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 [1–3]. 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
References
[1]
S. Tyagi, “Imaging intracellular RNA distribution and dynamics in living cells,” Nature Methods, vol. 6, no. 5, pp. 331–338, 2009.
[2]
G. Bao, J. R. Won, and A. Tsourkas, “Fluorescent probes for live-cell RNA detection,” Annual Review of Biomedical Engineering, vol. 11, pp. 25–47, 2009.
[3]
B. A. Armitage, “Imaging of RNA in live cells,” Current Opinion in Chemical Biology, vol. 15, no. 6, pp. 806–812, 2011.
[4]
A. K. Chen, M. A. Behlke, and A. Tsourkas, “Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA,” Nucleic Acids Research, vol. 36, no. 12, article e69, 2008.
[5]
P. Santangelo, N. Nitin, and G. Bao, “Nanostructured probes for RNA detection in living cells,” Annals of Biomedical Engineering, vol. 34, no. 1, pp. 39–50, 2006.
[6]
D. M. Kolpashchikov, “Binary probes for nucleic acid analysis,” Chemical Reviews, vol. 110, no. 8, pp. 4709–4723, 2010.
[7]
Y. Li, X. Zhou, and D. Ye, “Molecular beacons: an optimal multifunctional biological probe,” Biochemical and Biophysical Research Communications, vol. 373, no. 4, pp. 457–461, 2008.
[8]
K. J. Livak, S. J. A. Flood, J. Marmaro, W. Giusti, and K. Deetz, “Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization,” PCR Methods and Applications, vol. 4, no. 6, pp. 357–362, 1995.
[9]
M. K. Johansson, H. Fidder, D. Dick, and R. M. Cook, “Intramolecular dimers: a new strategy to flourescence quenching in dual-labeled oligonucleotide probes,” Journal of the American Chemical Society, vol. 124, no. 24, pp. 6950–6956, 2002.
[10]
M. S. Ellwood, M. Collins, E. F. Fritsch, et al., “Strand displacement applied to assays with nucleic acid probes,” Clinical Chemistry, vol. 32, no. 9, pp. 1631–1636, 1986.
[11]
L. E. Morrison, T. C. Halder, and L. M. Stols, “Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization,” Analytical Biochemistry, vol. 183, no. 2, pp. 231–244, 1989.
[12]
Q. Li, G. Luan, Q. Guo, and J. Liang, “A new class of homogeneous nucleic acid probes based on specific displacement hybridization,” Nucleic Acids Research, vol. 30, no. 2, p. E5, 2002.
[13]
D. Y. Zhang and E. Winfree, “Control of DNA strand displacement kinetics using toehold exchange,” Journal of the American Chemical Society, vol. 131, no. 47, pp. 17303–17314, 2009.
[14]
J. He, M. Rusckowski, Y. Wang et al., “Optical pretargeting of tumor with fluorescent MORF oligomers,” Molecular Imaging and Biology, vol. 9, no. 1, pp. 17–23, 2007.
[15]
M. Liang, X. Liu, D. Cheng et al., “Optical antisense tumor targeting in vivo with an improved fluorescent DNA duplex probe,” Bioconjugate Chemistry, vol. 20, no. 6, pp. 1223–1227, 2009.
[16]
D. S. Seferos, D. A. Giljohann, H. D. Hill, A. E. Prigodich, and C. A. Mirkin, “Nano-flares: probes for transfection and mRNA detection in living cells,” Journal of the American Chemical Society, vol. 129, no. 50, pp. 15477–15479, 2007.
[17]
S. Shakeel, S. Karim, and A. Ali, “Peptide nucleic acid (PNA)—a review,” Journal of Chemical Technology and Biotechnology, vol. 81, no. 6, pp. 892–899, 2006.
[18]
P. E. Nielsen, “Peptide Nucleic Acids (PNA) in chemical biology and drug discovery,” Chemistry and Biodiversity, vol. 7, no. 4, pp. 786–804, 2010.
