Abstract
RNA ligation allows the creation of large RNA molecules from smaller pieces. This can be useful in a number of contexts: to generate molecules that are larger than can be directly synthesized; to incorporate site-specific changes or RNA modifications within a large RNA in order to facilitate functional and structural studies; to isotopically label segments of large RNAs for NMR structural studies; and to construct libraries of mutant RNAs in which one region is extensively mutagenized or modified. The impediment to widespread use of RNA ligation is the low and variable efficiency of standard ligation strategies, which frequently preclude joining more than two pieces of RNA together.
We describe a method using RNA ligase (Rligation), rather than DNA ligase (Dligation), in a splint-mediated ligation reaction that joins RNA molecules with high efficiency. RNA ligase recognizes single-stranded RNA ends, which are held in proximity to one another by the splint. Monitoring the reaction is easily accomplished by denaturing gel electrophoresis and ethidium bromide staining. Using this technique, it is possible to generate a wide range of modified RNAs from synthetic oligoribonucleotides.
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References
Dönmez G, Hartmuth K, Lührmann R (2004) Modified nucleotides at the 5′ end of human U2 snRNA are required for spliceosomal E-complex formation. RNA 10:1925–1933
Crawford DJ, Hoskins AA, Friedman LJ, Gelles J, Moore MJ (2008) Visualizing the splicing of single pre-mRNA molecules in whole cell extract. RNA 14:170–179
Kim I, Lukavsky PJ, Puglisi JD (2002) NMR study of 100 kDa HCV IRES RNA using segmental isotope labeling. J Am Chem Soc 124:9338–9339
Moore MJ, Sharp PA (1992) Site-specific modification of pre-mRNA: the 2′-hydroxyl groups at the splice sites. Science 256:992–997
Kurschat WC, Müller J, Wombacher R, Helm M (2005) Optimizing splinted ligation of highly structured small RNAs. RNA 11: 1909–1914
Amitsur M, Levitz R, Kaufmann G (1987) Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA. EMBO J 6:2499–2503
Bain JD, Switzer C (1992) Regioselective ligation of oligoribonucleotides using DNA splints. Nucleic Acids Res 20:4372
Stark MR, Pleiss JA, Deras M, Scaringe SA, Rader SD (2006) An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs. RNA 12:2014–2019
Höbartner C, Micura R (2004) Chemical synthesis of selenium-modified oligoribonucleotides and their enzymatic ligation leading to an U6 SnRNA stem-loop segment. J Am Chem Soc 126:1141–1149
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
Wittenberg WL, Uhlenbeck OC (1985) Specific replacement of functional groups of uridine-33 in yeast phenylalanine transfer ribonucleic acid. Biochemistry 24:2705–2712
Arn EA, Abelson JN (1996) The 2″-5″ RNA ligase of Escherichia coli. Purification, cloning, and genomic disruption. J Biol Chem 271: 31145–31153
Scaringe SA (2001) RNA oligonucleotide synthesis via 5″-silyl-2-″orthoester chemistry. Methods 23:206–217
Sambrook J, Russell DW (2001) Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Acknowledgments
This work was supported by NSERC Discovery Grant 298521 and UNBC Office of Research awards to SDR.
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Stark, M.R., Rader, S.D. (2014). Efficient Splinted Ligation of Synthetic RNA Using RNA Ligase. In: Hertel, K. (eds) Spliceosomal Pre-mRNA Splicing. Methods in Molecular Biology, vol 1126. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-980-2_10
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DOI: https://doi.org/10.1007/978-1-62703-980-2_10
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-979-6
Online ISBN: 978-1-62703-980-2
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