Effects of PTO in long-term survival of corrected cells.

<p>(a) Varying numbers of PTO bonds 3′ to the mismatch, shown in gray, suggest 3-5PTO bonds as optimal for balancing toxicity and stable targeting frequencies, assayed 14-days after oligo transfection by flow cytometry. (b) Testing the position of PTO bonds, strand targeted and use of modified bases in long term survival. F5-20 used as non correcting control, F5-17, -29 and -30 are complementary to the non-transcribed strand, while F5-31, -32 are complementary to the transcribed. n = 4.</p

Induction of immune-related genes in oligo-transfected cells.

<p>(a)RT-qPCR relative quantification was performed for key immune-related genes, normalizing oligo-transfected to untransfected cells. (b) Mean Fluorescence Intensity (MFI) of total and EGFP+cells transfected with both F5-17 and F5-38 (Cy5-labeled) oligo at varying concentrations. Transfections were done in 24-well plates. n = 4 (c) Methylation of the CpG sequence present in the targeting oligo has no effect on the %EGFP+ cells. (d) Small-molecule inhibitors against key immune effectors, with DMSO as control, added 24 h after F5-17 oligo transfection assayed for EGFP+ cells at 48 h and 96 h. n = 4.</p

Chemically-modified base analogs.

<p>(a–b)Modified base-containing oligos complementary to the non-transcribed strand’s (a)first potential start codon TTG and (b) second potential start codon AAG, where each mismatched base X in the targeting oligo is shown in parenthesis. (c–d) RNAi targeting key mismatch repair proteins MLH1 and MSH2 for the (a)TTG and (b)AAG start codon targeted by oligos containing modified bases. Data was normalized relative to scr shRNA, thus in (c) each MMR component silencing produced a 2-fold improvement for the natural T base, while the improvement for FU was reduced. This is seen to a lesser degree in (d) comparing A and AM, while FA was further improved, suggesting it is more strongly recognized by MMR. n = 4.</p

Oligonucleotides used in this study.

<p>Sequences shown 5′ to 3′. PTO bonds shown as asterisks (*). dU = deoxyUridine, Fu = 2′-Fluorouracil, Fa = 2′-Fluoroadenine, Am = 2-Aminopurine, mC = 5-Methyl deoxyCytidine.</p

Oligo-mediated targeting reporter system.

<p>(a) Reporter consists of a HeLa cell line with two stably integrated copies of EGFP with a mutated TTG start codon and a second potential start codon (AAG) downstream, both shown in red. Each mutated start codon can be targeted by sense or antisense oligos. Representative oligos used in this study are highlighted, in duplex form, with the mismatch shown by an asterisk. Further detail can be found on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036697#pone-0036697-t001" target="_blank">Table 1</a>. (b) Sample flow cytometry dot plot of cells with no oligo (left) or transfected with F5-8 (right, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036697#pone-0036697-t001" target="_blank">Table 1</a>). The frequency of oligo-induced correction of a start codon can be estimated as the %EGFP+/Propidium Iodide- cells (c) %EGFP+ cells generated after different oligo transfections, with and without 20 mM thymidine, assayed 36 h post transfections. Oligo sequences are shown 5′ to 3′, and the PTO bonds are highlighted in gray. The control oligos F5-1 and F5-6 did not produce any significant proportion of EGFP+ cells, neither did F5-2, which is complementary to the transcribed strand and encodes an ATG-restoring mutation. The oligos targeting the first and second potential start codons on the non-transcribed strand, F5-3 and F5-5 respectively, did produce EGFP+ cells. Lipo = lipofectamine only control. n = 4.</p