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found that MCE modification can improve resistance to nuclease
digestion.15 However, nuclease resistance was not a key factor
because full-MCE did not result in improved exon skipping
compared to 5´-MCE. Previous studies have suggested that exon
skipping can be induced by the inhibition of the binding of exonic
splicing enhancers (ESEs) to the exon sequence of target pre-
31
mRNA.16, ESE binding motifs in mice were predicted using
RESCUE-ESE.32-33 In the complementary sequence of ASO, there
are four ESE binding motifs (see supplementary information).
Interestingly, the complementary sequence of MCE-modified
nucleotides in 5´-MCE overlapped with two of these binding
motifs, whereas there was no overlap for 3´-MCE. Thus, it might
be possible that ESE binding could be effectively inhibited by the
incorporation of MCE modifications in 5´-MCE. Further detailed
investigations are necessary to understand the precise effects of
MCE modifications. It should be noted that the exon skipping
efficiency of PMO was 36 ± 2.8%, which is comparable to that of
5´-MCE. Even a five-nucleotide substitution could greatly
improve the exon skipping efficiency compared with that of Me.
The exon skipping efficiency induced by MCE-modified SSO
containing s2TMCE (sT-MCE) was 47 ± 6.1%. The SSO with
s2TMCE was the most effective among the evaluated SSOs. This
tendency was consistent with the results of our previous report.19
Since the incorporation of MCE-modified nucleotides results in a
slight decrease in duplex stability, additional stabilizing
modifications can further improve the exon skipping effect.
ments/ucm534611.htm (accessed Nov 26, 2018).
10. Shimo, T.; Tachibana, K.; Saito, K.; Yoshida, T.; Tomita, E.;
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2015, 21, 270.
12. Le, B. T.; Adams, A. M.; Fletcher, S.; Wilton, S. D.; Veedu, R.
N., Mol. Ther. Nucleic Acids. 2017, 9, 155.
13. Yang, L.; Niu, H.; Gao, X.; Wang, Q.; Han, G.; Cao, L.; Cai,
C.; Weiler, J.; Yin, H., PLoS One 2013, 8, e61584.
14. Yamada, T.; Okaniwa, N.; Saneyoshi, H.; Ohkubo, A.; Seio,
K.; Nagata, T.; Aoki, Y.; Takeda, S.; Sekine, M., J. Org. Chem.
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15. Yamada, T.; Masaki, Y.; Okaniwa, N.; Kanamori, T.; Ohkubo,
A.; Tsunoda, H.; Seio, K.; Sekine, M., Org. Biomol. Chem.
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16. Aartsma-Rus, A.; van Vliet, L.; Hirschi, M.; Janson, A. A.;
Heemskerk, H.; de Winter, C. L.; de Kimpe, S.; van Deutekom,
J. C.; t Hoen, P. A.; van Ommen, G. J., Mol. Ther. 2009, 17,
548.
17. Popplewell, L. J.; Trollet, C.; Dickson, G.; Graham, I. R., Mol.
Ther. 2009, 17, 554.
18. Echigoya, Y.; Mouly, V.; Garcia, L.; Yokota, T.; Duddy, W.,
PLoS One 2015, 10, e0120058.
19. Masaki, Y.; Inde, T.; Nagata, T.; Tanihata, J.; Kanamori, T.;
Seio, K.; Takeda, S. i.; Sekine, M., MedChemComm 2015, 6,
630.
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2003, 68, 9971.
22. Saneyoshi, H.; Seio, K.; Sekine, M., J. Org. Chem. 2005, 70,
10453.
In summary, we successfully synthesized duplex stabilizing
nucleotides
with
an
MCE
modification,
2´-O-(N-
methylcarbamoylethyl)-5-methyl-2-thiouridine
(s2TMCE).
Selective 2-thiocarbonylation was achieved with the proper
precursor, which had a protection group at the 4-position and ester
group at the 2´-modification to avoid undesired thiocarbonylation.
The synthesized s2TMCE phosphoramidite derivative could be
incorporated into the oligonucleotide following standard methods.
MCE modification and s2TMCE residues in SSOs effectively
induced the exon skipping effect. Combining duplex stabilizing
modifications with the inhibitory modification of splicing factors
will be a useful strategy for the development of SSOs.
Acknowledgments
23. Arechavala-Gomeza, V.; Graham, I. R.; Popplewell, L. J.;
Adams, A. M.; Aartsma-Rus, A.; Kinali, M.; Morgan, J. E.;
Van Deutekom, J. C.; Wilton, S. D.; Dickson, G.; Muntoni, F.,
Hum. Gene Ther. 2007, 18, 798.
24. Aoki, Y.; Nakamura, A.; Yokota, T.; Saito, T.; Okazawa, H.;
Nagata, T.; Takeda, S., Mol. Ther. 2010, 18, 1995.
25. Kumar, R. K.; Davis, D. R., Nucleic Acids Res. 1997, 25, 1272.
26. Shohda, K.; Okamoto, I.; Wada, T.; Seio, K.; Sekine, M.,
Bioorg. Med. Chem. Lett. 2000, 10, 1795.
This work was supported by JSPS KAKENHI (Grant Numbers:
26810086, 17H04886) and in part by Health Sciences Research
Grants for Research on Psychiatric and Neurological Diseases and
Mental Health from the Ministry of Health, Labor and Welfare of
Japan. The authors thank Mr. Yusuke Iriyama (Nissan Chemical
Corporation) for providing oligonucleotide samples.
27. Rajeev, K. G.; Prakash, T. P.; Manoharan, M., Org. Lett. 2003,
5, 3005.
References and notes
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