TY - JOUR

T1 - Knockdown of Circular RNAs Using LNA-Modified Antisense Oligonucleotides

AU - Løvendorf, Marianne Bengtson

AU - Holm, Anja

AU - Petri, Andreas

AU - Thrue, Charlotte Albæk

AU - Uchida, Shizuka

AU - Venø, Morten T.

AU - Kauppinen, Sakari

N1 - Publisher Copyright: © Copyright 2023, Mary Ann Liebert, Inc., publishers 2023.

PY - 2023

Y1 - 2023

N2 - Circular RNAs (circRNAs) constitute an abundant class of covalently closed noncoding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys, as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. In this study, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We found that, while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes. In summary, our results demonstrate that LNA-modified ASOs complementary to backsplice-junction sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

AB - Circular RNAs (circRNAs) constitute an abundant class of covalently closed noncoding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys, as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. In this study, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We found that, while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes. In summary, our results demonstrate that LNA-modified ASOs complementary to backsplice-junction sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

KW - antisense oligonucleotide

KW - circRNAs

KW - knockdown

KW - locked nucleic acid

U2 - 10.1089/nat.2022.0040

DO - 10.1089/nat.2022.0040

M3 - Journal article

C2 - 36445751

AN - SCOPUS:85147389966

VL - 33

SP - 45

EP - 57

JO - Nucleic Acid Therapeutics

JF - Nucleic Acid Therapeutics

SN - 2159-3337

IS - 1

ER -