2019

Molecules

Rocznik: 2019 | Tom: 24 | Numer: 16

artykuł naukowy

angielski

EN 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.

3020

10.3390/molecules24163020

https://www.mdpi.com/1420-3049/24/16/3020/pdf

CC BY (uznanie autorstwa)

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08.2019

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