The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization in response to shear flow can provide unprecedented opportunities for the molecular analysis of atherosclerosis. We carried out an extensive study of the design of molecular beacons to target BMP-4 mRNA, which plays important roles in proatherogenic development in response to unstable flow conditions. Specifically, we selected an optimal molecular beacon design, and found that the fluorescent intensity from targeting BMP-4 mRNA correlated well with the GFP signal after up-regulating BMP-4 and co-expressing GFP using adenovirus. The knock-down of BMP-4 mRNA using siRNA significantly reduced the beacon signal, further demonstrating detection specificity. We found that, due to target accessibility, molecular beacons designed with different target sequences gave very different signal levels, and establishing molecular beacon design rules has significant implications to live cell mRNA detections, especially to the studies of BMP-4 mRNA in endothelial cells under shear flow.
Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons
