Figure 1.
(A) The SOD-1 minigene contains the fourth and fifth exons of the SOD-1 gene and intervening intron. The intron was reduced to 250 nucleotides after the central 845 nucleotides were eliminated by PCR. The resulting minigene encodes a 555-nucleotide pre-mRNA and 305-nucleotide mRNA. (B) The minigene derived from the SOD-1 cDNA template was cloned into pcDNA3.1 vector containing both T7 and CMV promoters for, respectively, T7 and Pol II polymerase transcription and a BGH polyadenylation signal. The T7 transcribed SOD-1 minigene mRNA was PAGE purified and either 5′-labeled with 32P or spiked into the denatured nuclear extract. C) The SOD-1 genomic DNA derived SOD-1 minigene was cloned into the same vector and added to the nuclear extract for Pol II transcription and splicing into the mRNA. The unspliced pre-mRNA was degraded using an antisense ASO targeting the intron region and excess E. coli RNase H1. Levels of the SOD-1 minigene pre-mRNA and mRNA were quantified by qRT-PCR using primers complementary to the vector regions immediately upstream and downstream of the SOD-1 minigene RNA region (dark lines) and probes complementary to, respectively, the intron region or exon-exon junction (grey lines).
Table 1.
Minigene specific qRT/PCR primers and probes.
Figure 2.
ASO binding to naked 32P-labeled SOD-1 minigene mRNA.
Denaturing PAGE analysis of digestion reactions with ASOs and without ASO (labeled UTC). The bands corresponding to the RNase H1 cleavage products from on-target binding are labeled with ASO number in blue and off-target ASO cleavage products are labeled with red ASO numbers. The position of the off-target ASO hybridization in the SOD-1 minigene mRNA was determined by comparing the size of the off-target cleavage bands with the size of the on-target cleavage bands (joined by red lines).
Figure 3.
ASO binding to the SOD-1 minigene mRNA spiked into denatured nuclear extract.
(A) The binding profile of the ASOs for the mRNA in denatured extract (orange line) compared with the predicted target site accessibilities (green line). ASO binding is reported as percent untreated mRNA control (left y-axis). A greater mRNA reduction (lower percent control) correlates with tighter ASO binding. Target site accessibilities are reported as unpaired probabilities (right y-axis). Greater probabilities predict that the target region is single stranded and accessible to ASO, and lower probabilities suggest that the target region is involved in mRNA structure. (B) Prediction of ASO target site accessibility within the SOD-1 minigene mRNA using RNAP-fold. The scanning window size (w) and maximum allowed distance between the base-pairs (L) within the mRNA were set to, respectively, 80 and 40 ribonucleotides. The binding accessibility of each ASO for the target mRNA was calculated based on the length of the ASO target site (u), specifically 20 ribonucleotides.
Table 2.
Binding affinities of ASOs for the oligoribonucleotide targets and SOD-1 minigene mRNA spiked into the nuclear extract.
Figure 4.
ASO binding to the SOD-1 minigene mRNA transcribed and spliced in the nuclear extract.
The binding profile of the ASOs to mRNA transcribed and spliced in the nuclear extract (blue line) compared with the mRNA in denatured extract (orange line). The proteins bound to the mRNA at each ASO target site are listed in Figure S1. Here, proteins bound are listed below the ASO number by class: RNA-binding proteins (R), E-complex proteins (E), H-complex proteins (H) and exon-junction proteins (EJ). ASO binding is reported as percent untreated mRNA control. The mean and errors reported are based on three trials.
Table 3.
Binding affinities of ASOs for the SOD-1 minigene mRNA spiked into the denatured nuclear extract and mRNA transcribed and spliced in the nuclear extract.
Table 4.
Binding affinities of the ASOs for the on- and off-target oligoribonucleotides.
Figure 5.
On- and off-target ASO binding to the SOD-1 minigene mRNA.
