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

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.

Design and evaluation of antisense sequence length for modified mouse U7 small nuclear RNA to induce efficient pre-messenger RNA splicing modulation in vitro

“…According to previous reports, designing an ASO to have an appropriate length is the key to enhancing splicing modulation efficiency [ 21 , 22 ]. In fact, previous reports suggest that high T m values contribute to the effectiveness of splicing modulation [ 19 – 21 ].…”

“…However, few reports have investigated the effect of the length of the antisense sequence [ 18 ]. This may be important, as previous reports on ASOs revealed that selecting the proper ASO length is the key to enhancing splicing modulation efficiency [ 19 – 22 ]. In fact, LNA-modified ASOs are known to have an optimal length for effective splicing modulation [ 21 ].…”

Evaluation of Chemically Modified Nucleic Acid Analogues for Splice Switching Application

“…Results demonstrated that, compared to the fully 2′-OMe-PS controls, 18- mer and 16-mer α-L-LNA/2′-OMe-PS mixmers displayed slightly better exon skipping efficiency. 50 In addition, other LNA analogues including AmNA, GuNA, and cEt also have been recently reported in constructing chimeric ASOs for inducing efficient splice switching, 51 , 52 thereby expanding the scope of optimizing bridged nucleic acid modified splice modulating ASOs by incorporating novel analogues.…”