Abstract
Methods for the detection of biologically relevant interactions by highly precise catalytic control elements based on hairpin ribozymes, and their subsequent analysis are described. These include ribozyme design, catalytic performance in real time as a function of fluorescence signal amplification, and applications for sensing protein and nucleic acid interactions in high-throughput formats. Detailed instructions for two of our main reporter ribozyme formats that either follow repressible or inducible regulatory mechanisms are provided. We have shown that these techniques can be applied for detecting diverse target molecules including microRNAs, or protein—protein interactions. These reporter systems thus represent a general way to obtain signal-amplifying sensors for diverse applications in molecular profiling.
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Ferre-D’Amare, A. R. (2004) The hairpin ribozyme Biopolymers 73, 71–78.
Zhuang, X., Kim, H., Pereira, M. J., Babcock, H. P., Walter, N. G., and Chu, S. (2002) Correlating structural dynamics and function in single ribozyme molecules Science 296, 1473–1476.
Wilson, T. J., Nahas, M., Ha, T., and Lilley, D. M. (2005) Folding and catalysis of the hairpin ribozyme Biochem. Soc. Trans. 33, 461–465.
Esteban, J. A., Banerjee, A. R., and Burke, J. M. (1997) Kinetic mechanism of the hairpin ribozyme. Identification and characterization of two nonexchangeable conformations J. Biol. Chem. 272, 13,629–13,639.
Ferre-D’amare, A. R. and Rupert, P. B. (2002) The hairpin ribozyme: from crystal structure to function Biochem. Soc. Trans. 30, 1105–1109.
Fedor, M. J. (2002) The catalytic mechanism of the hairpin ribozyme Biochem. Soc. Trans. 30, 1109–1115.
Esteban, J. A., Walter, N. G., Kotzorek, G., Heckman, J. E., and Burke, J. M. (1998) Structural basis for heterogeneous kinetics: reengineering the hairpin ribozyme Proc. Natl. Acad. Sci. USA 95, 6091–6096.
Walter, N. G., Burke, J. M., and Millar, D. P. (1999) Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction Nat. Struct. Biol. 6, 544–549.
Najafi-Shoushtari, S. H., Mayer, G., and Famulok, M. (2004) Sensing complex regulatory networks by conformationally controlled hairpin ribozymes Nucleic Acids Res. 32, 3212–3219.
Hartig, J. S., Grüne, I., Najafi-Shoushtari, S. H., and Famulok, M. (2004) Sequence-Specific Detection of MicroRNAs by Signal-Amplifying Ribozymes J. Am. Chem. Soc. 126, 722–723.
Hartig, J. S., Najafi-Shoushtari, S. H., Grüne, I., Yan, A., Ellington, A. D., and Famulok, M. (2002) Protein-dependent ribozymes report molecular interactions in real time Nat. Biotechnol. 20, 717–722.
Najafi-Shoushtari, S. H. and Famulok, M. (2005) Competitive regulation of modular allosteric aptazymes by a small molecule and oligonucleotide effector RNA 11, 1514–1520.
Mathews, D. H., Sabina, J., Zuker, M., and Turner, D. H. (1999) Expanded Sequence Dependence of Thermodynamic Parameters Improves Prediction of RNA Secondary Structure J. Mol. Biol. 288, 911–940.
Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction Nucleic Acids Res. 31, 3406–3415.
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction Cold Spring Harb. Symp. Quant. Biol. 51Pt 1, 263–273.
Berzal-Herranz, A., Joseph, S., Chowrira, B. M., Butcher, S. E., and Burke, J. M. (1993) Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme Embo. J. 12, 2567–2573.
Romero-Lopez, C., Barroso-delJesus, A., Puerta-Fernandez, E., and Berzal-Herranz, A. (2004) Design and optimization of sequence-specific hairpin ribozymes Methods Mol. Biol. 252, 327–338.
Ecker, D. J., Vickers, T. A., Bruice, T. W., et al. (1992) Pseudo-half-knot formation with RNA Science 257, 958–961.
Tinoco, I., Jr. (1997) From RNA hairpins to kisses to pseudoknots Nucleic Acids Symp. Ser. 36, 49–51.
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© 2008 Humana Press Inc., Totowa, NJ
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Najafi-Shoushtari, S.H., Famulok, M. (2008). Modular Reporter Hairpin Ribozymes for Analyzing Molecular Interactions. In: Marx, A., Seitz, O. (eds) Molecular Beacons: Signalling Nucleic Acid Probes, Methods, and Protocols. Methods in Molecular Biology, vol 429. Humana Press. https://doi.org/10.1007/978-1-60327-040-3_17
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DOI: https://doi.org/10.1007/978-1-60327-040-3_17
Publisher Name: Humana Press
Print ISBN: 978-1-58829-700-6
Online ISBN: 978-1-60327-040-3
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