Peptide-based Delivery One of the critical issues in molecular imaging and therapeutics is the ability to deliver molecular probes and drug molecules into cells, tissues and animals (including human). Specifically, in measuring the expression level of RNAs in living cells using synthetic probes, it is essential to deliver these probes into cells through the plasma membrane (for mRNA detection) and to cell nuclei through the nuclear membrane (for nuclear RNA detection). In performing targeted therapy, it is essential to deliver drug molecules into specific cells (such as cancer cells). It is possible that the same delivery strategy could be used to internalize both imaging probes and therapeutic agents into diseased cells. To perform live-cell studies of gene expression using molecular beacons and other nanoprobes, we have developed two delivery methods. The first is a reversible membrane permeabilization method in which ~30 nm pores on the cell membrane are formed by streptolysin O (SLO) in serum-free media, allowing the influx of molecular beacons (Fig. 2a). The cell membrane is then resealed by removing the SLO-beacon mixture and adding normal media with serum. The second is to conjugate cell penetrating peptides (CPPs e.g. TAT-1) to molecular beacons (Fig. 2b) for probe internalization. Both methods could give nearly 100% delivery efficiency. Three conjugation strategies were used in constructing peptide-linked molecular beacons: a stable chemical linker (Fig. 2b), a disulfide bridge, and streptavidin-biotin linkage. We have detected human GAPDH and Survivin mRNAs in live HDF cells using all three designs (Figs. 2c, 2d and 2e, respectively), demonstrating the filamentous intracellular localization of GAPDH mRNA. We have also demonstrated that cellular delivery of molecular beacons using the peptide-based approach has far better performance compared with conventional transfection methods. We are currently establishing the tissue delivery and mRNA detection capabilities using CPPs, primarily for cancer diagnosis. The peptide-linked molecular beacons approach promises to open new and exciting opportunities in sensitive gene detection and quantification in vivo.