Recent advances in RNA functional studies highlights the pivotal role of these molecules in cell physiology. Diverse methods have been implemented to measure the expression levels of various RNA species, using either purified RNA or fixed cells. Despite the fact that fixed cells offer the possibility to observe the spatial distribution of RNA, assays with capability to real-time monitoring RNA transport into living cells are needed to further understand the role of RNA dynamics in cellular functions. Molecular beacons (MBs) are stem-loop hairpin-structured oligonucleotides equipped with a fluorescence quencher at one end and a fluorescent dye (also called reporter or fluorophore) at the opposite end. This structure permits that MB in the absence of their target complementary sequence do not fluoresce. Upon binding to targets, MBs emit fluorescence, due to the spatial separation of the quencher and the reporter. Molecular beacons are promising probes for the development of RNA imaging techniques; nevertheless much work remains to be done in order to obtain a robust technology for imaging various RNA molecules together in real time and in living cells. The present work concentrates on the different requirements needed to use successfully MB for cellular studies, summarizing recent advances in this area. 1. Introduction A wealth of experimental evidence accumulated to this date illustrates the wide range of cellular functions conducted by ribonucleic acids (RNAs). In many occasions these functions involve RNA transport from one cellular compartment to another or into the same compartment to specialized regions. In order to accomplish this, RNA molecules are under a specific and selective control via expression levels and/or stability in a spatial-temporal manner. Different methods developed to purify RNA [1–3] from cell populations or tissues have provided relevant information about the relative concentration of RNA in cells, or cellular compartments, yet these methods provide limited information about the spatial-temporal distribution of RNAs and their dynamic transport. The integral understanding of cellular process in which RNA is involved, requires a method that reveals RNA localization in real time in a subcellular context in living cells. The information obtained from this type of assays promise to impulse the advancement in molecular biology, medical research, and diagnostics. Many methods have been developed to measure RNA expression levels between different cell populations, such as the polymerase chain reaction (PCR) [4], northern blot [5],
References
[1]
V. Gli？in, R. Crkvenjakov, and C. Byus, “Ribonucleic acid isolated by cesium chloride centrifugation,” Biochemistry, vol. 13, no. 12, pp. 2633–2637, 1974.
[2]
C. Auffray and F. Rougeon, “Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA,” European Journal of Biochemistry, vol. 107, no. 2, pp. 303–314, 1980.
[3]
P. Chomczynski and N. Sacchi, “Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction,” Analytical Biochemistry, vol. 162, no. 1, pp. 156–159, 1987.
[4]
R. K. Saiki, S. Scharf, and F. Faloona, “Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia,” Science, vol. 230, no. 4732, pp. 1350–1354, 1985.
[5]
J. C. Alwine, D. J. Kemp, B. A. Parker et al., “Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper,” Methods in Enzymology, vol. 68, pp. 220–242, 1979.
[6]
V. E. Velculescu, L. Zhang, B. Vogelstein, and K. W. Kinzler, “Serial analysis of gene expression,” Science, vol. 270, no. 5235, pp. 484–487, 1995.
[7]
L. Diatchenko, Y. F. C. Lau, A. P. Campbell et al., “Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 12, pp. 6025–6030, 1996.
[8]
P. Liang and A. B. Pardee, “Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction,” Science, vol. 257, no. 5072, pp. 967–971, 1992.
[9]
N. Lisitsyn and M. Wigler, “Cloning the differences between two complex genomes,” Science, vol. 259, no. 5097, pp. 946–951, 1993.
[10]
M. D. Adams, M. Dubnick, A. R. Kerlavage et al., “Sequence identification of 2,375 human brain genes,” Nature, vol. 355, no. 6361, pp. 632–634, 1992.
[11]
M. Schena, D. Shalon, R. W. Davis, and P. O. Brown, “Quantitative monitoring of gene expression patterns with a complementary DNA microarray,” Science, vol. 270, no. 5235, pp. 467–470, 1995.
[12]
G. J. Bassell, C. M. Powers, K. L. Taneja, and R. H. Singer, “Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts,” Journal of Cell Biology, vol. 126, no. 4, pp. 863–876, 1994.
[13]
M. C. Vaccaro, S. Gigliotti, F. Graziani et al., “A transient asymmetric distribution of XNOA 36 mRNA and the associated spectrin network bisects Xenopus laevis stage I oocytes along the future A/V axis,” European Journal of Cell Biology, vol. 89, no. 7, pp. 525–536, 2010.
[14]
Y. Shinmyo, T. Mito, T. Matsushita et al., “Caudal is required for gnathal and thoracic patterning and for posterior elongation in the intermediate-germband cricket Gryllus bimaculatus,” Mechanisms of Development, vol. 122, no. 2, pp. 231–239, 2005.
[15]
R. Krause, M. Hemberger, M. Messerschmid et al., “Molecular cloning and characterization of murine Mpgc60, a gene predominantly expressed in the intestinal tract,” Differentiation, vol. 63, no. 5, pp. 285–294, 1998.
[16]
M. Mione, Z. Lele, C. T. Kwong, M. L. Concha, and J. D. Clarke, “Expression of pcp4a in subpopulations of CNS neurons in zebrafish,” Journal of Comparative Neurology, vol. 495, no. 6, pp. 769–787, 2006.
[17]
S. Behrens, B. M. Fuchs, F. Mueller, and R. Amann, “Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit?” Applied and Environmental Microbiology, vol. 69, no. 8, pp. 4935–4941, 2003.
[18]
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.
[19]
C. J. Yang, H. Lin, and W. Tan, “Molecular assembly of superquenchers in signaling molecular interactions,” Journal of the American Chemical Society, vol. 127, no. 37, pp. 12772–12773, 2005.
[20]
S. Tyagi and F. R. Kramer, “Molecular beacons: probes that fluoresce upon hybridization,” Nature Biotechnology, vol. 14, no. 3, pp. 303–308, 1996.
[21]
S. D. Bélanger, M. Boissinot, C. Ménard, F. J. Picard, and M. G. Bergeron, “Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the Smart Cycler,” Journal of Clinical Microbiology, vol. 40, no. 4, pp. 1436–1440, 2002.
[22]
S. D. Bélanger, M. Boissinot, N. Clairoux, F. J. Picard, and M. G. Bergeron, “Rapid detection of Clostridium difficile in feces by real-time PCR,” Journal of Clinical Microbiology, vol. 41, no. 2, pp. 730–734, 2003.
[23]
A. Beloukas, D. Paraskevis, M. Psichogiou, and A. Hatzakis, “The role of HIV-1 DNA as an additional marker of HIV-1 infection,” Current HIV Research, vol. 7, no. 3, pp. 255–265, 2009.
[24]
M. G. Bergeron and D. Ke, “New DNA-based PCR approaches for rapid real-time detection and prevention of group B streptococcal infections in newborns and pregnant women,” Expert Reviews in Molecular Medicine, vol. 3, no. 27, pp. 1–14, 2001.
[25]
W. Chen, G. Martinez, and A. Mulchandani, “Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella,” Analytical Biochemistry, vol. 280, no. 1, pp. 166–172, 2000.
[26]
G. Bonnet, S. Tyagi, A. Libchaber, and F. R. Kramer, “Thermodynamic basis of the enhanced specificity of structured DNA probes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 11, pp. 6171–6176, 1999.
[27]
A. Tsourkas, M. A. Behlke, S. D. Rose, and G. Bao, “Hybridization kinetics and thermodynamics of molecular beacons,” Nucleic Acids Research, vol. 31, no. 4, pp. 1319–1330, 2003.
[28]
I. Tapp, L. Malmberg, E. Rennel, M. Wik, and A. C. Syvanen, “Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5'-nuclease TaqMan？ assay and molecular beacon probes (BioTechniques (2000) 28 (732-738)),” BioTechniques, vol. 28, no. 4, pp. 732–738, 2000.
[29]
M. M. Mhlanga and L. Malmberg, “Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR,” Methods, vol. 25, no. 4, pp. 463–471, 2001.
[30]
S. A. Marras, F. R. Kramer, and S. Tyagi, “Genotyping SNPs with molecular beacons,” Methods in Molecular Biology, vol. 212, pp. 111–128, 2003.
[31]
L. B. Barreiro, R. Henriques, and M. M. Mhlanga, “High-throughput SNP genotyping: combining tag SNPs and molecular beacons,” Methods in Molecular Biology, vol. 578, pp. 255–276, 2009.
[32]
L. G. Kostrikis, S. Tyagi, M. M. Mhlanga, D. D. Ho, and F. R. Kramer, “Spectral genotyping of human alleles,” Science, vol. 279, no. 5354, pp. 1228–1229, 1998.
[33]
L. G. Kostrikis, S. Shin, and D. D. Ho, “Genotyping HIV-1 and HCV strains by a combinatorial DNA melting assay (COMA),” Molecular Medicine, vol. 4, no. 7, pp. 443–453, 1998.
[34]
G. Orrù, G. Faa, S. Pillai et al., “Rapid PCR real-time genotyping of M-Malton α1-antitrypsin deficiency alleles by molecular beacons,” Diagnostic Molecular Pathology, vol. 14, no. 4, pp. 237–242, 2005.
[35]
G. Orru, F. Coghe, G. Faa et al., “Rapid multiplex real-time PCR by molecular beacons for different BRAF allele detection in papillary thyroid carcinoma,” Diagnostic Molecular Pathology, vol. 19, no. 1, pp. 1–8, 2010.
[36]
K. Szuhai, E. Sandhaus, S. M. Kolkman-Uljee et al., “A novel strategy for human papillomavirus detection and genotyping with sybrgreen and molecular beacon polymerase chain reaction,” American Journal of Pathology, vol. 159, no. 5, pp. 1651–1660, 2001.
[37]
B. A. J. Giesendorf, J. A. M. Vet, S. Tyagi, E. J. M. G. Mensink, F. J. M. Trijbels, and H. J. Blom, “Molecular beacons: a new approach for semiautomated mutation analysis,” Clinical Chemistry, vol. 44, no. 3, pp. 482–486, 1998.
[38]
K. Frei, K. Szuhai, T. Lucas et al., “Connexin 26 mutations in cases of sensorineural deafness in eastern Austria,” European Journal of Human Genetics, vol. 10, no. 7, pp. 427–432, 2002.
[39]
D. R. Hodgson, C. A. Foy, M. Partridge, S. Pateromichelakis, and N. J. Gibson, “Development of a facile fluorescent assay for the detection of 80 mutations within the p53 gene,” Molecular Medicine, vol. 8, no. 5, pp. 227–237, 2002.
[40]
J. B. Biggins, J. R. Prudent, D. J. Marshall, M. Ruppen, and J. S. Thorson, “A continuous assay for DNA cleavage: the application of 'break lights' to enediynes, iron-dependent and nucleases,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 25, pp. 13537–13542, 2000.
[41]
J. J. Li, R. Geyer, and W. Tan, “Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA,” Nucleic acids research, vol. 28, no. 11, article e52, 2000.
[42]
C. D. Medley, T. J. Drake, J. M. Tomasini, R. J. Rogers, and W. Tan, “Simultaneous monitoring of the expression of multiple genes inside of single breast carcinoma cells,” Analytical Chemistry, vol. 77, no. 15, pp. 4713–4718, 2005.
[43]
X. H. Peng, Z. H. Cao, J. T. Xia et al., “Real-time detection of gene expression in cancer cells using molecular beacon imaging: new strategies for cancer research,” Cancer Research, vol. 65, no. 5, pp. 1909–1917, 2005.
