiation time. Our system shows potential for the construction of light-responsive dynamic DNA nanostructures and DNA circuits.
引文
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