Developing Modified Peptide Nucleic Acids to Regulate Dysregulated Splicing
Peptide Nucleic Acid (PNA) can stabilise the Tau Exon 10-Intron 10 self-regulatory RNA hairpin, known to regulate the alternative splicing of exon 10 in Microtubule-Associated Protein Tau (MAPT) transcript , and thus rescue the aberrant ratio of protein isoforms (4R/3R), preventing tauopathy. We used antisense PNA (ASPNA) and triplex-forming PNA (TFPNA) methods, and compared the effectiveness of both. We synthesised PNA oligomers using solid phase peptide synthesis (SPPS) and tested binding affinity to the RNA hairpin using polyacrylamide gel electrophoresis (PAGE). TFPNA is less costly and more efficient to synthesise, but has low binding affinity, whereas ASPNA costs more to synthesise but has a higher binding affinity.
Hypothesis—Binding a PNA strand to the Tau Exon 10-Intron 10 self-regulatory hairpin can rescue the aberrant ratio of 3R:4R isoforms and thus cure tauopathy.
KeywordsAntisense strands Alternative splicing Hairpins Hoogsteen base pair Missense mutation Peptide nucleic acid (PNA) Steric hindrance Isoform U1snRNP ASPNA TFPNA
This project would not have been possible without the guidance and help of individuals who have extended their invaluable assistance in the completion of this project:
• Our external mentor, Mr. Alan Ong, for his immense help and advice throughout the course of our research
• Our internal teacher-mentor, Mrs. Elizabeth Foo, for guiding us through our internal school deadlines and keeping us up to date
• Assistant Professor Chen Gang, for regularly sending us informative emails related to our research topic as well as allowing us to use his lab for research
• NTU, RI and MOE-GEB for giving us this unique opportunity to participate in the programme
• Our friends and family who have morally supported us throughout our project.
- 1.Svoboda, S., & Di Cara, A. (2006, April). Hairpin RNA: A Secondary Structure of Primary Importance. Retrieved February 25, 2017 from https://www.ncbi.nlm.nih.gov/pubmed/16568238.
- 2.Koh, W. (n.d.). Peptide Nucleic Acid (PNA) and Its Applications. Yuseong-gu, Daejeon: Panagene Inc.Google Scholar
- 8.Chen, G. (n.d.). RNA Folding and Therapeutics. Retrieved February 25, 2017 from http://www.ntu.edu.sg/home/rnachen/research.htm.
- 10.Fredericks, A. M., Cygan, K. J., Brown, B. A., & Fairbrother, W. G. (2015, June). RNA-Binding Proteins: Splicing Factors and Disease. Retrieved February 25, 2017 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496701/.
- 14.Gaillard, F. (n.d.). Neurofibrillary tangles | Radiology Reference Article. Retrieved August 06, 2017 from https://radiopaedia.org/articles/neurofibrillary-tangles.
- 15.Hoogsteen Base Pairing (Molecular Biology). (n.d.). Retrieved February 25, 2017 from http://what-when-how.com/molecular-biology/hoogsteen-base-pairing-molecular-biology/.
- 17.PNA: Peptide Nucleic Acid as a more stable alternative to DNA and RNA for many applications. (n.d.). Retrieved February 25, 2017 from http://www.crbdiscovery.com/newsletter/august2013.html.
- 18.Poulos, M. G., Batra, R., Charizanis, K., & Swanson, M. S. (2011, January). Developments in RNA Splicing and Disease. Retrieved February 25, 2017 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003463/.
- 19.Sanders, R. (2015, April 6). New Target for Anticancer Drugs: RNA. Berkeley News. Retrieved February 25, 2017 from http://news.berkeley.edu/2015/04/06/new-target-for-anticancer-drugs-rna/.
- 20.Wang, G., & Xu, X. S. (n.d.). Peptide Nucleic Acid (PNA) Binding-Mediated Gene Regulation. https://doi.org/10.1038/sj.cr.729020.
- 21.Warf, M. B., & Berglund, J. A. (2010, March). The Role of RNA Structure in Regulating pre-mRNA Splicing. Retrieved February 25, 2017 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834840/.
- 22.Jubilut, G. N., Cilli, E. M., Tominaga, M., Miranda, A., Okada, Y., & Nakaie, C. R. (2001, September). Evaluation of the Trifluoromethanosulfonic Acid/Trifluoroacetic Acid/Thioanisole Cleavage Procedure for Application in Solid-Phase Peptide Synthesis. Retrieved June 10, 2017 from https://www.ncbi.nlm.nih.gov/pubmed/11558592.