[44]
L. Yang, Z. Cao, Y. Lin, W. C. Wood, and C. A. Staley, “Molecular beacon imaging of tumor marker gene expression in pancreatic cancer cells,” Cancer Biology and Therapy, vol. 4, no. 5, pp. 561–570, 2005.
[45]
A. Wang, A. M. Salazar, M. V. Yates, A. Mulchandani, and W. Chen, “Visualization and detection of infectious coxsackievirus replication using a combined cell culture-molecular beacon assay,” Applied and Environmental Microbiology, vol. 71, no. 12, pp. 8397–8401, 2005.
[46]
P. Santangelo, N. Nitin, L. LaConte, A. Woolums, and G. Bao, “Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons,” Journal of Virology, vol. 80, no. 2, pp. 682–688, 2006.
[47]
H. Y. Yeh, M. V. Yates, A. Mulchandani, and W. Chen, “Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 45, pp. 17522–17525, 2008.
[48]
D. P. Bratu, B. J. Cha, M. M. Mhlanga, F. R. Kramer, and S. Tyagi, “Visualizing the distribution and transport of mRNAs in living cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 23, pp. 13308–13313, 2003.
[49]
D. P. Bratu, “Molecular beacons light the way: imaging native mRNAs in living cells,” Discovery Medicine, vol. 3, no. 19, pp. 44–47, 2003.
[50]
S. Tyagi and O. Alsmadi, “Imaging native β-actin mRNA in motile fibroblasts,” Biophysical Journal, vol. 87, no. 6, pp. 4153–4162, 2004.
[51]
B. Simon, M. Sandhu, and K. L. Myhr, “Live FISH: imaging mRNA in living neurons,” Journal of Neuroscience Research, vol. 88, no. 1, pp. 55–63, 2010.
[52]
X. Fang, J. J. Li, J. Perlette, W. Tan, and K. Wang, “Molecular beacons: novel fluorescent probes,” Analytical Chemistry, vol. 72, no. 23, pp. 747A–753A, 2000.
[53]
W. Tan, X. Fang, J. Li, and X. Liu, “Molecular beacons: a novel DNA probe for nucleic acid and protein studies,” Chemistry, vol. 6, no. 7, pp. 1107–1111, 2000.
[54]
S. A. Marras, F. R. Kramer, and S. Tyagi, “Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes,” Nucleic Acids Research, vol. 30, no. 21, article e122, 2002.
[55]
K. M. Parkhurst and L. J. Parkhurst, “Donor- acceptor distance distributions in a double-labeled fluorescent oligonucleotide both as a single strand and in duplexes,” Biochemistry？, vol. 34, no. 1, pp. 293–300, 1995.
[56]
S. Bernacchi and Y. Mély, “Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure,” Nucleic Acids Research, vol. 29, no. 13, article e62, 2001.
[57]
A. Tsourkas, M. A. Behlke, and G. Bao, “Structure-function relationships of shared-stem and conventional molecular beacons,” Nucleic Acids Research, vol. 30, no. 19, pp. 4208–4215, 2002.
[58]
E. McGown, M. Su, and R. Dennis, “Measurement of molecular beacons in the SPECTRAmax？ GEMINI spectrofluorometer,” Journal of Chemical Technology and Biotechnology, vol. 75, no. 10, pp. 942–944, 2000.
[59]
C. A. M. Seidel, A. Schulz, and M. H. M. Sauer, “Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-electron redox potentials and their Correlation with static and dynamic quenching efficiencies,” Journal of Physical Chemistry, vol. 100, no. 13, pp. 5541–5553, 1996.
[60]
T. Heinlein, J. P. Knemeyer, O. Piestert, J. Wolfrum, and M. Sauer, “Nucleobase-specific quenching of fluorescent dyes in DNA-hairpins,” The Journal of Physical Chemistry B, vol. 107, no. 31, pp. 7957–7964, 2003.
[61]
G. Luo, L. Zheng, X. Zhang, J. Zhang, P. Nilsson-Ehle, and N. Xu, “Genotyping of single nucleotide polymorphisms using base-quenched probe: a method does not invariably depend on the deoxyguanosine nucleotide,” Analytical Biochemistry, vol. 386, no. 2, pp. 161–166, 2009.
