mRNA/gRNA rate constants by Surface Plasmon Resonance.

<p>Representative SPR sensograms are shown with line fits. <b>A</b>. CYbU+gCYb-558. <b>B</b>. A6U+gA6-14. <b>C</b>. ND7-550+gND7-550. The association (k_on) and dissociation (k_off) rate constants represent the mean of a minimum of 3 runs (each run utilizing 3 different mRNA concentrations) and are listed with the error in parentheses. RU = resonance units.</p

Predicted secondary structures for the A6P1/gA6-14 and ND7-550/gND7-550 complexes.

<p>Sites where we observed gRNA-directed cleavages C1–C5, are indicated. ES = Editing Site. ES2* indicates the first editing site that is correctly cleaved in the A6P1/gA6-14 interaction. No cleavage at at the first editing site was observed for ND7-550. The gRNA-dependent cleavages for this substrate occurred at sites that are edited in the mature transcript (C2 - ES2, C3 - ES4 and C5 - ES6) and at sites that are not edited in the mature transcript (C1 and C4). The mRNA anchor binding sequence is shown in outline font.</p

ODN-directed accessibility assays.

<p><b>A</b>. Representative images of 8% denaturing polyacrylamide gels. Each reaction contained a pre-hybridized ³²P-labeled mRNA (A6U, CYbU, ND7UHR3, or ND7-550) that was digested with RNase H for 1, 15, and 30 minutes upon addition of a specific ODN (1∶1, 1∶5, 1∶10, or 1∶30 mRNA to ODN ratio). “NO”: no ODN control. The digested products (<) are indicated. <b>B</b>. Percentage of RNase H digestion products. For comparison purposes, the amount of digested A6U shown in each graph was kept constant at 1∶1 ratio. The CYbU mRNA was not included because no digested products were detected. These data are the average of three experiments.</p

Solution Structure Probing of ND7-550/gND7-550.

<p>Representative image of a denaturing polyacrylamide gel. The ND7-550 mRNA was 5′-end-labeled and renatured alone or with gND7-550. 0: no enzyme control. 1.5, 3, and 4.5 Units: amount of Mung Bean nuclease. MB and T1 are Mung Bean and RNase T1 digests for sequence mapping. The position of the ABS and the region where the gRNA U-tail is predicted to bind on the mRNA are indicated.</p

Oligodeoxyribonucleotides.

<p>Oligodeoxyribonucleotides.</p

mRNAs aligned with gRNAs and ODNs.

<p>The mRNA anchor binding site (underlined) is complementary to the gRNA anchor and ODN. Watson-Crick (|), non-Watson-Crick (:) base pairs, mismatches (#) and the first editing site (ES, arrowhead) are indicated. The mRNA sequences continue at the 3′-end, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012235#pone-0012235-g002" target="_blank">figure 2</a>.</p