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RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing.
Molecules 2019; 24(16)M

Abstract

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 3' 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.

Authors+Show Affiliations

NTU Institute for Health Technologies (HeathTech NTU), Interdisciplinary Graduate School, Nanyang Technological University, 50 Nanyang Drive, Singapore 637553, Singapore. Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore.Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore.School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.University Grenoble Alpes/CNRS, Département de Pharmacochimie Moléculaire, ICMG FR 2607, UMR 5063, 470 Rue de la Chimie, F-38041 Grenoble, France.Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore.Geriatic Education & Research Institute, 2 Yishun Central 2, Singapore 768024, Singapore.Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.University Grenoble Alpes/CNRS, Département de Pharmacochimie Moléculaire, ICMG FR 2607, UMR 5063, 470 Rue de la Chimie, F-38041 Grenoble, France.School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore. RNACHEN@ntu.edu.sg.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31434312

Citation

Ong, Alan Ann Lerk, et al. "RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing." Molecules (Basel, Switzerland), vol. 24, no. 16, 2019.
Ong AAL, Tan J, Bhadra M, et al. RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing. Molecules. 2019;24(16).
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 (Basel, Switzerland), 24(16), doi:10.3390/molecules24163020.
Ong AAL, et al. RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing. Molecules. 2019 Aug 20;24(16) PubMed PMID: 31434312.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing. AU - Ong,Alan Ann Lerk, AU - Tan,Jiazi, AU - Bhadra,Malini, AU - Dezanet,Clément, AU - Patil,Kiran M, AU - Chong,Mei Sian, AU - Kierzek,Ryszard, AU - Decout,Jean-Luc, AU - Roca,Xavier, AU - Chen,Gang, Y1 - 2019/08/20/ PY - 2019/07/09/received PY - 2019/08/14/revised PY - 2019/08/19/accepted PY - 2019/8/23/entrez PY - 2019/8/23/pubmed PY - 2019/8/23/medline KW - PNA KW - RNA structure KW - antisense KW - exon inclusion KW - exon skipping KW - strand invasion JF - Molecules (Basel, Switzerland) JO - Molecules VL - 24 IS - 16 N2 - 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 3' 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. SN - 1420-3049 UR - https://www.unboundmedicine.com/medline/citation/31434312/RNA_Secondary_Structure-Based_Design_of_Antisense_Peptide_Nucleic_Acids_for_Modulating_Disease-Associated_Aberrant_Tau_Pre-mRNA_Alternative_Splicing L2 - http://www.mdpi.com/resolver?pii=molecules24163020 DB - PRIME DP - Unbound Medicine ER -