基于无机纳米材料和DNA纳米结构的研究及应用
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摘要
纳米技术的发展，推动了多个学科及领域的突破，特别是在材料，生物，医学，化学，电子学，光学等学科。纳米技术促进了多个学科进行交叉，融合，从而为解决问题提供了新方法和机遇。基于此，本论文开展了对纳米材料的研究及应用。这些材料包括零维的纳米金，一维的碳纳米管，二维的氧化石墨烯，三维的DNA纳米结构。论文涉及了生物分子与无机纳米材料的相互作用研究，功能化纳米传感器的构建，DNA纳米结构的设计及应用。主要研究结果如下：
（1）报道了一种新颖的纳米金组装方法，即通过poly A嵌段DNA将DNA探针组装到纳米金表面。通过比较，我们发现较之前的传统巯基组装策略，poly A双嵌段Au NPs-DNA具有一些独特的优势。第一，降低了探针合成成本；第二，poly A实现了对纳米金表面的饱和覆盖，从而抑制非特异性DNA吸附；第三，提高了杂交动力学速率。
（2）本部分工作系统考察了不同DNA结构，包括单标记ssDNA及部分杂交的dsDNA，多标记的RCA产物切割链，HCR长产物链和三维3-helix DNA结构，与GO的作用机制。借助于GO对荧光分子的超淬灭作用，表征了其标记的DNA链与GO的作用状态。从不同位置标记的ssDNA与GO作用时，荧光值随不同靶标DNA的加入而发生变化。我们证明ssDNA在GO表面的吸附状态：ssDNA的每个碱基都吸附在GO表面。从RCA介导的多标记荧光探针与GO作用时，荧光值随不同DNA靶标而发生变化，从而肯定了上述的结论。接着，从HCR长产物链和短产物链与GO的相互作用，我们认为DNA结构与GO的作用是一个动态的平衡吸附过程，当ssDNA在结构中比例增加，其结构与GO的作用力也会增加，表现为荧光值下降。最后，从三维DNA结构3-helix与GO的作用研究，发现三维DNA结构与GO的作用力比较弱，与之前的研究结果一致。
（3）该部分工作，首先讨论了GO与CEPs之间的作用机制，还探讨了GO对CPE1的超淬灭能力。在此基础上，我们提出了GO对CEPs/Fluo-Probe检测体系优化的可能性。通过实验，我们也如愿提高了CEPs/Fluo-Probe检测体系的信噪比。于此同时，CEPs/Fluo-Probe检测体系的检测限被提高了50pM。
（4）该部分工作主要研究了三种纳米材料，包括零维的纳米金，一维的碳纳米管，二维的氧化石墨烯，及其与DNA的相互作用，从而为理解它们的特征和设计传感策略提供帮助。荧光淬灭实验证明三种纳米材料（GO，SWNTs，AuNPs）对FAM-ssDNA都具有高的淬灭效率。同时基于淬灭作用，达到较高的单双链分辨率。通过比较发现GO单双链分辨能力最强。因此，基于GO的传感器展现了最好的检测限（0.2nM）和最好的重复性。于此类比，SWNTs组和AuNPs组的检测限分别为1nM和2nM。另外GO和SWNTs的传感器还表现出优良的SNP分析能力。更重要的是，我们通过研究NMs和NaCl浓度对作用力影响，分析了相关作用机制。
（5）本部分工作首先实现了3-helix和6-helix DNA结构的高产率合成，并表征及确定其大小和完整性。同时，我们研究了6-helix DNA结构的稳定性及抗降解能力。数据表明6-helix结构具有优异的稳定性。通过对结构上锁链的优化，我们实现了6-helix结构高打开效率及较快的打开速率。在此基础上，我们成功实现了细胞内6-helix结构的可控开关。另外，我们尝试载带CpG药物分子。但实验数据表明3-helix结构和6-helix结构本身就具有较强的免疫刺激活性，掩盖了CpG分子的刺激活性，其作用机制还待研究。
Development of nanotechnology, promote breakthroughs in a number of disciplines andfields, especially in materials, biology, medicine, chemistry, electronics, optics and otherdisciplines. Nanotechnology promote the cross and integration between several disciplines, so asto provide new methods and opportunities to solve the problems. Based on this, the paper carriedout the research and application based on nanomaterials. These materials include goldnanoparticles (zero-dimension), carbon nanotubes (one-dimension), graphene oxide (twodimension), DNA nanostructures (three-dimension). This paper involves the research aboutinteraction of biological molecules with inorganic nano-materials, nano-sensors based onfunctional nano-materials, design and application of DNA nanostructures. The main findings areas follows:
(1) In this section, we report a conceptually new strategy to prepare conjugates of DNA andgold nanoparticles (AuNPs). Based on our discovery that poly-adenine (polyA) can bind to thehighly curved surface of AuNPs with extraordinarily high affinity, we designed a diblockoligonucleotide with the anchoring block (ployA) and the recognition block (DNA sequence forrecognition) for conjugation with AuNPs. The significance of this type of diblockoligonucleotide-based conjugation strategy is described as follows:①Diblock oligonucleotidesare natural sequences that are essentially free of any modification.②PolyA effectively blocknon-specific DNA-gold binding.③Our new strategy provides favorable hybridization ability inboth kinetics and thermodynamics.