[19]
H. Fang, K. Zhang, G. Shen, K. L. Wooley, and J. S. A. Taylor, “Cationic shell-cross-linked knedel-Like (cSCK) nanoparticles for highly efficient PNA delivery,” Molecular Pharmaceutics, vol. 6, no. 2, pp. 615–626, 2009.
[20]
K. Zhang, H. Fang, G. Shen, J. S. A. Taylor, and K. L. Wooley, “Well-defined cationic shell crosslinked nanoparticles for efficient delivery of DNA or peptide nucleic acids,” Proceedings of the American Thoracic Society, vol. 6, no. 5, pp. 450–457, 2009.
[21]
F. Aktan, “iNOS-mediated nitric oxide production and its regulation,” Life Sciences, vol. 75, no. 6, pp. 639–653, 2004.
[22]
A. Pautz, J. Art, S. Hahn, S. Nowag, C. Voss, and H. Kleinert, “Regulation of the expression of inducible nitric oxide synthase,” Nitric Oxide, vol. 23, no. 2, pp. 75–93, 2010.
[23]
H. Fang, Y. Shen, and J. S. Taylor, “Native mRNA antisense-accessible sites library for the selection of antisense oligonucleotides, PNAs, and siRNAs,” RNA, vol. 16, no. 7, pp. 1429–1435, 2010.
[24]
K. Zhang, H. Fang, Z. Wang, J. S. A. Taylor, and K. L. Wooley, “Cationic shell-crosslinked knedel-like nanoparticles for highly efficient gene and oligonucleotide transfection of mammalian cells,” Biomaterials, vol. 30, no. 5, pp. 968–977, 2009.
[25]
H. Fang, X. Yue, X. Li, and J. S. Taylor, “Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells,” Nucleic Acids Research, vol. 33, no. 21, pp. 6700–6711, 2005.
[26]
R. Shrestha, Y. Shen, K. A. Pollack, J. S. Taylor, and K. L. Wooley, “Dual peptide nucleic acid- and peptide-functionalized shell cross-linked nanoparticles designed to target mRNA toward the diagnosis and treatment of acute lung injury,” Bioconjugate Chemistry, vol. 23, no. 3, pp. 574–585, 2012.
[27]
H. Kuhn, V. V. Demidov, B. D. Gildea, M. J. Fiandaca, J. C. Coull, and M. D. Frank-Kamenetskii, “PNA beacons for duplex DNA,” Antisense and Nucleic Acid Drug Development, vol. 11, no. 4, pp. 265–270, 2001.
[28]
I. A. Nazarenko, S. K. Bhatnagar, and R. J. Hohman, “A closed tube format for amplification and detection of DNA based on energy transfer,” Nucleic Acids Research, vol. 25, no. 12, pp. 2516–2521, 1997.
[29]
I. Nazarenko, R. Pires, B. Lowe, M. Obaidy, and A. Rashtchian, “Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes,” Nucleic Acids Research, vol. 30, no. 9, pp. 2089–2095, 2002.
[30]
M. R. Lewis and F. Jia, “Antisense imaging: and miles to go before we sleep?” Journal of Cellular Biochemistry, vol. 90, no. 3, pp. 464–472, 2003.
[31]
T. Noda and F. Amano, “Differences in nitric oxide synthase activity in a macrophage-like cell line, RAW264.7 cells, treated with lipopolysaccharide (LPS) in the presence or absence of interferon-γ (IFN-γ): possible heterogeneity of iNOS activity,” Journal of Biochemistry, vol. 121, no. 1, pp. 38–46, 1997.
[32]
C. Altmann, A. Andres-Hernando, R. H. McMahan, et al., “Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury,” American Journal of Renal Physiology, vol. 302, no. 4, pp. F421–F432, 2012.
[33]
H. W. Trask, R. Cowper-Sal-Lari, M. A. Sartor et al., “Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs,” RNA, vol. 15, no. 10, pp. 1917–1928, 2009.