[62]
S. A. Oladepo and G. R. Loppnow, “Self-quenching smart probes as a platform for the detection of sequence-specific UV-induced DNA photodamage,” Analytical and Bioanalytical Chemistry, vol. 397, no. 7, pp. 2949–2957, 2010.
[63]
N. Venkatesan, Y. J. Seo, and B. H. Kim, “Quencher-free molecular beacons: a new strategy in fluorescence based nucleic acid analysis,” Chemical Society Reviews, vol. 37, no. 4, pp. 648–663, 2008.
[64]
P. M. Holland, R. D. Abramson, R. Watson, and D. H. Gelfand, “Detection of specific polymerase chain reaction product by utilizing the 5' → 3' exonuclease activity of Thermus aquaticus DNA polymerase,” Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 16, pp. 7276–7280, 1991.
[65]
S. Tyagi, S. A. E. Marras, and F. R. Kramer, “Wavelength-shifting molecular beacons,” Nature Biotechnology, vol. 18, no. 11, pp. 1191–1196, 2000.
[66]
S. Tyagi, D. P. Bratu, and F. R. Kramer, “Multicolor molecular beacons for allele discrimination,” Nature Biotechnology, vol. 16, no. 1, pp. 49–53, 1998.
[67]
L. Tan, Y. Li, T. J. Drake et al., “Molecular beacons for bioanalytical applications,” Analyst, vol. 130, no. 7, pp. 1002–1005, 2005.
[68]
M. Zuker, “Mfold web server for nucleic acid folding and hybridization prediction,” Nucleic Acids Research, vol. 31, no. 13, pp. 3406–3415, 2003.
[69]
J. Perlette and W. Tan, “Real-time monitoring of intracellular mRNA hybridization inside single living cells,” Analytical Chemistry, vol. 73, no. 22, pp. 5544–5550, 2001.
[70]
J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York, NY, USA, 3rd edition, 2006.
[71]
C. J. Yang, M. Pinto, K. Schanze, and W. Tan, “Direct synthesis of an oligonucleotide-poly-(phenylene ethynylene) conjugate with a precise one-to-one molecular ratio,” Angewandte Chemie, vol. 44, no. 17, pp. 2572–2576, 2005.
[72]
H. Huang, K. Wang, W. Tan et al., “Design of a modular-based fluorescent conjugated polymer for selective sensing,” Angewandte Chemie, vol. 43, no. 42, pp. 5635–5638, 2004.
[73]
J. J. Li, R. Geyer, and W. Tan, “Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA,” Nucleic Acids Research, vol. 28, no. 11, article e52, 2000.
[74]
C. J. Yang, J. J. Li, and W. Tan, “Using molecular beacons for sensitive fluorescence assays of the enzymatic cleavage of nucleic acids,” Methods in Molecular Biology, vol. 335, pp. 71–81, 2006.
[75]
C. J. Yang, C. D. Medley, and W. Tan, “Monitoring nucleic acids using molecular beacons,” Current Pharmaceutical Biotechnology, vol. 6, no. 6, pp. 445–452, 2005.
[76]
C. Molenaar, S. A. Marras, J. C. Slats et al., “Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells,” Nucleic Acids Research, vol. 29, no. 17, article e89, 2001.
[77]
A. Tsourkas, M. A. Behlke, and G. Bao, “Hybridization of 2′-O-methyl and 2-deoxy molecular beacons to RNA and DNA targets,” Nucleic Acids Research, vol. 30, no. 23, pp. 5168–5174, 2002.
[78]
R. H. Kehlenbach, “In vitro analysis of nuclear mRNA export using molecular beacons for target detection,” Nucleic Acids Research, vol. 31, no. 11, article e64, 2003.