(2) We discussed the different behavior of different DNA structure on GO surface. In thisstudy, we researched on different DNA absorption state on the graphene surface, such as ssDNA,ssDNA including piece dsDNA, RCA products, DNA hairpins, long dsDNA with ssDNA distalend and3D DNA structure. Based on fluorescence assay of fluorescence restored, RCA systemand HCR system, it was proved that all of the nucleobases laid nearly flat on the GO surface andssDNA were stably adsorbed by the GO. And the fluorophores in double strand fragments couldleave the GO surface, but the surplus single strand DNA would be stably adsorbed on GOsurface even if those took the small percentage of the long DNA strand.
(3) we have demonstrated that the adsorption of CPEs on GO was related to the molecularstructure of CPEs and the functional groups present at the surface of GO. Based on thesuperquenching of cationic CPEs by GO and different absorption mechanism of ssDNA and dsDNA on GO, we have developed a sensitive homogeneous sensor for DNA detection anddemonstrated its improved S/N ratio and high specificity.
(4) In this work,three water soluble nanomaterials,0D AuNPs,1D SWNTs, and2D GO,and their characterized interaction with DNA were investingated detailed for well understandingtheir characters and designing sensing stratigies. The fluorescence quenching behavior of NMs,the performance of three NMs-based sensors, and the NMs-DNA interaction were well studiedhere. The highest differentiation of ssDNA and dsDNA were achieved by employingnanomaterials. Consequently, the GO-based sensor exhibited the best sensitivity (LOD:0.2nM),while1nM of LOD for SWNTs group and2nM for AuNPs group were presented. Worthy ofnoting, GO-and SWNTs-based sensors exhibited excellent discrimination of single mismatchbase pairs from perfect matched sequences. More importantly, the related mechanisms werecarefully analyzed through investigating the usage of NMs and the concentration of NaCl.
(5) In this section, we synthesised the3-helix and6-helix DNA structure with high yield,and characterized and determined its size and integrity. Meanwhile, we have studied the stabilityand resistance degradation of6-helix DNA structure. Data show that the stability and resistancedegradation of6-helix DNA structure were very well. By optimizing the structure of the chains,we achieved the high efficiency switching of6-helix structure. Base on these, we havesuccessfully achieved the controllable switching of6-helix structure in vivo. In addition, we triedto take CpG drug molecules into the cells with setting its in the interior of6-helix DNA structure.However, experimental data shown that3-helix structure and the6-helix structure itself hadstrong immune stimulating activity, which covered up the stimulating activity of CpG molecules.And its mechanism of this action has to be studied in the future.
（1）报道了一种新颖的纳米金组装方法，即通过poly A嵌段DNA将DNA探针组装到纳米金表面。通过比较，我们发现较之前的传统巯基组装策略，poly A双嵌段Au NPs-DNA具有一些独特的优势。第一，降低了探针合成成本；第二，poly A实现了对纳米金表面的饱和覆盖，从而抑制非特异性DNA吸附；第三，提高了杂交动力学速率。
（2）本部分工作系统考察了不同DNA结构，包括单标记ssDNA及部分杂交的dsDNA，多标记的RCA产物切割链，HCR长产物链和三维3-helix DNA结构，与GO的作用机制。借助于GO对荧光分子的超淬灭作用，表征了其标记的DNA链与GO的作用状态。从不同位置标记的ssDNA与GO作用时，荧光值随不同靶标DNA的加入而发生变化。我们证明ssDNA在GO表面的吸附状态：ssDNA的每个碱基都吸附在GO表面。从RCA介导的多标记荧光探针与GO作用时，荧光值随不同DNA靶标而发生变化，从而肯定了上述的结论。接着，从HCR长产物链和短产物链与GO的相互作用，我们认为DNA结构与GO的作用是一个动态的平衡吸附过程，当ssDNA在结构中比例增加，其结构与GO的作用力也会增加，表现为荧光值下降。最后，从三维DNA结构3-helix与GO的作用研究，发现三维DNA结构与GO的作用力比较弱，与之前的研究结果一致。
（3）该部分工作，首先讨论了GO与CEPs之间的作用机制，还探讨了GO对CPE1的超淬灭能力。在此基础上，我们提出了GO对CEPs/Fluo-Probe检测体系优化的可能性。通过实验，我们也如愿提高了CEPs/Fluo-Probe检测体系的信噪比。于此同时，CEPs/Fluo-Probe检测体系的检测限被提高了50pM。
（4）该部分工作主要研究了三种纳米材料，包括零维的纳米金，一维的碳纳米管，二维的氧化石墨烯，及其与DNA的相互作用，从而为理解它们的特征和设计传感策略提供帮助。荧光淬灭实验证明三种纳米材料（GO，SWNTs，AuNPs）对FAM-ssDNA都具有高的淬灭效率。同时基于淬灭作用，达到较高的单双链分辨率。通过比较发现GO单双链分辨能力最强。因此，基于GO的传感器展现了最好的检测限（0.2nM）和最好的重复性。于此类比，SWNTs组和AuNPs组的检测限分别为1nM和2nM。另外GO和SWNTs的传感器还表现出优良的SNP分析能力。更重要的是，我们通过研究NMs和NaCl浓度对作用力影响，分析了相关作用机制。
（5）本部分工作首先实现了3-helix和6-helix DNA结构的高产率合成，并表征及确定其大小和完整性。同时，我们研究了6-helix DNA结构的稳定性及抗降解能力。数据表明6-helix结构具有优异的稳定性。通过对结构上锁链的优化，我们实现了6-helix结构高打开效率及较快的打开速率。在此基础上，我们成功实现了细胞内6-helix结构的可控开关。另外，我们尝试载带CpG药物分子。但实验数据表明3-helix结构和6-helix结构本身就具有较强的免疫刺激活性，掩盖了CpG分子的刺激活性，其作用机制还待研究。
