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. 2021 May 17;49(19):10807–10817. doi: 10.1093/nar/gkab288

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© The Author(s) 2021. 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-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — Reinforcement of EF-Tu affinity of Phe-tRNA and npAA-tRNAs by T-stem engineering. (A) Sequence of the original tRNA^AsnE2 (#2) and T-stem variants #1–4. The affinities of tRNA #1–4 were designed to increase as the T-stem number increases from #1 to #4. (B–E) Quantification of the EF-Tu affinities of Phe-tRNA_GAC#1–4 (B, C) and ^MeF-tRNA_GAC#2–4 (D, E). Schematic representation of RNase A protection assay, the observed fraction of the ternary complex with the fitting curve to determine the K_D value (B, D), and the calculated ΔG value (C, E). Asterisk (*) indicates undetectably weak affinity (ΔG > −6 kcal/mol). Error bar indicates the fitting error. (F–J) The EF-Tu affinities of npAA-tRNA#1–4 and MALDI-TOF MS of P1-npAA examined for ^MeG (F), ^MeS (G), ^MeA (H), ^MeL (I) and ^AcK (J). Each dagger peak (†) corresponds to a byproduct containing Ile in place of npAA.