Figure 1.
Oligo-mediated targeting reporter system.
(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 Table 1. (b) Sample flow cytometry dot plot of cells with no oligo (left) or transfected with F5-8 (right, Table 1). 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.
Table 1.
Oligonucleotides used in this study.
Figure 2.
Chemically-modified base analogs.
(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.
Figure 3.
Varying PTOs in targeting oligos.
All oligos in this figure are complementary to the non-transcribed strand’s first potential start codon TTG, similar to F5-3 in Fig. 1. (a) Oligo toxicity and targeting efficiency as a function of PTO modifications. PTO bond position shown in gray. ‘Total alive cells’ was estimated as the total gated PI- cells in 200 uL from a 24-well plate 48 hrs post oligo transfection. (b) Correction efficiencies of new oligo designs with reduced PTO bonds and using FU modified base. n = 4.
Figure 4.
Proliferation defect in corrected cells.
Proliferation is inversely proportional to the MFI of the CellTrace Violet dye. Cells were treated with CellTrace Violet dye, then split and transfected with oligos 48 hrs later. Cells were fixed at each time point and run together on a LSRfortessa cell analyzer at the end of the time course (a) Flow cytometry dot plots showing relative proliferation of EGFP+ and EGFP- cells generated with different targeting oligos, at four different time points post-transfection. %EGFP shown as average±s.d. n = 6. (b) Proliferation index is calculated as the inverse MFI of CellTrace ×106 for each population. (c) Cells treated with anti-microtubule drugs Taxol and Vincristine 24 h after transfection with oligo F5-17 and 24 h exposure time, checked at 48 h and 96 h post-transfection. n = 4.
Figure 5.
Oligos complementary to the first start codon in the non-transcribed strand and containing a second mismatch, either an insertion (∧A) or substitution (-A), with silenced MSH2 and assayed 36 hrs after transfection. Targeting oligos shown in duplex 3′-’to 5′-, with PTO highlighted in gray and mismatches shown with an asterisk. F5-20 was used as non-correcting control. n = 4.
Figure 6.
Effects of PTO in long-term survival of corrected cells.
(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.
Figure 7.
Induction of immune-related genes in oligo-transfected cells.
(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.