The Research Of Biosensing And Molecular Logic Circuits Based On DNA Functional Nanostructure

The structure of DNA is prone to polymorphism. In addition to the well-known double helix, a number of alternative structures have been discovered in vivo and randomly selected in vitro, such as triple strands DNA, DNA Holliday junction, DNA G-quadruplex, DNA i-motif, as well as the specific steric structures of DNAzymes and DNA aptamers. These special DNA structures have some certain functions, for example, the particular sequences of DNAzymes and DNA aptamers can exert their activities by forming a specific steric structure with intramolecular hydrogen bonding, stacking interaction, electrostatic interaction, metal coordination and so on. In addition to these discovered and selected DNA nanostructures, self-assembled DNA nanostructures with controllable shapes and sizes can be created based on complementary base pairing by making use of structural DNA nanotechnology. All of these natural and artificial DNA nanostructures are the bases for constructing DNA-based nanodevices with great application potential. On the one hand, novel sensing machenism can be developed by utilizing the conformational changes of these functional DNA nanosturctures for detecting and analyzing a series of targets ranging from metal ions and small molecules to biomolecules, and even viruses or cells; on the other hand, self-assembled DNA nanostructures have provided new platforms for creating DNA-based intellegent nanodevices.Taking these two aspects into consideration, we made use of the peculiar properties of some functional DNA nanostructures(DNA hairpin, G-quadruplex, DNA hybrids) to develop label-free colorimetric analytical methods for the detection of enzymes involved in DNA metabolism and single nucleotide mismatches. Further, we obtained controllable self-assembled DNA nanostructures by taking advantage of structural DNA nanotechnology and these DNA nanostructures were employed as molecular computing modules for designing molecular logic circuits, which laid the foundation for creating DNA-based intellegent nanodevices. In order to achieve these goals, we have carried out the following works:(1) A universal method for colorimetric analyzing the activity of enzymes involved in DNA metabolism was developed based on G-quadruplex-hemin DNAzyme. This analytical method was termed DNAzyme-based enzyme activity assay(DEAA). In DEAA, simple DNA nanostructures, such as DNA hairpin structure and G-quadruplex, were employed, and we have designed a self-assembled DNA nanomachine, which can isothermally amplify the sequences of G-quadruplex in vitro for signal amplification, improving the detection sensitivity. DEAA is a lowcost, visual and universal technique for assay of the activity of multiple enzymes involved in DNA metabolism. Furthermore, DEAA can be readily extended to screening for inhibitors of enzymes and the colorimetric detection of single nucleotide mismatches. This research work showed the application potential of simple functional DNA nanostructures in biochemical analysis.(2) Structural DNA nanotechnology has provided approaches for the construction of complicated and exquisite three-dimensional(3D) DNA nanostructures, however, most of the existing strategies for the assembly of 3D DNA nanostructures are complex and require large numbers of different DNA strands. Aiming at these problems, we made use of the geometric symmetry of DNA nanostructure to modularly assemble fully addressable 3D DNA nanostructures with the least diverse DNA component strands, which significantly reduced the cost of 3D DNA nanostructures. As a case in point, an addressable DNA triangular nanoprism was assembled with seven kinds of DNA component strands. The self-assembled nanoprism has equilateral triangle bases with side length about 7.14 nm(21 bp) and vertical sides with length about 3.40 nm(10 bp), and its inner volume is approximately 75 nm3. Native polyacrylamide gel electrophoresis(PAGE) and fluorescence resonance energy transfer(FRET) studies were carried out to verify the self-assembly of the DNA triangular nanoprism. The thermal stability of the DNA triangular nanoprism was investigated. The melting curve reflected that the nanoprism started to open at 43 °C, indicating that the nanoprism was stable enough to be applicable at room temperature or physiological temperature(37 °C). Also, the stability of the prism structure in the human serum was studied. The DNA nanoprism could keep its structure intact for about six hours in 10%(v/v) human blood serum at 37 °C, which suggested that the DNA nanoprism could be applied in complex biological matrix.(3) Based on the results of the above work, we employed the DNA triangular nanoprism as a universal molecular computing module for the construction of molecular logic gates. We adopted an integration design strategy, the different parts of the DNA triangular nanoprism were assigned as one of the components of the logic device, respectively. Such an integration design strategy made the DNA triangular nanoprism a universal platform for building various molecular logic gates, such as binary basic logic gates(OR, AND, INHIBIT and XOR), combinatorial logic gates(INHIBIT-OR) and multi-valued logic gates(ternary INHIBIT gate). Moreover, a DNA computing system for identification of even numbers and odd numbers from natural numbers was successfully established by employing only this single prism and four single-stranded DNAs. In addition, we also demonstrated that the stimuli-responsive DNA nanoprism could execute logic operation steadily in complex biological matrix containing human serum.(4) Finally, we combined self-assembled DNA nanostructure with graphene oxide(GO) for novel DNA nanostructure composite materials, and employed this composite material as a versatile platform for designing molecular logic circuits. In this work, a DNA nanotripod was assembled via structural DNA nanotechnology. Then, the DNA nanotripod was used to regulate the interaction between a small fluorescent molecule ROX and GO for achieving a three-input majority logic gate. This three-input majority gate could be switched between AND and OR gates, making it a versatile building block for constructing more molecular logic gates and complex computing circuits. In order to demonstrate the versatility of the three-input majority gate, XOR and INHIBIT gates, as well as combinatorial logic gates were constructed. By these foundations, a molecular computing system was established for screening the composite numbers from natural numbers less than ten by making the best of the versatility of the platform. Also, we have demonstrated that this platform was able to stably execute logic operation in cell lysate.