AFFINITY
ELECTROPHORESIS
Electrophoretic analysis of
unique functional groups
Application of acryloylaminophenyl boronic
acid (APB)
Knowledge of the chemical nature of the 5' or 3'
terminus of RNA can often reveal the origin or even function of a particular
species. Aminophenylboronic acid linked directly to acrylamide
(acryloylaminophenyl boronic acid - APB) (1,2) has proved 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 .
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.
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.
Application of acryloylaminophenylmercuric
chloride (APM)
A number of tRNA modifications involve chemical
moieties, which are sufficiently different from other reactive groups in the
RNA molecule to permit group specific binding by appropriate affinity
electrophoretic gels. Acryloylaminophenylmercuric chloride (APM), a derivative
of acrylamide can be polymerized directly into an electrophoretic gel (3). With
these affinity electrophoretic gels it is possible to exploit the variation in
the chemical reactivity of the sulfur atom with respect to its chemical
environment and local conformation. There is a clear difference in the affinity
for s4U compared with another thiouridine derivative,
5-methylaminomethyl-2-thiouridine (mnm5s2U).
Modifications of bases in tRNAs
leading to the introduction of primary amino groups are not as common as
thio-substitutions. In fact, the only examples of this type are
aminocarboxypropyluridine (acp3U or X) and acp2C
(lysidine). Observations in our laboratory have shown such primary amine
side-chains, in general, may be readily thiolated with the bifunctional reagent
N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (resulting in, among other
things, a stabilization of the aminoacylester bond, in the case of charged
tRNA) and the corresponding tRNAs can subsequently be analyzed using the
mercurial affinity electrophoretic gel system (4).
Of the other modified bases present in tRNAs, several
possess structural features which are either analogous to ones already
demonstrated to interact with either APB or APM, or which can be subjected to
simple chemical modification in order to introduce a residue recognizable by
these gels. Thiolation of amino groups as described above may be extended to
free carboxyl moieties that have been previously linked to ethylenediamine (5).
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 described above. Phosphorothioate monoesters,
for instance, behave differently from phosphorothioate diesters during affinity
electrophoresis (3).
Since its introduction more than 20 years ago and
despite its commercial unavailability till now, the use of APM for a variety of
applications has been documented in over 100 publications.
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-thiouridine
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-thio-uridine
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-thio-uridine
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/SELEX
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.
Zaher, H.S. and Unrau, P.J. (2007) Selection of an improved RNA polymerase
ribozyme with superior extension and fidelity. RNA 13,1017-1026.
Burke,
D.H. and Rhee, S.S. (2010)
Assembly and activation of a kinase ribozyme. RNA. 16, 2349-2359.
SEQUENCE-DEPENDENT
ELECTROPHORESIS
Unlike structure-specific affinity
electrophoresis that is limited to rare or unnatural features in nucleic acids,
sequence-specific affinity electrophoresis is applicable wherever complementary
nucleic acid strands interact. Sequence-specific fractionation of nucleic acids
does not depend on the formation of a quasi covalent bond, as described above,
but demands an environment where specific base-pairing interactions can take
place. Assuming that conditions permitting such hybridisation in gels are the
same as in free solution one must be aware that conventional hybridisation
requirements are not reconcilable with affinity electrophoretic conditions.
It is, therefore, unlikely that a simple
immobilisation of one strand in an electrophoretic medium (creating an intrinsically
charged polymer, with the consequent problems of electroendosmosis) will
provide the means to follow hybridisation under stringent conditions (high salt
concentrations).
Peptide nucleic acids (PNA) are synthetic
chimeras of nucleobases linked to a peptide backbone. This spacing permits the
bases to form, among other possible structures, standard base pairs with
natural nucleic acids. The lack of the phosphodiester linkage, leading to an
electronically neutral species, has, however, important consequences for the
base-pairing potential of PNA. The greater stability of PNA-nucleic acid
duplexes, together with the salt independence of hybridisation has been
ascribed to the lack of backbone charge repulsion. These two factors, namely
electronic neutrality together with salt independent base pair specificity, are
the properties of PNA that make them ideal candidates as ligands in an affinity
electrophoretic partnership.
Specific
base-pairing between synthetic short single stranded oligonucleotides and a
gel-entrapped complementary PNA has been demonstrated and quantified in this
system recently (59).
Single base mismatched hybrids could readily be distinguished from fully
complementary in their melting temperatures and hence in their migration
behaviour. The application of this concept has been extended to double stranded DNA of PCR-product length (60-90 bp) with the
intention of detecting or screening for known naturally occurring and
clinically significant point mutations.
The
potential power of this approach has been tested by entrapping a PNA 11-mer
specific for a common mutation in the human gene responsible for hereditary
hemochromatosis into a non-denaturing polyacrylamide gel. Electrophoretic
fractionation of fluorescent PCR products from wild type and from mutant
samples gives rise to signals, detected with a standard fluorescent DNA
sequencing system, that are in accord with a distinct and specific retardation
of a single base mismatched mutant sequence by the PNA matrix (60).
While the detection of this and other
mutations may also be possible by other electrophoretic means, such as SSCP,
this requires considerable experience in both experimental design and
interpretation. Numerous PCR-based mutation detection systems exist but these
either involve subsequent manual electrophoretic evaluation, limiting the
throughput, or in the case of an automated commercial system require
specialised equipment. Furthermore, conventional PCR techniques rely on the
presence or absence of a particular electrophoretic pattern or signal and, in
contrast to the sequence-specific retardation by the PNA-gel, do not directly
identify the actual nucleotides involved in the mutation. Mismatch-detection
hybridisation methods, while amenable to automation using yet-to-be developed
chip array technology, will not permit real-time analysis of samples since a
finite time for hybridisation and wash steps must be allowed. The proposed affinity
electrophoretic analysis that has been extended further for capillary
electrophoresis systems (61) is now being developed for the
multiplex use of several mutation-specific PNAs simultaneously, to introduce a
fully automated, highly sensitive procedure of high potential throughput for a
routine screening of clinically relevant PCR products.