(A) Orange line represents the ASO binding profile for the mRNA spiked into the denatured nuclear extract obtained using exon 4-specific primers (red arrows). Black line represents the ASO binding profile for the mRNA spiked into the denatured nuclear extract obtained using exon 5-specific primers (blue arrows). ASO binding is reported as percent untreated mRNA control. The mean and standard errors reported are based on three experiments. On-target reduction of mRNA was observed for ASOs 18–28 targeting exon 4 when the exon 4-specific primer/probe set was used (orange line). No reduction of the mRNA was observed for these ASOs using the exon 5 primer/probe set (black line) indicating that these ASOs did not exhibit off-target binding to the exon 5 region of the minigene mRNA. On-target reduction of mRNA was observed for ASOs 37–51 targeting exon 5 when the exon 5-specific primer/probe set was used (black line). Reduction of mRNA was observed for ASOs 37, 38, 40, and 82 using the exon 4-specific primer/probe set (orange line) indicating that these ASOs exhibited off-target binding to the exon 4 region of the minigene mRNA. (B) Blue line represents the ASO binding profile for the mRNA transcribed and spliced in the nuclear extract using exon 4-specific primers (red arrows). Black line represents the ASO binding profile for the mRNA transcribed and spliced in the nuclear extract using exon 5-specific primers (blue arrows). ASO binding is reported as percent untreated mRNA control. The mean and standard errors are based on three experiments. On-target reduction of mRNA was observed for ASOs 18–28 targeting exon 4 when the exon 4-specific primer/probe set was used (blue line). No reduction of the mRNA was observed for these ASOs using the exon 5 primer/probe set (black line) indicating that these ASOs did not exhibit off-target binding to the exon 5 region of the minigene mRNA. On-target reduction of mRNA was observed for ASOs 37–51 targeting exon 5 when the exon 5-specific primer/probe set was used (black line). Reduction of mRNA was observed in the presence of ASOs 37, 38, 40, and 82 using the exon 4-specific primer/probe set (blue line) indicating that these ASOs exhibited off-target binding to the exon 4 region of the minigene mRNA.
Table 5.
Binding affinities of the ASOs for the on- and off-target sites of the SOD-1 minigene mRNA spiked into the denatured nuclear extract.
Table 6.
Binding affinities of the ASOs for the on- and off-target sites of the SOD-1 minigene mRNA transcribed and spliced in the nuclear extract.
Figure 6.
A) ASO activity for tetracycline-inducible SOD-1 minigene.
Exons 4 and 5 and a truncated intron 4 were cloned into the vector pcDNA4/TO allowing for tetracycline regulated expression of the minigene and zeocin selection of stable cell lines. SOD/TO cells were transfected with ASOs. Following ASO treatment reduction of SOD1 minigene or endogenous SOD1 was evaluated by qRT/PCR using primers and probes specific for the minigene or endogenous SOD1. Data are presented as percent expression spliced mRNA relative to mock-treated control cells. The error bars represent the mean and standard errors of at least three experiments.
Figure 7.
Effect of E. coli RNase H overexpression on on-target antisense activity.
HEK 293 cells harboring the SOD/TO minigene or the SOD/TO minigene and pcDNA3.1-RHA were treated with ASOs at concentration between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was measured by qRT/PCR. Data are presented as percent mock-transfected control for SOD/TO (solid line) and SOD/TO-RHA cells (dashed line).
Figure 8.
Effect of E. coli RNase H overexpression on off-target antisense activity.
HEK 293 cells harboring the minigene or the SOD 282_DL minigene and pcDNA3.1-RHA were treated with ASOs at concentrations between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was determined by qRT/PCR. Data are presented as percent mock-transfected control for SOD 282_DL (solid line) and SOD 282_DLH cells (dashed line).
Figure 9.
Cellular off-target cleavage is observed only with RNase H1 overexpression.
A modified RLM-RACE protocol was used to determine sites of target-specific and off-target cleavage. A) Exon 5 target-specific RACE cleavage products. (B) Quantitative RACE of exon 5 target-specific cleavage products for SOD/TO cells (black bars) and SOD/TO-RHA cells (gray bars). Results are given as threshold cycle (cT) for the amplification reaction with or without overexpression of RNase H. C) Exon 4 off-target RACE cleavage products. D) Quantitative RACE of exon 4 off-target cleavage products (cT). E) Human RNase H1 was overexpressed in HeLa SOD/TO cells by infecting with adenoviral human RNase H1 for 48 hours [4]. Cells were then treated with 50 nM ASO 38, and RLM_RACE was performed as described above. 293 cells with or without E. coli RNase H; HeLa cells with or without human RNase H1. F) RACE products were gel purified and sequenced using gene specific RACE primers. Arrows indicate the predominant cleavage site.