RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing
Alternative splicing of tau pre-mRNA is regulated by a 5' splice site (5'ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant spli...
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sg-ntu-dr.10356-935332020-11-01T04:44:32Z RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing Ong, Alan Ann Lerk Tan, Jiazi Bhadra, Malini Dezanet, Clément Patil, Kiran M. Chong, Mei Sian Kierzek, Ryszard Decout, Jean-Luc Roca, Xavier Chen, Gang School of Physical and Mathematical Sciences Interdisciplinary Graduate School (IGS) School of Biological Sciences RNA Structure Strand Invasion Science::Chemistry::Biochemistry Alternative splicing of tau pre-mRNA is regulated by a 5' splice site (5'ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5'ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 30 arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5'ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5' arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5'ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent. MOE (Min. of Education, S’pore) Published version 2019-09-16T08:00:15Z 2019-12-06T18:41:01Z 2019-09-16T08:00:15Z 2019-12-06T18:41:01Z 2019 Journal Article Ong, A. A. L., Tan, J., Bhadra, M., Dezanet, C., Patil, K. M., Chong, M. S., ... Chen, G. (2019). RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing. Molecules, 24(16), 3020-. doi:10.3390/molecules24163020 1420-3049 https://hdl.handle.net/10356/93533 http://hdl.handle.net/10220/49939 10.3390/molecules24163020 en Molecules © 2019 The Authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 14 p. application/pdf |
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RNA Structure Strand Invasion Science::Chemistry::Biochemistry Ong, Alan Ann Lerk Tan, Jiazi Bhadra, Malini Dezanet, Clément Patil, Kiran M. Chong, Mei Sian Kierzek, Ryszard Decout, Jean-Luc Roca, Xavier Chen, Gang RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing |
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Alternative splicing of tau pre-mRNA is regulated by a 5' splice site (5'ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5'ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 30 arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5'ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5' arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5'ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent. |
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School of Physical and Mathematical Sciences |
author_facet |
School of Physical and Mathematical Sciences Ong, Alan Ann Lerk Tan, Jiazi Bhadra, Malini Dezanet, Clément Patil, Kiran M. Chong, Mei Sian Kierzek, Ryszard Decout, Jean-Luc Roca, Xavier Chen, Gang |
format |
Article |
author |
Ong, Alan Ann Lerk Tan, Jiazi Bhadra, Malini Dezanet, Clément Patil, Kiran M. Chong, Mei Sian Kierzek, Ryszard Decout, Jean-Luc Roca, Xavier Chen, Gang |
author_sort |
Ong, Alan Ann Lerk |
title |
RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing |
title_short |
RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing |
title_full |
RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing |
title_fullStr |
RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing |
title_full_unstemmed |
RNA secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mRNA alternative splicing |
title_sort |
rna secondary structure-based design of antisense peptide nucleic acids for modulating disease-associated aberrant tau pre-mrna alternative splicing |
publishDate |
2019 |
url |
https://hdl.handle.net/10356/93533 http://hdl.handle.net/10220/49939 |
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1683493298570788864 |