Abstract
Polyacrylamide gel electrophoresis is a widely used technique for RNA analysis and purification. The polyacrylamide matrix is highly versatile for chemical derivitization, enabling facile exploitation of thio-mercury chemistry without the need of tedious manipulations and/or expensive coupling reagents, which often give low yields and side products. Here, we describe the use of [(N-acryloylamino)phenyl]mercuric chloride in three-layered polyacrylamide gels to detect, separate, quantify, and analyze sulfur-containing RNAs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sprinzl M et al (1987) Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res 15(Suppl):r53–r188
Hafner M et al (2010) PAR-CliP–a method to identify transcriptome-wide the binding sites of RNA binding proteins. J Vis Exp 2(41): 2034
Chowrira B, Burke J (1992) Extensive phosphorothioate substitution yields highly active and nuclease resistant hairpin ribozymes. Nucleic Acids Res 20(11):2835–2840
Breaker RR et al (2003) A common speed limit for RNA-cleaving ribozymes and deoxy-ribozymes. RNA 9:949–957
Dahm SC, Uhlenbeck OC (1991) Role of different metal ions in the hammerhead RNA cleavage reaction. Biochemistry 30(39): 9464–9469
Scott EC, Uhlenbeck OC (1999) A re-investigation of the thio effect at the hammerhead cleavage site. Nucleic Acids Res 27:479–484
Dean NM, McKay RA (1994) Inhibition of protein kinase C-alpha expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides. Proc Natl Acad Sci USA 91:11762–11766
Cochrane JC, Strobel SA (2004) Probing RNA structure and function by nucleotide analog interference mapping. Curr Protoc Nucleic Acid Chem, Chapter 6:Unit 6.9
Czworkowski J, Odom OW, Hardesty B (1991) Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia Coli. Biochemistry 30:4821–4830
Milligan JF, Uhlenbeck OC (1989) Determination of RNA-protein contacts using thiophosphate substitutions. Biochemistry 28(7):2849–2855
Schatz D, Leberman R, Eckstein F (1991) Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts. Proc Natl Acad Sci USA 88:6132–6136
Rudinger J et al (1992) Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase. Proc Natl Acad Sci USA 89:5882–5886
Kreutzer R, Kern D, Giegé R, Rudinger J (1995) Footprinting of tRNA(Phe) transcripts from Thermus thermophilus HB8 with the homologous phenylalanyl-tRNA synthetase reveals a novel mode of interaction. Nucleic Acids Res 23(22):4598–4602
Igloi G (1988) Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis. Biochemistry 27:3842–3849
Rhee SS, Burke DH (2004) Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry. Anal Biochem 325:137–143
Saran D et al (2005) A trans acting ribozyme that phosphorylates exogenous RNA. Biochemistry 44:15007–15016
Saran D (2006) Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme. Nucleic Acids Res 34:3201–3208
Cho B-R, Burke DH (2006) Structural stabilization of the Kin.46 self-kinasing ribozyme through topological rearrangement. RNA 12:2118–2125
Saran D, Burke DH (2007) Synthesis of photo-cleavable and non-cleavable substrate-DNA and substrate-RNA conjugates for the selection of nucleic acid catalysts. Bioconjugate Chem 18:275–279
Cho B-R, Burke DH (2007) Conformational dynamics of self-thiophosphorylating RNA. Bull Korean Chem Soc 28:463–466
Biondi E et al (2010) Convergent donor and acceptor substrate utilization among kinase ribozymes. Nucleic Acids Res 38(19): 6785–6795
Rhee SS, Burke DH (2010) Active site assembly within a kinase ribozyme. RNA 16:2349–2359
Biondi E, Maxwell AWR, Burke DH (2012) A small ribozyme with dual-site kinase activity. Nucleic Acids Res: in press
Biondi E, Sawyer AW, Maxwell AWR, Burke DH (2012) Unusual dependence on both pH and Cu2+ in a dual-site kinase ribozyme. (submitted)
Acknowledgments
This work was supported by National Science Foundation grant CHE-1057506 to Donald H. Burke.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Biondi, E., Burke, D.H. (2012). Separating and Analyzing Sulfur-Containing RNAs with Organomercury Gels. In: Jin, H., Gassmann, W. (eds) RNA Abundance Analysis. Methods in Molecular Biology, vol 883. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-839-9_8
Download citation
DOI: https://doi.org/10.1007/978-1-61779-839-9_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-838-2
Online ISBN: 978-1-61779-839-9
eBook Packages: Springer Protocols