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Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function: Reconnaissance for designing functional conjugates
Stockholm University, Faculty of Science, Department of Neurochemistry.
2008 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that one protein isoform is disease-related while another isoform is desirable. Splice switching opens the door to new drug targets, and antisense oligonucleotides (asONs), designed to switch splicing, are effective drug candidates. Cellular uptake of oligonucleotides(ONs) is poor, therefore utilization of cell-penetrating peptides (CPPs), well recognized for intracellular cargo delivery, is a promising approach to overcome this essential issue. Most CPPs are internalized by endocytosis, although the mechanisms involved remain controversial.

Here, evaluation of CPP-mediated ON delivery over cellular membranes has been performed. A protocol that allows for convenient assessment of CPP-mediated cellular uptake and characterization of corresponding internalization routes is established. The protocol is based on both fluorometric uptake measurements and a functional splice-switching assay, which in itself is based on biological activity of conveyed ONs. Additionally, splice switching ONs (SSOs) have been optimized for high efficiency and specificity. Data suggest that SSO activity is improved for chimeric phosphorothioate SSOs containing locked nucleic acid (LNA) monomers. It is striking that the LNA monomers in such chimeric constructs give rise to low mismatch discrimination of target pre-mRNA, which highlight the necessity to optimize sequences to minimize risk for off-target effects.

The results are important for up-coming work aimed at developing compounds consisting of peptides and novel synthetic nucleic acids, making these entities winning allies in the competition to develop therapeutics regulating protein expression patterns.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi , 2008. , 45 p.
Keyword [en]
Cell-penetrating peptide, nucleic acids, alternative splicing, mismatch discrimination
National Category
URN: urn:nbn:se:su:diva-7491ISBN: 978-91-7155-580-9OAI: diva2:198406
Available from: 2008-04-14 Created: 2008-04-14 Last updated: 2015-04-21Bibliographically approved
List of papers
1. Assessing the delivery efficacy and internalization route of cell-penetrating peptides
Open this publication in new window or tab >>Assessing the delivery efficacy and internalization route of cell-penetrating peptides
2007 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 2, no 8, 2043-2047 p.Article in journal (Refereed) Published
Abstract [en]

Developing efficient delivery vectors for bioactive molecules is of great importance within both traditional and novel drug development, such as oligonucleotide (ON)-based therapeutics. To address delivery efficiency using cell-penetrating peptides (CPPs), we here present a protocol based on splice correction utilizing both neutral and anionic antisense ONs, either covalently conjugated via a disulfide bridge or non-covalently complexed, respectively, that generates positive readout in the form of luciferase expression. The decisive advantage of using splice correction for evaluation of CPPs is that the ON induces a biological response in contrast to traditionally used methods, for example, fluorescently labeled peptides. An emerging number of studies emphasize the role of endocytosis in translocation of CPPs, and this protocol is also utilized to determine the relative contribution of different endocytic pathways in the uptake of CPPs, which provides valuable information for future design of novel, more potent CPPs for bioactive cargoes.

National Category
urn:nbn:se:su:diva-20686 (URN)10.1038/nprot.2007.302 (DOI)000253139400024 ()17703217 (PubMedID)
Available from: 2007-11-28 Created: 2007-11-28 Last updated: 2015-04-21Bibliographically approved
2. Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers
Open this publication in new window or tab >>Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers
Show others...
2008 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 412, 307-313 p.Article in journal (Refereed) Published
Abstract [en]

The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced beta-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2'-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.

locked nucleic acid (LNA), mismatch, RNA, splice-switching activity, splice-switching oligonucleotide
National Category
urn:nbn:se:su:diva-13896 (URN)10.1042/BJ20080013 (DOI)000256491900012 ()
Available from: 2008-05-16 Created: 2008-05-16 Last updated: 2015-04-21Bibliographically approved

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