A targeted, self-delivered, and photocontrolled molecular beacon for mRNA detection in living cells.

The spatiotemporal dynamics of specific mRNA molecules are difficult to image and detect inside living cells, and this has been a significant challenge for the chemical and biomedical communities. To solve this problem, we have developed a targeted, self-delivered, and photocontrolled aptamer-based molecular beacon (MB) for intracellular mRNA analysis. An internalizing aptamer connected via a double-stranded DNA structure was used as a carrier probe (CP) for cell-specific delivery of the MB designed to signal target mRNA. A light activation strategy was employed by inserting two photolabile groups in the CP sequence, enabling control over the MB's intracellular function. After the probe was guided to the target cell via specific binding of aptamer AS1411 to nucleolin on the cell membrane, light illumination released the MB for mRNA monitoring. Consequently, the MB is able to perform live-cell mRNA imaging with precise spatiotemporal control, while the CP acts as both a tracer for intracellular distribution of the MB before photoinitiation and an internal reference for mRNA ratiometric detection.

[1]  P. Meltzer,et al.  Increased manganese superoxide dismutase expression suppresses the malignant phenotype of human melanoma cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[3]  T. Hazelrigg,et al.  Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis , 1994, Nature.

[4]  Y. Shav-Tal,et al.  Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells , 2010, Nature Cell Biology.

[5]  D. Shangguan,et al.  Aptamers evolved from live cells as effective molecular probes for cancer study , 2006, Proceedings of the National Academy of Sciences.

[6]  M. Rosbash,et al.  Messenger and heterogeneous nuclear RNA in HeLa cells: differential inhibition by cordycepin. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Weihong Tan,et al.  DNA branch migration reactions through photocontrollable toehold formation. , 2013, Journal of the American Chemical Society.

[8]  H. Lusic,et al.  Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and Xenopus embryos. , 2010, Journal of the American Chemical Society.

[9]  Stochasticity of Manganese Superoxide Dismutase mRNA Expression in Breast Carcinoma Cells by Molecular Beacon Imaging , 2005, Chembiochem : a European journal of chemical biology.

[10]  D. Scherman,et al.  A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Surong Zhang,et al.  Fluorescence resonance energy transfer in near-infrared fluorescent oligonucleotide probes for detecting protein–DNA interactions , 2008, Proceedings of the National Academy of Sciences.

[12]  Yong Wang,et al.  Cell type–specific delivery of siRNAs with aptamer-siRNA chimeras , 2006, Nature Biotechnology.

[13]  Diana P Bratu,et al.  Visualizing the distribution and transport of mRNAs in living cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Daniel St Johnston,et al.  The intracellular localization of messenger RNAs , 1995, Cell.

[15]  D Bopp,et al.  The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. , 1988, The EMBO journal.

[16]  Piet Herdewijn,et al.  Increased uptake of antisense oligonucleotides by delivery as double stranded complexes. , 2004, Biochemical pharmacology.

[17]  Y. Rojanasakul,et al.  Novel non-endocytic delivery of antisense oligonucleotides. , 2000, Advanced drug delivery reviews.

[18]  R. Jansen,et al.  mRNA localization: message on the move , 2001, Nature Reviews Molecular Cell Biology.

[19]  E. Spicer,et al.  The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells. , 2008, Cancer research.

[20]  Gang Bao,et al.  Fluorescent probes for live-cell RNA detection. , 2009, Annual review of biomedical engineering.

[21]  Jonas W Perez,et al.  Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells. , 2010, Journal of the American Chemical Society.

[22]  Yi Lu,et al.  Reversible cell-specific drug delivery with aptamer-functionalized liposomes. , 2009, Angewandte Chemie.

[23]  David A. Rand,et al.  Measurement of single-cell dynamics , 2010, Nature.

[24]  Liat Rosenfeld,et al.  Single-allele analysis of transcription kinetics in living mammalian cells , 2010, Nature Methods.