[79]
R. Shah and W. S. El-Deiry, “p53-dependent activation of a molecular beacon in tumor cells following exposure to doxorubicin chemotherapy,” Cancer Biology and Therapy, vol. 3, no. 9, pp. 871–875, 2004.
[80]
V. Vijayanathan, T. Thomas, L. H. Sigal, and T. J. Thomas, “Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon,” Antisense and Nucleic Acid Drug Development, vol. 12, no. 4, pp. 225–233, 2002.
[81]
K. Petersen, U. Vogel, E. Rockenbauer et al., “Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms,” Molecular and Cellular Probes, vol. 18, no. 2, pp. 117–122, 2004.
[82]
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.
[83]
C. J. Yang, L. Wang, Y. Wu et al., “Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons,” Nucleic Acids Research, vol. 35, no. 12, pp. 4030–4041, 2007.
[84]
X. Fang, J. J. Li, and W. Tan, “Using molecular beacons to probe molecular interactions between lactate dehydrogenase and single-stranded DNA,” Analytical Chemistry, vol. 72, no. 14, pp. 3280–3285, 2000.
[85]
R. Yang, J. Jin, Y. Chen et al., “Carbon nanotube-quenched fluorescent oligonucleotides: probes that fluoresce upon hybridization,” Journal of the American Chemical Society, vol. 130, no. 26, pp. 8351–8358, 2008.
[86]
N. Graf, M. G？ritz, and R. Kr？mer, “A metal-ion-releasing probe for DNA detection by catalytic signal amplification,” Angewandte Chemie, vol. 45, no. 24, pp. 4013–4015, 2006.
[87]
A. K. Chen, M. A. Behlke, and A. Tsourkas, “Sub-cellular trafficking and functionality of 2′-O-methyl and 2′-O-methyl-phosphorothioate molecular beacons,” Nucleic Acids Research, vol. 37, no. 22, article e149, 2009.
[88]
W. J. Rhee and G. Bao, “Slow non-specific accumulation of 2'-deoxy and 2'-O-methyl oligonucleotide probes at mitochondria in live cells,” Nucleic Acids Research, vol. 38, no. 9, article e109, 2010.
[89]
A. K. Chen, M. A. Behlke, and A. Tsourkas, “Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells,” Nucleic Acids Research, vol. 35, no. 16, article e105, 2007.
[90]
Y. Wu, C. J. Yang, L. L. Moroz, and W. Tan, “Nucleic acid beacons for long-term real-time intracellular monitoring,” Analytical Chemistry, vol. 80, no. 8, pp. 3025–3028, 2008.
[91]
Y. Kim, C. J. Yang, and W. Tan, “Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem,” Nucleic Acids Research, vol. 35, no. 21, pp. 7279–7287, 2007.
[92]
R. W. Dirks, C. Molenaar, and H. J. Tanke, “Visualizing RNA molecules inside the nucleus of living cells,” Methods, vol. 29, no. 1, pp. 51–57, 2003.
[93]
M. A. Barry and A. Eastman, “Identification of deoxyribonuclease II as an endonuclease involved in apoptosis,” Archives of Biochemistry and Biophysics, vol. 300, no. 1, pp. 440–450, 1993.
[94]
R. V. Giles, C. J. Ruddell, D. G. Spiller, J. A. Green, and D. M. Tidd, “Single base discrimination for ribonuclease H-dependent antisense effects within intact human leukaemia cells,” Nucleic Acids Research, vol. 23, no. 6, pp. 954–961, 1995.
[95]
R. V. Giles, D. G. Spiller, J. Grzybowski, R. E. Clark, P. Nicklin, and D. M. Tidd, “Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells,” Nucleic Acids Research, vol. 26, no. 7, pp. 1567–1575, 1998.
[96]
I. Walev, S. C. Bhakdi, F. Hofmann et al., “Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 6, pp. 3185–3190, 2001.
[97]
M. Becker-Hapak, S. S. McAllister, and S. F. Dowdy, “TAT-mediated protein transduction into mammalian cells,” Methods, vol. 24, no. 3, pp. 247–256, 2001.
