TRANSFECTION ACTIVITY ENHANCEMENT OF A GENE ENCODING DNA ORIGAMI-LIKE NANOSTRUCTURE
Author
Tabatabai, Seyed Meghdad
Term
4. semester
Education
Publication year
2023
Submitted on
2023-08-07
Pages
87
Abstract
DNA-origami giver mulighed for præcist programmerbare nanostrukturer, som kan komprimere genetisk materiale og dermed fungere som platform for genlevering. Dette speciale undersøger, hvordan transfektionsaktiviteten af en tidligere designet genkodende DNA-origami-lignende nanostruktur kan øges ved hjælp af ikke-virale, positivt ladede bærere. Arbejdet omfatter fremstilling af plasmid- og skabelon-DNA (dobbelt- og enkeltstrenget via PCR og asymmetrisk PCR), foldning og oprensning af nanostrukturen, samt dekoration med lavmolekylær polyethylenimin (PEI), poly-L-lysin (PLL) og TAT, en cellepenetrerende peptidsekvens syntetiseret ved Fmoc-fastfasepeptidsyntese. PEI blev desuden delvist hydrofobt modifieret med fedtsyrer for at fremme celleoptagelse. Bærernes egenskaber og DNA-kompleksering blev karakteriseret (agarosegelelektroforese, HPLC, massespektrometri, gel-retardation og ninhydrinassay), og effekt og toksicitet blev vurderet in vitro i HeLa-celler (bl.a. med Alamar Blue og transfektionsforsøg). Resultaterne viste beskeden transfektionsaktivitet for plasmid-DNA og for en dobbeltstrenget skabelon med de afprøvede bærere, mens der ikke sås signifikant aktivitet med den enkeltstrengede skabelon eller den foldede nanostruktur. Fundene peger på begrænsninger i transkription fra enkeltstrenget DNA og understreger behovet for at designe promotorsekvenser, der er bedre egnet til ssDNA-transkription og efterfølgende translation.
DNA origami enables precisely programmable nanoscale architectures that can compact genetic material and serve as a platform for gene delivery. This thesis investigates how to enhance the transfection activity of a previously designed gene-encoding DNA origami-like nanostructure using non-viral, positively charged carriers. The work includes preparation of plasmid and scaffold DNA (double- and single-stranded via PCR and asymmetric PCR), folding and purification of the nanostructure, and decoration with low–molecular weight polyethyleneimine (PEI), poly-L-lysine (PLL), and TAT, a cell-penetrating peptide synthesized by Fmoc solid-phase peptide synthesis. PEI was further hydrophobically modified with fatty acids to promote cellular uptake. Carrier properties and DNA complexation were characterized (agarose gel electrophoresis, HPLC, mass spectrometry, gel retardation, and ninhydrin assays), and efficacy and toxicity were assessed in vitro in HeLa cells (including Alamar Blue viability and transfection assays). The results showed modest transfection activity with the selected carriers for plasmid DNA and a double-stranded scaffold, while no significant activity was observed for the single-stranded scaffold or the folded nanostructure. These findings highlight limitations in transcription from single-stranded DNA and point to the need for promoter designs better suited to ssDNA transcription and subsequent translation.
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