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. 2007 Oct 11;35(20):6870–6883. doi: 10.1093/nar/gkm778

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© 2007 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — Semi-synthesis of Ala-tRNA^Ala analogues. (A) The protected dinucleotide pdCpA-Ala-pentenoyl was ligated to a 74-nt in vitro transcript by the T4 RNA ligase. The product of the reaction was deprotected leading to an acylated-RNA molecule identical to Ala-tRNA^Ala except for the presence of a 3′deoxycytidine instead of a cytidine at position 75. Nucleotide substitutions were introduced in the 74-nt in vitro transcript using the PCR approach described above except that one of the two mutagenic primers lacked the two 5′ bases specifying bases 75 and 76 of the transcript. (B) Schematic representation of the main steps for synthesis of the dinucleotide pdCpA-Ala-pentenoyl. (C) Analysis of the T4 ligation products by denaturing polyacrylamide electrophoresis. Lane 1, 76-nt transcript with the wild-type tRNA^Ala sequence; lane 2, 74-nt in vitro transcript; lane 3, ligation of the 74-nt transcript with pdCpA-Ala-pentenoyl.