View All Posters - View Poster

P2003
Cell-Permeable Fluorogenic Oligonucleotides Single Cell RNA Profiling, Antisense & siRNA Delivery, & Live Cell Imaging
Presenter Akira Komoriya, OncoImmunin, Inc., USA
Additional Authors: Beverly Z. Packard
The intracellular delivery technology that has been the foundation for a generation of highly cell permeable and specific fluorogenic protease substrates, e.g., PhiPhiLux®, the CaspaLux®es, CyToxiLux®, and GranToxiLux®, has been extended to the oligonucleotide arena. Application of the technology yields the following characteristics for reporter probes at the live single cell level  (1) Probes are highly cell permeable with an achieved intracellular concentration of ca. 2 µM (2) High cell permeability is uniform for all cells and tissues (3) Judicious selection of fluorophores allows simultaneous determination at the single cell level of multiple target enzyme activities (4) Lack of probe cytotoxicity enables continuous observation over several days and (5) Live cell assays based on this technology have been shown to be suitable for high-content and high-throughput drug candidate screening. The same characteristics are preserved when the peptide moiety of a protease probe is replaced with an equally non-cell permeable oligonucleotide moiety. Specifically, intracellular transport efficiency and uniformity have been shown via confocal microscopy as well as by FMAT 8200 analyses. Furthermore, using a series of oligonucleotides with wobble sequences of lengths ranging from 15 to 40 nucleotides, the observed cell permeability and probe fluorogenesis were found to be sequence independent. For example, when target cells were incubated with DNA oligonucleotide probes designed to hybridize with target beta-actin RNA, the intensity of the fluorescence was higher than those derived from the scrambled sequence oligonucleotide and/or a set of olignucleotides probes with totally randomized sequences. Thus, this drug delivery technology does not require use of viral vectors, lipids, salts, detergents and/or electric stimulation. Homogeneous, live cell assays for RNA profiling, antisense and siRNA delivery, as well as imaging at the single cell level can now be developed.