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. 2015 Apr 27;43(10):5145–5157. doi: 10.1093/nar/gkv277

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — In vitro deamination assays. (A) Sequence of the in vitro transcribed pre-tRNA^Val_(AAC) showing the 5′-leader, the 3′-trailer and the mature tRNA sequences. The expected site for EndoV cleavage is also indicated (triangle) and the anticodon of the tRNA is underlined. (B) Chromatogram obtained after sequencing of the anticodon loop of in vitro transcribed pre-tRNA^Val_(AAC) incubated ((+) hetADAT) or not ((−) hetADAT) with purified human hetADAT. (C) EndoV assay for detection of inosine on in vitro transcribed pre-tRNA^Val_(AAC) that has been previously incubated ((+) hetADAT) or not ((−) hetADAT) with purified human hetADAT. Pre-tRNA^Val was ³²P-radiolabelled after EndoV treatments allowing for detection of all the cleavage products. Expected tRNA lengths: 95 nt for full length pre-tRNA^Val_(AAC); 54 nt for 3′-tRNA arm after EndoV cleavage if inosine is present at position 34 of the tRNA; 41 nt for 5′-tRNA arm after EndoV cleavage if inosine is present at position 34 of the tRNA. ³²P-radiolabelled RNA decade marker was used to estimate the molecular sizes.