Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells.

A strategy is presented for the live cell imaging of messenger RNA using hairpin DNA-functionalized gold nanoparticles (hAuNP). hAuNP improve upon technologies for studying RNA trafficking by their efficient internalization within live cells without transfection reagents, improved resistance to DNase degradation, low cytotoxicity, and the incorporation of hairpin DNA molecular beacons to confer high specificity and sensitivity to the target mRNA sequence. Furthermore, the targeted nanoparticle-beacon construct, once bound to the target mRNA sequence, remains hybridized to the target, enabling spatial and temporal studies of RNA trafficking and downstream analysis. Targeted hAuNP exhibited high specificity for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA in live normal HEp-2 cells and respiratory syncytial virus (RSV) mRNA in live RSV-infected HEp-2 cells with high target to background ratios. Multiplexed fluorescence imaging of distinct mRNAs in live cells and simultaneous imaging of mRNAs with immunofluorescently stained protein targets in fixed cells was enabled by appropriate selection of molecular beacon fluorophores. Pharmacologic analysis suggested that hAuNP were internalized within cells via membrane-nanoparticle interactions. hAuNP are a promising approach for the real-time analysis of mRNA transport and processing in live cells for elucidation of biological processes and disease pathogenesis.

[1]  K. Mullis,et al.  Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. , 1985, Science.

[2]  R. Singer,et al.  Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts , 1994, The Journal of cell biology.

[3]  Michael R. Green,et al.  Localization of pre-mRNA splicing in mammalian nuclei , 1994, Nature.

[4]  M. Player,et al.  Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Hesketh,et al.  Nuclear Import of Metallothionein Requires Its mRNA to Be Associated with the Perinuclear Cytoskeleton* , 1999, The Journal of Biological Chemistry.

[6]  G. Molema,et al.  Serum as a modulator of lipoplex‐mediated gene transfection: dependence of amphiphile, cell type and complex stability , 2000, The journal of gene medicine.

[7]  J. Krijnse Locker,et al.  Microtubule-dependent organization of vaccinia virus core-derived early mRNAs into distinct cytoplasmic structures. , 2001, Molecular biology of the cell.

[8]  K. Aktories,et al.  Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Libchaber,et al.  Single-mismatch detection using gold-quenched fluorescent oligonucleotides , 2001, Nature Biotechnology.

[10]  J. Hesketh,et al.  Influence of metallothionein-1 localization on its function. , 2001, The Biochemical journal.

[11]  M. Giacca,et al.  Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins* , 2003, Journal of Biological Chemistry.

[12]  V. Puri,et al.  Selective caveolin-1-dependent endocytosis of glycosphingolipids. , 2003, Molecular biology of the cell.

[13]  Yangqing Xu,et al.  Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons. , 2003, Analytical chemistry.

[14]  Gang Bao,et al.  Hybridization kinetics and thermodynamics of molecular beacons. , 2003, Nucleic acids research.

[15]  Gang Bao,et al.  Dual FRET molecular beacons for mRNA detection in living cells. , 2004, Nucleic acids research.

[16]  David Ish-Horowicz,et al.  Differential cytoplasmic mRNA localisation adjusts pair-rule transcription factor activity to cytoarchitecture in dipteran evolution , 2004, Development.

[17]  Shuming Nie,et al.  Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells. , 2004, Nucleic acids research.

[18]  D. Ish-Horowicz,et al.  inscuteable mRNA Localization Is Dynein-Dependent and Regulates Apicobasal Polarity and Spindle Length in Drosophila Neuroblasts , 2004, Current Biology.

[19]  C. Lingwood,et al.  Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity. , 2004, Glycobiology.

[20]  G. Bao,et al.  Nanostructured Probes for RNA Detection in Living Cells , 2006, Annals of Biomedical Engineering.

[21]  Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape. , 2005 .

[22]  T. Klar,et al.  Gold nanoparticles quench fluorescence by phase induced radiative rate suppression. , 2005, Nano letters.

[23]  J. Elisseeff,et al.  Noninvasive Mitochondrial Imaging in Live Cell Culture , 2005, Photochemistry and photobiology.

[24]  James E Crowe,et al.  Progression of respiratory syncytial virus infection monitored by fluorescent quantum dot probes. , 2005, Nano letters.

[25]  Gang Bao,et al.  Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons. , 2005, Journal of biomedical optics.

[26]  L. Pelkmans,et al.  Assembly and trafficking of caveolar domains in the cell , 2005, The Journal of cell biology.

[27]  T. Kirchhausen,et al.  Dynasore, a cell-permeable inhibitor of dynamin. , 2006, Developmental cell.

[28]  C. Okamoto,et al.  Membrane transduction of oligoarginine in HeLa cells is not mediated by macropinocytosis. , 2006, Molecular pharmaceutics.

[29]  G. Bao,et al.  Live-Cell Characterization and Analysis of a Clinical Isolate of Bovine Respiratory Syncytial Virus, Using Molecular Beacons , 2006, Journal of Virology.

[30]  Chad A. Mirkin,et al.  Oligonucleotide-Modified Gold Nanoparticles for Intracellular Gene Regulation , 2006, Science.

[31]  Evan W. Miller,et al.  Preparation and use of Coppersensor-1, a synthetic fluorophore for live-cell copper imaging , 2006, Nature Protocols.

[32]  Jan Ellenberg,et al.  λN-GFP: an RNA reporter system for live-cell imaging , 2007, Nature Methods.

[33]  C. Mirkin,et al.  Oligonucleotide loading determines cellular uptake of DNA-modified gold nanoparticles. , 2007, Nano letters.

[34]  Chad A Mirkin,et al.  Nano-flares: probes for transfection and mRNA detection in living cells. , 2007, Journal of the American Chemical Society.

[35]  S. Kelley,et al.  Cyanine dye conjugates as probes for live cell imaging. , 2007, Bioorganic & medicinal chemistry letters.

[36]  S. Bullock.  Translocation of mRNAs by molecular motors: think complex? , 2007, Seminars in cell & developmental biology.

[37]  Antony K. Chen,et al.  Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA , 2008, Nucleic acids research.

[38]  L. Weisman.  Respiratory syncytial virus (RSV) prevention and treatment: past, present, and future. , 2009, Cardiovascular & hematological agents in medicinal chemistry.

[39]  C. Holt,et al.  Subcellular mRNA Localization in Animal Cells and Why It Matters , 2009, Science.

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