[98]
E. L. Snyder and S. F. Dowdy, “Protein/peptide transduction domains: potential to deliver large DNA molecules into cells,” Current Opinion in Molecular Therapeutics, vol. 3, no. 2, pp. 147–152, 2001.
[99]
J. S. Wadia and S. F. Dowdy, “Protein transduction technology,” Current Opinion in Biotechnology, vol. 13, no. 1, pp. 52–56, 2002.
[100]
H. Brooks, B. Lebleu, and E. Vivès, “Tat peptide-mediated cellular delivery: back to basics,” Advanced Drug Delivery Reviews, vol. 57, no. 4, pp. 559–577, 2005.
[101]
J. S. Wadia and S. F. Dowdy, “Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer,” Advanced Drug Delivery Reviews, vol. 57, no. 4, pp. 579–596, 2005.
[102]
N. Nitin, P. J. Santangelo, G. Kim, S. Nie, and G. Bao, “Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells,” Nucleic Acids Research, vol. 32, no. 6, article e58, 2004.
[103]
N. Nitin and G. Bao, “NLS peptide conjugated molecular beacons for visualizing nuclear RNA in living cells,” Bioconjugate Chemistry, vol. 19, no. 11, pp. 2205–2211, 2008.
[104]
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.
[105]
T. M. Klein, E. D. Wolf, R. Wu, and J. C. Sanford, “High-velocity microprojectiles for delivering nucleic acids into living cells,” Nature, vol. 327, no. 6117, pp. 70–73, 1987.
[106]
N. S. Yang, J. Burkholder, B. Roberts, B. Martinell, and D. McCabe, “In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment,” Proceedings of the National Academy of Sciences of the United States of America, vol. 87, no. 24, pp. 9568–9572, 1990.
[107]
E. F. Fynan, R. G. Webster, D. H. Fuller, J. R. Haynes, J. C. Santoro, and H. L. Robinson, “DNA vaccines: protective immunizations by parenteral, mucosal, and gene- gun inoculations,” Proceedings of the National Academy of Sciences of the United States of America, vol. 90, no. 24, pp. 11478–11482, 1993.
[108]
J. A. Wolff, P. Williams, G. Acsadi, S. Jiao, A. Jani, and W. Chong, “Conditions affecting direct gene transfer into rodent muscle in vivo,” BioTechniques, vol. 11, no. 4, pp. 474–485, 1991.
[109]
C. Condon, S. C. Watkins, C. M. Celluzzi, K. Thompson, and L. D. Falo, “DNA-based immunization by in vivo transfection of dendritic cells,” Nature Medicine, vol. 2, no. 10, pp. 1122–1128, 1996.
[110]
D. H. Mathews, J. Sabina, M. Zuker, and D. H. Turner, “Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure,” Journal of Molecular Biology, vol. 288, no. 5, pp. 911–940, 1999.
[111]
S. F. Altschul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, “Basic local alignment search tool,” Journal of Molecular Biology, vol. 215, no. 3, pp. 403–410, 1990.
[112]
P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Research, vol. 32, no. 6, article e57, 2004.
[113]
A. Tsourkas, M. A. Behlke, Y. Xu, and G. Bao, “Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons,” Analytical Chemistry, vol. 75, no. 15, pp. 3697–3703, 2003.
[114]
K. Wang, Z. Tang, C. J. Yang et al., “Molecular engineering of DNA: molecular beacons,” Angewandte Chemie, vol. 48, no. 5, pp. 856–870, 2009.
[115]
T. J. Drake, C. D. Medley, A. Sen, R. J. Rogers, and W. Tan, “Stochasticity of manganese superoxide dismutase mRNA expression in breast carcinoma cells by molecular beacon imaging,” ChemBioChem, vol. 6, no. 11, pp. 2041–2047, 2005.
[116]
H. Y. Yeh, M. V. Yates, A. Mulchandani, and W. Chen, “Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 45, pp. 17522–17525, 2008.
