%0 Journal Article
%T Imaging mRNA Expression in Live Cells via PNA¡¤DNA Strand Displacement-Activated Probes
%A Zhenghui Wang
%A Ke Zhang
%A Karen L. Wooley
%A John-Stephen Taylor
%J Journal of Nucleic Acids
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/962652
%X Probes for monitoring mRNA expression in vivo are of great interest for the study of biological and biomedical problems, but progress has been hampered by poor signal to noise and effective means for delivering the probes into live cells. Herein we report a PNA¡¤DNA strand displacement-activated fluorescent probe that can image the expression of iNOS (inducible nitric oxide synthase) mRNA, a marker of inflammation. The probe consists of a fluorescein labeled antisense PNA annealed to a shorter -labeled DNA which quenches the fluorescence, but when the quencher strand is displaced by the target mRNA the fluorescence is restored. DNA was used for the quencher strand to facilitate electrostatic binding of the otherwise netural PNA strand to a cationic shell crosslinked knedel-like (cSCK) nanoparticle which can deliver the PNA¡¤DNA duplex probe into cells with less toxicity and greater efficiency than other transfection agents. RAW 264.7 mouse macrophage cells transfected with the iNOS PNA¡¤DNA probe via the cSCK showed a 16 to 54-fold increase in average fluorescence per cell upon iNOS stimulation. The increase was 4 to 7-fold higher than that for a non-complementary probe, thereby validating the ability of a PNA¡¤DNA strand displacement-activated probe to image mRNA expression in vivo. 1. Introduction There has been great interest in developing real-time fluorescent imaging agents for mRNA expression in vivo that are based on antisense oligodeoxynucleotides and analogs [1¨C3]. There are two main problems in getting such systems to work well. The first is to deliver the agents efficiently into the cytoplasm, and the second is to minimize background signal from unbound probe. The main problem with getting nucleic acids and analogs into the cytoplasm is that they are membrane impermeable, thereby requiring the use of a physical, chemical, or biochemical device or agent [4]. Many mRNA-imaging studies have used microinjection, electroporation, or pore forming agents such as streptolysin O (SLO), but such agents would be unsuitable for in vivo work. Others have made use of cell-penetrating peptides, or transfection agents, but these often result in endocytosis and trapping of the probe in endosomes which reduces the amount of probes in the cytoplasm and can lead to nonspecific background signal. To reduce the background signal from unbound probe, probes have been designed to emit fluorescence only in the presence of target mRNA by a variety of strategies. Among these are molecular beacons, binary and dual FRET probes, strand-displacement probes, quenched
%U http://www.hindawi.com/journals/jna/2012/962652/