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Title
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Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding.
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Authors
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P.Burgstaller,
M.Kochoyan,
M.Famulok.
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Ref.
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Nucleic Acids Res, 1995,
23,
4769-4776.
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PubMed id
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Abstract
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In a recent study, an RNA aptamer for the specific recognition of the amino acid
L-arginine was evolved from an in vitro selected L-citrulline binding parent
sequence [M. Famulok (1994) J. Am. Chem. Soc. 116, 1698-1706]. We have now
carried out a structural analysis of these aptamers by using chemical
modification experiments. Footprinting experiments and a damage selection
approach were performed to identify those positions protected from modification
in the presence of the amino acids and modifications that interfere with the
binding of the ligand. It is shown that of the two bulged regions present in
both aptamers one can be modified without loss of binding activity whereas in
the other bulge nearly every position is shown to be involved in the recognition
of the ligands. This might be indicative for non-canonical base pairing to occur
within the non-Watson-Crick paired regions which might be stabilized by the
complexed amino acid. Binding to the cognate amino acid significantly enhances
the conformational stability of the RNA. We also tested the sensitivity of both
aptamers towards lead (II) ion induced cleavage and identified a hypersensitive
cleavage site within the invariant bulged region. Lead cleavage is inhibited by
the complexed amino acid, indicating a conformational change of the aptamer upon
ligand binding. NMR titration data obtained with both aptamers and their cognate
ligands confirm the proposed conformational changes and indicate the formation
of a 1:1 complex of RNA:amino acid.
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