Abstract
Splint ligation of RNA, whereby specific RNA molecules are ligated together, can be carried out using T4 DNA ligase and a bridging DNA oligonucleotide complementary to the RNAs. This method takes advantage of the property of T4 DNA ligase to join RNA molecules when they are in an RNA:DNA hybrid. Splint ligation is a useful tool for the introduction of modified nucleotides into RNA molecules, insertion of a radiolabel into a specific position within an RNA and for the assembly of smaller synthetic RNAs into longer RNA molecules. Such modifications enable a wide range of experiments to be carried out with the modified RNA including structural studies, co-immunoprecipitations, and the ability to map sites of RNA:RNA and RNA:protein interactions.
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References
Moore MJ, Sharp PA. Site-specific modification of pre-mRNA: the 2′-hydroxyl groups at the splice sites. Science. 1992;256:992–7.
O’Keefe RT, Newman AJ. Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing. EMBO. 1998;17:565–74.
Juzumiene DI, Wollenzien P. Arrangement of the central pseudoknot region of 16S rRNA in the 30S ribosomal subunit determined by site-directed 4-thiouridine crosslinking. RNA. 2001;7:71–84.
O’Keefe RT, Norman C, Newman AJ. The invariant U5 snRNA loop 1 sequence is dispensable for the first catalytic step of pre-mRNA splicing in yeast. Cell. 1996;86:679–89.
Dix I, Russel CS, O’Keefe RT, Newman AJ, Beggs JD. Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae. RNA. 1998;4:1675–86.
Alvi RK, Lund M, O’Keefe RT. ATP-dependent interaction of yeast U5 snRNA loop1 with the 5′ splice site. RNA. 2001;7:1013–23.
McGrail JC, Tatum E, O’Keefe RT. Mutation in the U2 snRNA influences exon interactions of the U5 snRNA loop 1 during pre-mRNA splicing. EMBO. 2006;25:3813–22.
McGrail JC, O’Keefe RT. The U1, U2 and U5 snRNAs crosslink to the 5′ exon during yeast pre-mRNA splicing. Nucleic Acids Res. 2008;36:814–25.
Tzakos AG, Easton LE, Lukavsky PJ. Perparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies. Nat Protoc. 2007;2:2139–47.
Kleppe K, van de Sande JH, Khorana HG. Polynucleotide ligase-catalyzed joining of deoxyribo-oligonucleotides on ribopolynucleotide templates and of ribo-oligonucleotides on deoxyribopolynucleotide templates. Proc Natl Acad Sci USA. 1970;67:68–73.
Fareed GC, Wilt EM, Richardson CC. Enzymatic breakage and joining of deoxyribonucleic acid. J Biol Chem. 1971;246:925–32.
Sano H, Feix G. Ribonucleic acid ligase activity of deoxyribonucleic acid ligase from phage T4 infacted Escherichia coli. Biochemistry. 1974;13:5110–5.
Acknowledgments
This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and The Wellcome Trust.
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Kershaw, C.J., O’Keefe, R.T. (2013). Splint Ligation of RNA with T4 DNA Ligase. In: Conn, G. (eds) Recombinant and In Vitro RNA Synthesis. Methods in Molecular Biology, vol 941. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-113-4_19
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DOI: https://doi.org/10.1007/978-1-62703-113-4_19
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-113-4
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