Cell Penetrating Peptides Mediated DNA-Free Transfection in Arabidopsis and Chinese Cabbage

Abstract Background: The third-generation genome editing system CRISPR/Cas had shown strong application prospects in crop genetic improvement. However, this technology largely depends on genetic transformation. Public concerns on GMO (genetically modified organisms) safety, as well as related regulations, have restricted the application. Therefore, establishing DNA-free transfection system is important to promote CRISPR/Cas genome editing in agriculture.Results：In this paper, cell penetrating peptides fusion protein (CPP-mCherry) was found to be effective on DNA-free transfection. DNA sequences of nine tandem arginine (R9), one cysteine (cys), reporter mCherry and histidine label were sequentially constructed into pET 45B+ expression vector and transformed into Escherichia coli BL21(DE3) strain. CPP-mCherry fusion protein can be induced by 1mM IPTG for at least 1 hour in 28 °C. CPP-mCherry fusion protein can be obtained by 200W ultrasonication, then purified by Ni column and MWCO dialysis. The Arabidopsis thaliana root tips and leaves, as well as Chinese cabbage microspores and 3-week-old microspore embryo can be used as transfected recipient. Concentration can be selected between 10-100μg/ml and incubated overnight at room temperature. R9-cys-mCherry protein can be translocated into the nucleus of microspore. The transfection efficiency of root tips reached 100% and of microspore and MDE was 8.13% and 94.79%, respectively. Conclusions: Here, a CPP mediated DNA-free transfection system was built in dicots. These results lay a technical foundation of DNA-free genome editing.

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Designed filamentous cell penetrating peptides: probing supramolecular structure-dependent membrane activity and transfection efficiency

Chemical Communications ◽

10.1039/c5cc02699g ◽

2015 ◽

Vol 51 (59) ◽

pp. 11757-11760 ◽

Cited By ~ 17

Author(s):

Dawei Xu ◽

Derek Dustin ◽

Linhai Jiang ◽

Damien S. K. Samways ◽

He Dong

Keyword(s):

Supramolecular Structure ◽

Transfection Efficiency ◽

Supramolecular Assembly ◽

Cell Penetrating Peptides ◽

Cationic Peptides ◽

Single Chain ◽

Membrane Activity ◽

Highly Efficient ◽

Cell Penetrating ◽

Filamentous Cell

In this work, we will demonstrate the supramolecular assembly of single-chain cationic peptides into stable macromolecular filamentous nanostructures and investigate their supramolecular structure-dependent membrane activity for the development of highly efficient therapeutic carriers.

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Synergistic effects of conjugating cell penetrating peptides and thiomers on non-viral transfection efficiency

Biomaterials ◽

10.1016/j.biomaterials.2011.11.046 ◽

2012 ◽

Vol 33 (7) ◽

pp. 2321-2326 ◽

Cited By ~ 29

Author(s):

Deni Rahmat ◽

Mohammad I. Khan ◽

Gul Shahnaz ◽

Duangkamon Sakloetsakun ◽

Glen Perera ◽

...

Keyword(s):

Transfection Efficiency ◽

Synergistic Effects ◽

Cell Penetrating Peptides ◽

Cell Penetrating ◽

Viral Transfection

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Reversible activation of pH-sensitive cell penetrating peptides attached to gold surfaces

Chemical Communications ◽

10.1039/c4cc07278b ◽

2015 ◽

Vol 51 (2) ◽

pp. 273-275 ◽

Cited By ~ 13

Author(s):

Joe E. Baio ◽

Denise Schach ◽

Adrian V. Fuchs ◽

Lars Schmüser ◽

Nils Billecke ◽

...

Keyword(s):

Fusion Protein ◽

Cell Penetrating Peptides ◽

Ph Sensitive ◽

Sensitive Cell ◽

Gold Surfaces ◽

Transition Mechanism ◽

Coil Transition ◽

Cell Penetrating ◽

Covalently Bound

Here, we demonstrate that a fusion protein mimic, designed to achieve a reversible, pH-driven helix-coil transition mechanism, retain its functionality when covalently bound to a surface.

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Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2012.01.041 ◽

2012 ◽

Vol 418 (3) ◽

pp. 469-474 ◽

Cited By ~ 34

Author(s):

Helene L. Åmand ◽

Bengt Nordén ◽

Kristina Fant

Keyword(s):

Transfection Efficiency ◽

Cell Penetrating Peptides ◽

Dimer Formation ◽

Cell Penetrating

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Cell-Penetrating Peptides and DNA-Free Genome Editing in Plants

International Journal of Plant Animal and Environmental Sciences ◽

10.26502/ijpaes.202116 ◽

2021 ◽

Vol 11 (03) ◽

Author(s):

Han Wu ◽

Zhipeng Zhang ◽

Fulai Ke ◽

Pengxiang Jia ◽

Kai Zhu ◽

...

Keyword(s):

Genome Editing ◽

Cell Penetrating Peptides ◽

Cell Penetrating

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Internalisation and Biological Activity of Nucleic Acids Delivering Cell-Penetrating Peptide Nanoparticles Is Controlled by the Biomolecular Corona

Pharmaceuticals ◽

10.3390/ph14070667 ◽

2021 ◽

Vol 14 (7) ◽

pp. 667

Author(s):

Annely Lorents ◽

Maria Maloverjan ◽

Kärt Padari ◽

Margus Pooga

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Transfection Efficiency ◽

Protein Corona ◽

Blood Stream ◽

Cell Penetrating Peptides ◽

Biological Efficiency ◽

Cell Penetrating

Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is the formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g., blood stream. In this study, we compared protein corona of CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g., by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be considered while developing delivery systems for nucleic acids.

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Internalisation and biological activity of nucleic acids delivering cell-penetrating peptide nanoparticles is controlled by the biomolecular corona

10.1101/2021.03.26.437157 ◽

2021 ◽

Author(s):

Annely Lorents ◽

Maria Maloverjan ◽

Kärt Padari ◽

Margus Pooga

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Transfection Efficiency ◽

Protein Corona ◽

Blood Stream ◽

Cell Penetrating Peptides ◽

Biological Efficiency ◽

Cell Penetrating

Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g. blood stream. In this study, we compared CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles, and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g. by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be taken into account while developing delivery systems for nucleic acids.

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Divalent metal ions boost effect of nucleic acids delivered by cell-penetrating peptides

10.1101/2021.12.09.471911 ◽

2021 ◽

Author(s):

Maria Maloverjan ◽

Kart Padari ◽

Aare Abroi ◽

Ana Rebane ◽

Margus Pooga

Keyword(s):

Nucleic Acids ◽

Therapeutic Potential ◽

Transfection Efficiency ◽

Cell Penetrating Peptides ◽

In Vivo Studies ◽

Divalent Metal Ions ◽

Reporter Protein ◽

Cell Penetrating ◽

Novel Strategy

Cell-penetrating peptides (CPPs) are promising tools for transfection of various substances, including nucleic acids, into cells. The aim of current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed of CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed an effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed with addition of the ions, without increasing cytotoxicity. We propose a novel strategy of preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, in the aim to increase CPP-mediated transfection efficiency and likelihood of using CPPs in clinics.

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