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Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Vise andre og tillknytning
2009 (engelsk)Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, nr 12, s. 2509-2517Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.

sted, utgiver, år, opplag, sider
2009. Vol. 1788, nr 12, s. 2509-2517
Emneord [en]
cell-penetrating peptide, oligonucleotide delivery, pyrenebutyrate, cellular translocation, locked nucleic acid, splice switching
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi; biofysik
Identifikatorer
URN: urn:nbn:se:su:diva-31144DOI: 10.1016/j.bbamem.2009.09.014ISI: 000272581500005OAI: oai:DiVA.org:su-31144DiVA, id: diva2:275743
Prosjekter
Multidisciplinary BIOVINNOVA-SAMBIOTilgjengelig fra: 2009-11-06 Laget: 2009-11-05 Sist oppdatert: 2017-12-12bibliografisk kontrollert
Inngår i avhandling
1. Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
Åpne denne publikasjonen i ny fane eller vindu >>Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
2009 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Atypical gene expression has a major influence on the disease profile of several severe human disorders. Oligonucleotide (ON) based therapeutics has opened an avenue for compensating deviant protein expression by acting on biologically important nucleic acids, mainly RNAs. Antisense ONs (asONs) can be designed to target complementary specific RNA sequences and thereby to influence the corresponding protein synthesis. However, cellular uptake of ONs is poor and is, together with the target specificity of the asONs, the major limiting factor for the development of ON based therapeutics.

In this thesis, the mechanisms of well-characterized cell-penetrating peptides (CPPs) are evaluated and CPPs are adapted for cellular ON-delivery. The functionality of ON derivatives in cells is investigated and by optimization of asONs, targeting pre-messenger RNA, high efficiency and specificity is achieved. The optimization of the asONs is based on sequence design and through the choice of nucleic acid analogue composition. It is concluded that asONs, partly composed of locked nucleic acids are attractive for splice-switching applications but these mixmers must be designed with limited number of locked nucleic acid monomers to avoid risk for off-target activity. A protocol allowing for convenient characterization of internalization routes for CPPs is established and utilized. A mechanistic study on cellular CPP uptake and translocation of associated ON cargo reveals the importance of the optimal combination of for example charge and hydrophobicity of CPPs for efficient cellular uptake. Formation of non-covalent CPP:ON complexes and successful cellular delivery is achieved with a stearylated version of the well-recognized CPP, transportan 10.

The results illustrate that CPPs and ON derivatives have the potential to become winning allies in the competition to develop therapeutics regulating specific protein expression patterns involved in the disease profile of severe human disorders.

sted, utgiver, år, opplag, sider
Stockholm: Department of Neurochemistry, Stockholm University, 2009. s. 64
Emneord
cell-penetrating peptide, splice-switching oligonucleotide, oligonucleotide derivative
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-31226 (URN)978-91-7155-950-0 (ISBN)
Disputas
2009-12-22, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Prosjekter
VINNOVA-SAMBIO Multidisciplinary BIO
Merknad
At the time of doctoral defense, the following papers were unpublished and had s status as follows: Paper 4: Accepted. Peper 5: In press.Tilgjengelig fra: 2009-11-30 Laget: 2009-11-08 Sist oppdatert: 2018-01-12bibliografisk kontrollert
2. Biophysical studies of peptides with functions in biotechnology and biology
Åpne denne publikasjonen i ny fane eller vindu >>Biophysical studies of peptides with functions in biotechnology and biology
2012 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles).

The resorufin-based arsenical hairpin binder (ReAsH) bound to a short peptide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to type II β-turn was determined by NMR structure calculations (Paper I).

Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV.

The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency.

Endosomal escape is a crucial challenge for the CPP applications. We modeled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs.

Dynorphin A (Dyn A) peptide mutations are associated with neurodegenerative disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2012. s. 75
Emneord
Genetic fluorescence label, Biarsenical tetracysteine motif, Cell-penetrating peptides, Large unilamellar vesicles, Pyrenebutyrate, Endosomal escape, Membrane perturbation, Bacteriorhodopsin, Dynorphin
HSV kategori
Forskningsprogram
biofysik
Identifikatorer
urn:nbn:se:su:diva-66948 (URN)978-91-7447-417-6 (ISBN)
Disputas
2012-02-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Tilgjengelig fra: 2012-01-23 Laget: 2011-12-22 Sist oppdatert: 2013-04-09bibliografisk kontrollert

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