All-in-One Plasmids

APPs are engineered plasmids that encode all fluorescent probes needed to visualize both an mRNA molecule and the protein being translated from it. By carefully tuning probe expression ratios within a single plasmid, APPs eliminate the need to balance multiple separate constructs, which has been a major challenge in single-molecule translation experiments. Researchers need only co-transfect two plasmids—a reporter and an APP—to reliably detect individual translation sites with high signal-to-noise in living cells.
The system is modular and supports multiple fluorescent colors, simultaneous tracking of different mRNAs or proteins in the same cell, and optional immobilization of mRNA for long-term observation. Using APPs, it is possible to achieve continuous imaging for up to 30 minutes with minimal photobleaching. The measured average elongation rate (~6.5 codons per second) aligns well with genome-wide benchmarks while revealing significant variability between individual mRNAs—information that is difficult to access using existing techniques.