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

Erle M Holgersen1,2, Shreshth Gandhi1,2, Yongchao Zhou1,2

  • 1Deep Genomics, Inc., Toronto, Canada.

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Summary
This summary is machine-generated.

Steric-blocking oligonucleotides (SBOs) can cause unintended gene expression changes. RNA sequencing reveals splicing changes are hybridization-driven, while expression changes have varied causes, limiting the predictive power of in silico methods.

Keywords:
off-target effectssplice-switchingsteric-blocking oligonucleotides

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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.