Knockdown of Circular RNAs Using LNA-Modified Antisense Oligonucleotides.

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• Circular RNAs (circRNAs) are abundant noncoding RNAs with largely unknown functions.
• Investigating circRNA function is challenging due to sequence overlap with linear transcripts.
• Specific depletion of circRNAs without affecting host gene expression is a key hurdle.

Purpose of the Study:

• To evaluate locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) for circRNA knockdown.
• To assess the specificity and efficacy of ASOs in circRNA loss-of-function studies.

Main Methods:

• Design and application of LNA-modified antisense oligonucleotides (ASOs) targeting circRNAs.
• Utilized RNase H-dependent gapmer ASOs and design-optimized gapmer ASOs.
• Assessed knockdown efficiency and specificity in vitro.

Main Results:

• Most RNase H-dependent gapmer ASOs effectively knocked down target circRNAs.
• Some gapmers inadvertently reduced linear parent transcript levels.
• Design-optimized gapmer ASOs demonstrated potent and specific circRNA knockdown with minimal host gene effects.

Conclusions:

• LNA-modified ASOs complementary to backsplice-junction sequences enable robust circRNA knockdown.
• Optimized ASOs are a valuable tool for exploring circRNA biological roles.
• This method facilitates loss-of-function studies in cultured cells and animal models.