(693e) Using the Cell-Free Gene Expression System for Highly Accurate Microrna Detection

Early diagnosis and the monitoring of diseases and infections is a crucial medical practice to avoid aggressive and malignant disease progression and to retain the quality of one’s health. However, many patients struggle to monitor their health regularly or there are no proper methods to detect certain illnesses early enough. Recently, numerous studies have confirmed that the dysregulation of microRNA (miRNA) can be correlated with the development and specific stage of major diseases. These miRNAs serve as the perfect biomarker, as they circulate in the blood during cell-to-cell communication in highly protective packaging. The current gold standard and other technologies that detect this biomarker suffer from high costs, high variability, and inaccuracies. These flaws stem from the same methods, resulting in low differentiation between the thousands of different forms of miRNA resulting in low accuracy.

The cell-free gene expression system is the remedy to these flawed methods, providing a cellular environment and finely tuned genetic circuitry capabilities programmed by the user. Here we have programmed an ultrasensitive and highly accurate miRNA sensing platform using the E. coli-based cell-free gene expression system. This platform can differentiate a 1 nucleotide difference between similar miRNA when the gold standard, RT-qPCR, could not. The genetic circuitry component allows for meticulous amplification steps to be added after the precise miRNA has been detected, removing false positives, and increasing sensitivity. Our circuit also has an extremely low leak compared to others that does not trigger the amplification cascade the same as the sample, allowing for low background noise. With the cell-free system’s tolerance to toxic substances, the circuit can perform from a blood sample with minimal purification, lowering cost. The system is also able to be freeze-dried, making it inexpensive to transport and store. Given these advantages and our findings, the cell-free gene expression system is the optimal tool for miRNA sensing and profiling.