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Volume 22
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Article

Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids

by
Takenori Shimo
,
Yusuke Nakatsuji
,
Keisuke Tachibana
and
Satoshi Obika
*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(7), 3526; https://doi.org/10.3390/ijms22073526
Submission received: 15 February 2021 / Revised: 20 March 2021 / Accepted: 26 March 2021 / Published: 29 March 2021
(This article belongs to the Special Issue Precision Nucleic Acid Therapeutics)
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Abstract

Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (Tm) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same Tm values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower Tm values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.
Keywords: exon skipping; splice-switching oligonucleotide; chemical modification; bridged nucleic acids (BNAs); locked nucleic acids (LNAs); amido-bridged nucleic acids (AmNAs); guanidine-bridged nucleic acids (GuNAs); dystrophin exon skipping; splice-switching oligonucleotide; chemical modification; bridged nucleic acids (BNAs); locked nucleic acids (LNAs); amido-bridged nucleic acids (AmNAs); guanidine-bridged nucleic acids (GuNAs); dystrophin

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MDPI and ACS Style

Shimo, T.; Nakatsuji, Y.; Tachibana, K.; Obika, S. Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids. Int. J. Mol. Sci. 2021, 22, 3526. https://doi.org/10.3390/ijms22073526

AMA Style

Shimo T, Nakatsuji Y, Tachibana K, Obika S. Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids. International Journal of Molecular Sciences. 2021; 22(7):3526. https://doi.org/10.3390/ijms22073526

Chicago/Turabian Style

Shimo, Takenori, Yusuke Nakatsuji, Keisuke Tachibana, and Satoshi Obika. 2021. "Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids" International Journal of Molecular Sciences 22, no. 7: 3526. https://doi.org/10.3390/ijms22073526

APA Style

Shimo, T., Nakatsuji, Y., Tachibana, K., & Obika, S. (2021). Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids. International Journal of Molecular Sciences, 22(7), 3526. https://doi.org/10.3390/ijms22073526

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