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

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• Steric-blocking oligonucleotides (SBOs) are tools to modulate gene expression by targeting RNA.
• Off-target binding of SBOs to near-complementary sites can lead to unintended biological effects.
• Evaluating these off-target effects is crucial for the safe and effective use of SBOs.

Purpose of the Study:

• To assess off-target differential splicing and expression events induced by SBOs.
• To evaluate the efficacy of in silico prediction methods for SBO off-target effects.
• To compare in silico predictions with experimental RNA sequencing data.

Main Methods:

• RNA sequencing (RNA-seq) was employed to analyze differential splicing and expression.
• 81 SBOs were tested for off-target splicing effects and 46 for differential expression.
• In silico screens, including edit distance and machine learning models, were evaluated.

Main Results:

• Differential splicing events were primarily driven by hybridization, while differential expression events were more common and multifactorial.
• In silico screens with edit distance showed low sensitivity and high false discovery rates.
• Machine learning improved prediction but still failed to detect most off-target events at low FDR.

Conclusions:

• In silico methods currently have limited utility for predicting SBO off-target effects.
• Experimental validation using RNA-seq is the recommended approach for assessing SBO off-target impacts.
• Further development is needed to improve the accuracy of computational prediction tools.