*4.4. Peptide Competition Assays*

The peptide competition assays were conducted using Biacore 8K (GE Healthcare, Marlborough, MA, USA). The untagged RTA was immobilized on a CM5 chip to around 4000 RU in flow cell 2 for all eight channels by amine coupling. The flow cell 1 was activated and blocked. The A-B-A injection was used. The surface was first injected for 1 minute with peptide P6 at 0, 125, 250, and 500 μM. Then, yeas<sup>t</sup> ribosomes (20 nM) mixed with the same concentrations of P6 were injected on the surface for 2 min. Ribosome disassociation was allowed for another 2 min. The flow rate was 30 μL/min. The surface was regenerated by three one-minute injections of 2 M NaCl and one 1 min injection of running buffer containing 2% DMSO. The running buffer was the same as peptide–RTA interaction buffer with 0.005% of surfactant P20. The binding levels of ribosome at "A-B-A binding later" were compared.

## *4.5. Inhibition of RTA Depurination*

The inhibition of depurination activity was measured by qRT-PCR using the untagged RTA [31]. The final RTA concentration was 1.0 nM for yeas<sup>t</sup> ribosomes and 0.2 nM for rat liver ribosomes with both ribosome concentrations at 60 nM. The same buffer used to examine the interaction of RTA with peptides was used. The final peptide concentrations varied dependent on the level of inhibition by the peptides. RTA was mixed with the peptides at room temperature for several minutes. The ribosomes were added to start the reaction. The depurination reaction was incubated at 25 ◦C for 5 min and was stopped by adding an equal volume of RNA extraction buffer. RNA was purified from the depurinated ribosomes using a previously published method [22]. The percentage of depurination was determined by qRT-PCR [31]. In each set of measurements, the ribosome and peptide mixture without toxin was used as 100% and the mixture with ribosome and toxin but without the peptide was used as 0%. The percentage of inhibition was plotted against the peptide concentration and the IC50 was calculated by fitting the data with the Michaelis–Menten function using Origin Pro 9.1.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/10/9/371/s1, Figure S1: Inhibition of depurination activity of RTA on yeas<sup>t</sup> ribosomes by peptides P10–P3. The IC50 values were determined as in Figure 6 and are shown in Table 2. Figure S2: Inhibition of depurination activity of RTA on rat liver ribosomes by peptides P10, P9, and P8. The IC50 values were determined as in Figure 6 and are shown in Table 2.

**Author Contributions:** X.-P.L., J.N.K. and N.E.T. planned experiments, X.-P.L. and J.N.K. performed experiments, X.-P.L., J.N.K. and N.E.T. interpreted data, X.-P.L. and N.E.T. wrote the paper. All authors reviewed the paper.

**Funding:** This work was supported by National Institutes of Health grants AI072425 and AI127980 to Nilgun E. Tumer.

**Acknowledgments:** We thank Yijun Zhou for critical reading of the paper. We thank Karen Chave (Northeast Biodefense Center Protein Synthesis Core, U54-AI057158-Lipkin) for purification of RTA variants from *E. coli*.

**Conflicts of Interest:** The authors declare no conflict of interest.
