Toehold Switch Sensor for miRNA Cancer Biomarker

Abstract

Colorectal cancer (CRC) is the third most common cancer in the United States, yet existing screening and diagnostic methods remain expensive, invasive, and require many resources, discouraging early testing. Abnormal microRNA (miRNA) expression has been shown to be a cancer biomarker, with miR-29a being upregulated in the blood of CRC patients. To address this gap, we sought to develop a low-cost, portable, and minimally invasive assay for CRC miR-29a detection in blood plasma. We first had to construct our toehold switch. The toehold switch was assembled using Gibson Assembly and transformed into E. coli, isolated through plasmid miniprep, and verified through Oxford Nanopore long-read. After sequencing the biosensor, we determined we have successfully assembled the toehold switch and will begin testing of its efficacy. We are now currently testing the sensitivity and specificity of our toehold switch construct reporter for our target miR-29a biomarker. Afterwards, we will optimize the performance of our toehold switch reporter & integrate it into a one pot TRAP system, a Thermally Responsive Alkane Partition System, to streamline the reactions leading to Green Fluorescent Protein (GFP) expression in a positive test. However, due to time constraints we were unable to complete all the cell-free testing of our miR-29a and toehold switch and get to the TRAP system but we have gotten paradoxical results as our different constructs with and without the presence of miRNA had values that were significantly different, potentially indicating the success of our toehold.