Menu

tRNA Probes

Detection of RNA Modification

Identification of Unique Functional Groups by Electrophoretic Analysis

A number of tRNA modifications involve chemical moieties that are sufficiently different from other reactive groups in the RNA molecule to permit group-specific binding by appropriate affinity electrophoretic gels.

Detection of modified 2'- or 3'-ribose hydroxyl at RNA ends

The chemical nature of the 5'- or 3'-terminus of RNA can often reveal the origin or even function of a particular RNA species. The direct linkage of aminophenylboronic acid to acrylamide (1,2) has proven to be a reliable electrophoretic medium for the analysis of all ribose modifications found at either end of naturally occurring RNAs or as functional groups in modified bases.

Item

Description

Quantity per tube

Cost per tube

APB
Acryloylaminophenylboronic acid 500 mg $90
 
References:

1. Igloi, G.L. and Kössel, H. (1985) Affinity electrophoresis for monitoring terminal phosphorylation and the presence of queuosine in RNA. Application of polyacrylamide containing a covalently bound boronic acid. Nucleic Acids Res. 13, 6881-6898.

2. Igloi, G.L. and Kössel, H. (1987) The use of boronate affinity electrophoresis gels for studying both ends of RNA. Methods Enzymol. 155, 433-448. 

3. Matts, J.A., Sytnikova, Y., Chirn, G., Igloi, G.L. and Lau, N.C. (3014) Small RNA library construction from minute biological samples. Methods Molec. Biol. 1093, 123-136.

Selected applications:

Lien, J-M., Petcu, D. J., Aldrich, C.E. and Mason, W.S. (1987) Initiation and Termination of Duck Hepatitis B Virus DNA Synthesis during Virus Maturation.  J. Virol. 61, 3832-3840.

Förster, C.I., Chakraburtty, K. and Sprinzl, M. (1993) Discrimination between initiation and elongation of protein biosynthesis in yeast: identity assured by a nucleotide modification in the initiator tRNA. Nucleic Acids Res. 21, 5679-5683.

DiMaria, P., Palic, B., Debrunner-Vossbrinck, B. A., Lapp, J. and Vossbrinck, C. R. (1996) Characterization of the highly divergent U2 RNA homolog in the microsporidian Vairimorpha necatrix. Nucleic Acids Res. 24, 515–522.

 

Detection of Thiol and other modified bases

Acryloylaminophenylmercuric chloride (APM), a derivative of acrylamide, can be polymerized directly into an electrophoretic gel (3). With this affinity electrophoretic gel, it is possible to exploit the variation in the chemical reactivity of the sulfur atom with respect to its chemical environment and local conformation. The different reactivity of the sulfur in a wide range of different chemical and physical environments results in different mobilities in the mercury/acrylamide system. There is a clear difference in the affinity for s4U compared with another thiouridine derivative, 5-methylaminomethyl-2-thiouridine (mnm5s2U). Also, phosphorothioate monoesters, for instance, behave differently from phosphorothioate diesters during affinity electrophoresis.

Modifications of bases in tRNAs leading to the introduction of primary amino groups are not as common as thio-substitutions. Examples of this type are aminocarboxypropyluridine (acp3U or X) and acp2C (lysidine). Observations in our laboratory have shown such primary amine side-chains may be readily thiolated with the bifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and the corresponding tRNAs can subsequently be analyzed using the mercurial affinity electrophoretic gel system (4). In addition, the SPDP modification will react with the alpha amino group of an aa-tRNA and thereby stabilize its aminoacylester bond. Thiolation of amino groups may, conceivably, also be extended to free carboxyl moieties that have been previously linked to ethylenediamine (5). Thus, modified bases can be detected in tRNAs if they possess structural features that are either analogous to ones already demonstrated to interact with either APB or APM or that can be subjected to simple chemical modification in order to introduce a residue recognizable by these gels.

Since its introduction more than 20 years ago and despite its commercial unavailability till now, the successful use of APM for a variety of applications has been documented in over 100 publications. 

