RNA Sensors Based On DNA Molecular Machine And Autonomous RNA Transcription

In this study, three RNA sensors were constructed based on the coupling of traditional DNA molecular machine and/or autonomous RNA transcription to realize the high-sensitivity detection of RNA. Three DNA cycling amplification system or DNA polymerization-RNA transcription ligation systems were designed as different coupling mechanisms to achieve the signal amplifications:in these mechanisms, the target RNA strands were took as the input, and after a collection of autonomic cycling reactions, reporting RNA or DNA strands with much amplified concentrations were outputted and detected; amplification of the target RNA can thus be realized.In the amplification system1, DNA polymerization and RNA transcription were coupled to construct the amplification system. The fluorescence of the complex of malachite green and its RNA aptamer was employed to produce fluorescence signal. The molecular machine worked on a straight-srand probe, which consisted of the complementary segment of the target DNA, the complementary segment of the promoter of T7RNA polymerase (for transcription) and a template sequence for the aptamer sequence of malachite green. When the target DNA was inputted, it binded with the probe as a primer to start a DNA polymerization, which formed a dsDNA which would in turn invoked RNA transcription, in which RNA strands with the sequence of the aptamer of malachite green, which bound to malachite green to produce fluorescence for measurement. In this system, the optimal condition for the DNA polymerization-RNA transcription was explored, and the relationship between the fluorescent signal and the concentration of the inputted DNA was measured.In the amplification system2, another amplification system was constructed based on the coupled reaction of DNA polymerization and RNA transcription; different form the previous one, this system involved the repeatance of the DNA polymerization and RNA transcription, forming a cycling system. In this amplification system, a probe with two strands was used, with one strand for the production of the signal DNA strand, while the other for the recognition of the target RNA and the repeatance of the DNA polymerization and RNA transcription cycle. In addition to the DNA polymerization and RNA transcription in the amplification system1, these two reactions were repeated due to the cycling of the target RNA strand on the second strand; and the cycling increased the thoughput of the molecular machine, thus bringing forth even more amplification. In this system, the optimal condition for the DNA polymerization-RNA transcription was explored, and the relationship between the fluorescent signal and the concentration of the inputted RNA was measured.In the amplification system3, an amplification system was constructed based on the coupled reaction of DNA polymerization and restrict nicking of specific DNA sequences. This system involved the repeatance of the DNA polymerization and nicking, forming a cycling system. In this amplification system, a probe with two strands was used, with one strand for the production of the signal DNA strand, while the other for the recognition of the target RNA and the repeatance of the DNA polymerization and restricted nicking. These two reactions were repeated due to the cycling of the target RNA strand on the second strand; and the cycling increased the thoughput of the molecular machine, thus bringing forth even more amplification. In this system, the optimal condition for the DNA polymerization-RNA transcription was explored, and the relationship between the fluorescent signal and the concentration of the inputted RNA was measured.