Development of nanotechnology, promote breakthroughs in a number of disciplines andfields, especially in materials, biology, medicine, chemistry, electronics, optics and otherdisciplines. Nanotechnology promote the cross and integration between several disciplines, so asto provide new methods and opportunities to solve the problems. Based on this, the paper carriedout the research and application based on nanomaterials. These materials include goldnanoparticles (zero-dimension), carbon nanotubes (one-dimension), graphene oxide (twodimension), DNA nanostructures (three-dimension). This paper involves the research aboutinteraction of biological molecules with inorganic nano-materials, nano-sensors based onfunctional nano-materials, design and application of DNA nanostructures. The main findings areas follows:
(1) In this section, we report a conceptually new strategy to prepare conjugates of DNA andgold nanoparticles (AuNPs). Based on our discovery that poly-adenine (polyA) can bind to thehighly curved surface of AuNPs with extraordinarily high affinity, we designed a diblockoligonucleotide with the anchoring block (ployA) and the recognition block (DNA sequence forrecognition) for conjugation with AuNPs. The significance of this type of diblockoligonucleotide-based conjugation strategy is described as follows:①Diblock oligonucleotidesare natural sequences that are essentially free of any modification.②PolyA effectively blocknon-specific DNA-gold binding.③Our new strategy provides favorable hybridization ability inboth kinetics and thermodynamics.
(2) We discussed the different behavior of different DNA structure on GO surface. In thisstudy, we researched on different DNA absorption state on the graphene surface, such as ssDNA,ssDNA including piece dsDNA, RCA products, DNA hairpins, long dsDNA with ssDNA distalend and3D DNA structure. Based on fluorescence assay of fluorescence restored, RCA systemand HCR system, it was proved that all of the nucleobases laid nearly flat on the GO surface andssDNA were stably adsorbed by the GO. And the fluorophores in double strand fragments couldleave the GO surface, but the surplus single strand DNA would be stably adsorbed on GOsurface even if those took the small percentage of the long DNA strand.
(3) we have demonstrated that the adsorption of CPEs on GO was related to the molecularstructure of CPEs and the functional groups present at the surface of GO. Based on thesuperquenching of cationic CPEs by GO and different absorption mechanism of ssDNA and dsDNA on GO, we have developed a sensitive homogeneous sensor for DNA detection anddemonstrated its improved S/N ratio and high specificity.
(4) In this work,three water soluble nanomaterials,0D AuNPs,1D SWNTs, and2D GO,and their characterized interaction with DNA were investingated detailed for well understandingtheir characters and designing sensing stratigies. The fluorescence quenching behavior of NMs,the performance of three NMs-based sensors, and the NMs-DNA interaction were well studiedhere. The highest differentiation of ssDNA and dsDNA were achieved by employingnanomaterials. Consequently, the GO-based sensor exhibited the best sensitivity (LOD:0.2nM),while1nM of LOD for SWNTs group and2nM for AuNPs group were presented. Worthy ofnoting, GO-and SWNTs-based sensors exhibited excellent discrimination of single mismatchbase pairs from perfect matched sequences. More importantly, the related mechanisms werecarefully analyzed through investigating the usage of NMs and the concentration of NaCl.
(5) In this section, we synthesised the3-helix and6-helix DNA structure with high yield,and characterized and determined its size and integrity. Meanwhile, we have studied the stabilityand resistance degradation of6-helix DNA structure. Data show that the stability and resistancedegradation of6-helix DNA structure were very well. By optimizing the structure of the chains,we achieved the high efficiency switching of6-helix structure. Base on these, we havesuccessfully achieved the controllable switching of6-helix structure in vivo. In addition, we triedto take CpG drug molecules into the cells with setting its in the interior of6-helix DNA structure.However, experimental data shown that3-helix structure and the6-helix structure itself hadstrong immune stimulating activity, which covered up the stimulating activity of CpG molecules.And its mechanism of this action has to be studied in the future.
引文
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