Novel Biosensing Technologies Based On DNA Self-assembly For Detection Of MiRNA

Micro RNAs(mi RNAs or mi Rs) are involved in various cellular processes, including differentiation, proliferation, apoptosis and stress response via negatively regulating target m RNAs and inhibiting translation of protein. As a new promising biomarker, mi RNAs plays crucial roles in early diagnosis, prognosis and clinical monitoring of cancer diseases. Conventional methods including real-time quantitative PCR(RT-q PCR), microarray, and northern blot are generally used for profiling the expression of mi RNAs. Although these methods have several advantages, they still suffer from some shortcomings in practical applications. For instance, tedious steps and time consuming, low sensitivity and applicability, which affect the performance and reproducibility of these analytical methods, limiting their potential applications in the ultrasensitive bioanalysis of mi RNA. In this work, by integrating the latest researches of analytical chemistry, molecular biology, and clinical laboratory diagnostics, and employing the function nucleic acids and DNA self-assembly technology as breakthrough point, as well as combining with the point of care testing, two kinds of novel biosensing technology have been developed for rapid, isothermal detection of mi RNAs. This dissertation mainly contains the following two parts:1. A colorimetric biosensor for detection of attomolar micro RNA with a functional nucleic acid-based amplification machineA functional nucleic acid-based amplification machine was designed for simple and label-free ultrasensitive colorimetric biosensing of mi RNA. The amplification machine was composed of a complex of trigger template and C-rich DNA modified molecular beacon(MB) and G-rich DNA(GDNA) as the probe, polymerase and nicking enzyme, and a dumbbell-shaped amplification template. The presence of target mi RNA triggered MB mediated strand displacement to cyclically release nicking triggers, which led to a toehold initiated rolling circle amplification to produce large amounts of GDNAs. The formed GDNAs could stack with hemin to form G-quadruplex/hemin DNAzyme, a well-known horseradish peroxidase(HRP) mimic, for catalyzing a colorimetric reaction. The modified MB improved the stringent target recognition and reduced background signal. The proposed sensing strategy showed very high sensitivity and selectivity with a wide dynamic range from 10 a M to 1.0 n M, and enabled successful visual analysis of trace amount of mi RNA in real sample by the naked eye. This rapid and highly efficient signal amplification strategy provided a simple and sensitive platform for mi RNA detection. It would be a versatile and powerful tool for clinical molecular diagnostics.2. Catalytic Hairpin Assembly Actuated DNA Nanotweezer for Logic Gate Building and Sensitive Enzyme-free Biosensing of Micro RNAsA traget-switched DNA nanotweezer is designed for AND logic gate operation and enzyme-free detection of micro RNAs(mi RNAs) by catalytic hairpin assembly(CHA) and proximity-dependent DNAzyme formation. The double crossover motif-based nanotweezer consists of an arched structure as the set strand for target inputs and two split G-rich DNAs at the termini of two arms for signal output. Upon a CHA, small amount of binary target inputs can switch numerous open nanotweezers to a closed state, which leads to the formation of proximity-dependent DNAzyme in the presence of hemin to produce a highly sensitive biosensing system. The binary target inputs can be used for successful building of AND logic gate, which is validated by polyacrylamide gel electrophoresis, surface plasmon resonance and the biosensing signal. The developed biosensing system shows a linear response of the output chemiluminescence signal to input binary mi Rs with a detection limit of 30 f M. It can be used for mi R analysis in complex sample matrix. This system provides a simple and reusable platform for logic gate operation and enzyme-free, highly sensitive and specific multianalysis of mi RNAs.