DNA Molecular Machine Controlled Isothermal Assembly Of Gold Nanoparticles

DNA is a double helix biopolymer consisting of four types of repeating units,adenine (A), thymine (T), guanine (G), and cytosine (C), which is generally regarded as carriers for storage and replication of genetic information in life body. As a reaction motif with precise molecular structure, DNA is widely used in construction of molecular device, DNA molecular computing, diagnostics, and biosensing. In particular, construction of DNA catalytic networks using the toehold-mediated DNA strand displacement reaction have been attracted more attentions of people. In these catalytic systems, the DNA target is used as catalyst, which can catalyze the running of DNA molecular machines through a series of DNA strand displacement reactions,and can be recyclable in these systems.Gold nanoparticles (AuNPs) are attracting considerable interest due to their unique physical and chemical properties. Since the pioneering work of surface modified AuNPs using thiolated DNA in the mid-1990s by Mirkin et al., These DNA functionalized AuNPs have been widely applied at present in various fields,including in vitro and in vivo detection, cell transfection, gene regulation, and programmed nanostructure assembly. In the classical DNA-AuNPs assembly systems, two ends of the DNA linker is complementary with two types of DNA probes on AuNP surface respectively to form visual aggregate, thus realizing the assembly of AuNPs. But this classical 'direct-linkage-strategy'only has single function and cannot control the assembly process of AuNPs in more precise way. In particular, this simple strategy cannot be used in the DNA-AuNPs assembly system with complex logic functions. Therefore, this weakness limits further applications of the DNA-AuNPs assembly system.In this thesis, through combing the DNA-AuNPs assembly system with toehold-mediated DNA strand displacement system, we constructed a series of DNA strand displacement based systems to tune the isothermal assembly processes of AuNPs in more accurate method. Firstly, we studied the property of DNA strand displacement reaction on surface of DNA modified AuNP. We found that there was a sharp reaction speed jump when the toehold length of invading strand on AuNP surface was increased from 7 to 8, which was caused by the high DNA grafting density on AuNP surface. This study help us to a better understanding the property of DNA strand displacement reaction on AuNP surface and will be conductive to design and application of isothermal assembly of DNA-AuNPs system.Multiple inputs DNA logic gate can execute more complex logical operation compared to simple DNA logic gate, and have potential to detect multiple DNA gene sequences simultaneously in DNA diagnostics. In chapter three, we constructed two logic gates ([(A AND B) AND (C AND D)] and [(A OR B) AND (C OR D)]) with multiple inputs and outputs with expected outcomes through utilizing the combination of AuNPs and AuNRs with DNA nanodevices using sequence design.The catalyst strands were used as input signals in this system, because of two non-overlapping double hump-like UV spectra peaks of AuNPs and AuNRs, whereas the maximum changes of absorbance value in AuNPs and AuNRs were used as output signals. This strategy was very intuitive, and the results were easier to be distinguished by detecting the surface plasmon resonance maximum changes in the value of the reaction solution with the UV-vis spectrometer. The system also demonstrated promising potential in multiple inputs and outputs logic operations construction.In principle, DNA-AuNPs assembly systems that are driven by toehold mediated DNA strand displacement reaction can be further combined into multiple-layer sophisticated DNA catalytic circuitries. The challenges for construction of multiple-layer DNA-AuNPs assembly system driven by DNA molecular machine are complex interactions between multiple nucleic acid strands and the negative effect of leakage in multiple-layer system. In chapter four, cascaded two- and three-layer circuits of AuNPs assembly driven by DNA molecular machine were fabricated. Combined with experiments and computational modeling, the dynamic behaviors and leakage performances of the circuits have been analyzed. Our studies indicate that both the DNA sequence and the molar ratio of the machines have important effects on integrating DNA-AuNP circuitry into complex systems with robust behavior. We believe that the construction of complex DNA-AuNP circuit guided by predictive computer simulation will contribute toward advancing the implementation of complex DNA circuits.In the research of chapter five, we noticed that because of the specificity of the DNA strand grafted on AuNP surface, the modified DNA-AuNP conjugate only can be used for detection of specific DNA target, which increased the cost and complexity of the DNA target detection. Therefore, we constructed a two-layer DNA catalytic network which is consisted of an upstream DNA circuit and a downstream DNA-AuNPs assembly system based on our previous work. In this system, the DNA linker released from the upstream DNA circuit can trigger the downstream DNA-AuNPs assembly. Through the analysis of theory and experimental optimization, we chose a best toehold design for this system, which showed perfect performance in single nucleotide polymorphism (SNP) discrimination. Negligible changes were observed in both the UV-vis absorbance and color image for the systems with the spurious targets' own single-base mismatch at different positions,insertion, and deletion in either the invading toehold region or the branch migration region; whereas, the system with a correct target completed the reaction in ?6 h.Moreover, DNA-AuNPs conjugates in this strategy can be used in other DNA targets detection without redesign, which will save laborious work and cost.In the above classical toehold mediated strand displacement systems,DNA-AuNPs assembly kinetic rate mainly depends on the length and sequence composite of the toehold. However, the functions of other regulators (pH,temperature, and light) are suppressed. This potential restriction limits the precise regulation of the DNA-AuNPs assembly system. Therefore, in the last chapter we integrated a pH-triggered C+-GC triplex structure with a toehold mediated strand displacement reaction based DNA circuit to engineer assembly of the DNA-AuNPs conjugates for an additional environmental controlling factor. Thus, the pH-responsive triplex DNA based nanomachine can regulate reaction rate of the catalytic DNA-AuNPs assembly at different pH conditions in an efficient manner.In summary, we constructed a series of DNA molecular machines controlled AuNPs assembly systems in this thesis. And these systems can tune the isothermal assembly processes of AuNPs in many ways compared to the traditional simple method. In addition, our systems show great advantages and potentails in multiple-input DNA logic gates, DNA detection, SNP discrimination, and pH sensors.