Transcriptome-Wide Off-Target Effects of Steric-Blocking Oligonucleotides

Abstract

Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to RNA transcripts and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization driven, whereas differential expression events are more common and driven by other mechanisms (including spurious experimental variation). We further evaluated the performance of in silico screens for off-target splicing events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate (FDR) of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR of 90%. Despite these large improvements in performance, this approach does not detect the majority of events at an FDR <99%. Our results suggest that in silico methods are currently of limited use for predicting the off-target effects of SBOs, and experimental screening by RNA-seq should be the preferred approach.

Keywords: off-target effects; splice-switching; steric-blocking oligonucleotides.

FIG. 1.

Quantification of percent spliced in for each exon. Overlapping transcripts are used to identify a set of upstream and downstream splice sites, and spliced reads mapping between these and the exon boundaries are counted as inclusion reads (green). Reads mapping directly between the upstream and downstream splice sites are counted as exclusion reads (blue). Reads where one or both ends does not match an annotated splice junction are not counted (gray).

FIG. 2.

Proportion of potential binding sites resulting in a splicing (A) and expression (B) change, broken down by edit distance and effect size. The numbers above the bars give the number of significant (q < 0.05) events with absolute dPSI >0.5 (A) or fold change <0.5 (B) and the total number of events by edit distance. Error bars show 95% binomial proportion confidence intervals. dPSI, difference in percent spliced in.

FIG. 3.

Enrichment of hits compared with a background of hits at edit distance 6+ at different dPSI cutoffs, broken down by region. Error bars show 95% bootstrap confidence intervals.

FIG. 4.

Performance of different predictors at identifying off-target splicing events with a change in PSI of at least 0.5.

FIG. 5.

(A) Illustration of the gradient-boosted tree model trained to prioritize in silico hits at an edit distance of five or lower. (B) Performance when evaluated on a test set of unseen SBOs and exons. Significant splicing changes with dPSI >0.2 were labeled positives. SBOs, steric-blocking oligonucleotides.