Item

Description

Quantity per tube

Cost per tube

APM
Acryloylaminophenylmercuric chloride  50 mg $80
  
References:

3. Igloi, G.L. (1988) Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organo-mercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis. Biochemistry 27, 3842-3849.

4. Igloi, G.L. (1992) Affinity electrophoretic detection of primary amino groups in nucleic acids: application to modified bases of tRNA and to aminoacylation. Anal. Biochem. 206, 363-368.

5. Gornicki. P., Nurse, K., Hellmann, W., Boublik, M., and Ofengand, J. (1984) High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 30 S ribosomal subunit. J. Biol. Chem. 259, 10493-10498.

Selected Applications:

5-taurinomethyl-2-uridine

Suzuki, T., Suzuki, T., Wada, T. Saigo, K., and Watanabe, K. (2002) Taurine as a constituent of mitochondrial tRNAs: new insights into the functions of taurine and human mitochondrial diseases. EMBO J. 21, 6581–6589.
 

2-thiouridine

Ashraf, S. S., Sochaka, E., Cain, R., Guenther, R., Malkiewicz, A. and Agris P. F. (1999) Single atom modification (O →S) of tRNA confers ribosome binding. RNA 5,188-194.

Dewez, M., Bauer, F., Dieu, M., Raes, M., Vandenhaute, J, and Hermand, D. (2008) The conserved Wobble uridine tRNA thiolase Ctu1–Ctu2 is required to maintain genome integrity. Proc. Natl. Acad. Sci. USA 105, 5459-5464.
 

5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U)

Kaneko, T., Suzuki, T., Kapushoc, S. T., Rubio, M-A., Ghazvini, J., Watanabe, K., Simpson, L. and Suzuki, T. (2003) Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism. EMBO J. 22, 657–667.
 

Deoxy-4-thiouridine

Vitorino, D. Santos, D., Vianna, A.-L., Fourrey, J.-L., and Favre, A. (1993) Folding of DNA substrate-hairpin ribozyme domains: use of deoxy 4-thiouridine as an intrinsic photolabel. Nucleic Acids Res. 21, 201-207.
 

2-thioribothymidine

Shigi, N., Suzuki, T., Tamakoshi, M., Oshima, T., and Watanabe, K. (2002) Conserved Bases in the TΨC Loop of tRNA Are Determinants for Thermophile-specific 2-Thiouridylation at Position 54. J. Biol. Chem. 277, 39128-39135.
 

Thiocarboxylated Protein

Van der Veen, A. G., Schorpp, K., Schlieker, C., Buti, L., Damon, J. R., Spooner, E., Ploegh, H. L., and Jentsch, S. (2011) Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier. Proc. Natl. Acad. Sci. USA 108, 1763-1770.
 

tRNA thiolation

Leidel, S., Pedrioli, P.G., Bucher, T., Brost, R., Costanzo, M., Schmidt, A., Aebersold, R., Boone, C., Hofmann, K. and Peter, M. (2009) Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA. Nature 458, 228-232 .

Miranda, H.V., Nembhard, N., Su, D., Hepowit, N., Krause, D.J., Pritz, J.R., Phillips, C., Söll, D. and Maupin-Furlow, J.A. (2011) E1- and ubiquitin-like proteins provide a direct link between protein conjugation and sulfur transfer in archaea. Proc. Natl Acad. Sci. USA. 108, 4417-4422.
 

Ribozymes

Vaish, N.K., Heaton, P. A., Fedorova, O., and Eckstein, F. (1998) In vitro selection of a purine nucleotide-specific hammerheadlike ribozyme. Proc. Natl. Acad. Sci. USA 95, 2158-2162.

Zaker, H. S., and Unrau, P. J. (2007) Selection of an improved RNA polymerase ribozyme with superior extension and fidelity. RNA 13, 1017-1026.

Van der Burke, D. H., and Rhee, S. S. (2010) Assembly and activation of a kinase ribozyme. RNA 16, 2349-2359 .