DNA Machine For DNA Amplification And Its Application To The Determination Of Other Biomolecules

DNA, life’s information carrier, has recently emerged as a versatile material for constructing self-assembled synthetic molecular structures and devices. DNA machines are viewed as biomolecular assemblies that perform machine-like functions at the molecular level. In this work, combined DNA recognition, aptamer recognition with nanoparticles (NPs), different DNA-based machines were constructed for amplified analysis of nucleic acid, low-molecular-weight substrates and proteins. The experimental protocols could be summarized as follows:1. A novel autonomous bio-bar-code DNA machine that is driven by template-dependent DNA replication is developed to exponentially amplify special DNA sequences. Combined with a DNA aptamer recognition element, the DNA machine can be further applied in the aptamer-based, amplified analysis of small molecules. As a model analyte, adenosine triphosphate (ATP) is determined by using the DNA machine system in combination with a DNA aptamer recognition strategy and differential pulse anodic stripping voltammetry (DPASV). Under the optimum conditions, detection limits as low as2.8×10-17M (3s) for target DNA and4.7×10-9M (3s) for ATP are achieved. The satisfactory determination of ATP in K562leukemia cell and Ramos Burkitt’s lymphoma cell reveal that this protocol possesses good selectivity and practicality. As a promising biomolecular device, this DNA machine may have an even broader application in the rapidly developing field of nanobiotechnology.2. A novel autonomous DNA machine was constructed for amplified electrochemical analysis of two DNAs. The DNA machine operates in a two-cycle working mode to amplify DNA recognition events; the working mode is assisted by two different nicking endonucleases (NEases). Two bio-barcode probes, a ZnS NP-DNA probe and a CdS NP-DNA probe, were used to trace two target DNAs. The detection system was based on a sensitive DPASV method for the simultaneous detection of Zn2+and Cd2+tracers, which were obtained by dissolving the two probes. Under the optimised conditions, detection limits as low as5.6×10-17M (3s) and4.1×10-17M (3s) for the two target DNAs were achieved. It has been proven that the DNA machine system can simultaneously amplify two target DNAs by more than four orders of magnitude within30min at room temperature. In addition, in combination with an aptamer recognition strategy, the DNA machine was further used in the aptamer-based amplification analysis of ATP and lysozyme. With the amplification of the DNA machine, detection limits as low as5.6×10-9M (3s) for ATP and5.2×10-13M (3s) for lysozyme were simultaneously obtained. The satisfactory determination of ATP and lysozyme in Ramos cells reveals the good selectivity and feasibility of this protocol. The DNA machine is a promising tool for ultrasensitive and simultaneous multianalysis because of its remarkable signal amplification and simple machine-like operation.3. A simple and versatile strategy was developed for the construction of DNA-templated NP assemblies using click DNA ligation. Maleic acid/sorbic acid Diels-Alder cycloaddition reaction is applied in the click DNA ligation. Sorbic acid-prelabelled DNA nanowires with micron-scale length were synthesized and acted as templates for the construction of NP assemblies. Au NPs were orderly assembled onto the DNA nanowires using the click DNA ligation to form DNA-templated Au NP chains. DNA-templated CdTe quantum dots (QDs) chains were also synthesized using the method, and were further functionalized with cell-binding aptamer to construct a novel linear-shaped cellular probe for cancer cell imaging. The click DNA ligation method offers a convenient and efficient way to synthesize DNA-templated NP assemblies, and may become a powerful tool in other DNA-based studies.4. A novel double-color fluorescence signal method was presented for visual detection of nucleic acid using rolling circle amplification (RCA). Combined with a RCA reaction, green DNA/CdTe QD probe was used for producing green control dots before the reactions for target DNA. Target DNA hybridized to capture DNA near the green control dots, and then triggerd another RCA reaction. Orange DNA/CdTe QD probe was used to tracing target DNA, and produce orange signal dots. The DNA hybridization event could be judged using the combination of green control dots and orange signal dots. The strategy presented a novel platform for single-molecule DNA detection, thus possessed powerful potentials for further applications.