Compaction of DNA into an Origami-like Structure for Efficient Gene Delivery
Author
Giarentis, Konstantinos
Term
4. term
Education
Publication year
2022
Submitted on
2022-06-02
Pages
75
Abstract
This master's thesis explored building a DNA origami-like structure—folded DNA designed to take a defined shape—that could be delivered into living mammalian cells to make them produce a fluorescent protein. An initial attempt used the plasmid pHAGE-EF1αL-eGFP-W, but poor sequencing quality made it unsuitable. A new design was then created using the TagRFP gene from the pTagRFP-C vector as a template, with the help of caDNAno2 and the vHelix plugin for Maya. The structure’s stability was evaluated by computer simulation (oxDNA2). A long scaffold DNA strand was produced by asymmetric PCR, and an attempt was made to produce ten single-stranded DNA fragments required for folding. Assembly (annealing) was assessed by agarose gel electrophoresis (AGE) and atomic force microscopy (AFM). Finally, different DNA origami annealing protocols were tested for their transfection efficiency in HeLa cell cultures, alongside a pTagRFP-C plasmid without a delivery carrier and the same plasmid mixed with poly-L-lysine.
Denne kandidatafhandling undersøgte at bygge en DNA-origami-lignende struktur—foldet DNA designet til at få en bestemt form—som kunne bringes ind i levende pattedyrsceller og få dem til at producere et fluorescerende protein. Et første forsøg brugte plasmidet pHAGE-EF1αL-eGFP-W, men dårlig sekventeringskvalitet gjorde det uegnet. Derefter blev der udviklet et nyt design med TagRFP-genet fra pTagRFP-C-vektoren som skabelon ved hjælp af caDNAno2 og vHelix-pluginet til Maya. Strukturens stabilitet blev vurderet med computersimulering (oxDNA2). En lang stillads-DNA-streng blev fremstillet med asymmetrisk PCR, og der blev forsøgt at producere ti enkeltstrengede DNA-fragmenter, som var nødvendige for foldningen. Samlingen (annealing) blev vurderet med agarosegelelektroforese (AGE) og atomkraftmikroskopi (AFM). Til sidst blev forskellige annealeringsprotokoller for DNA-origamien testet for deres transfektionseffektivitet i HeLa-cellekulturer, sammen med et pTagRFP-C-plasmid uden leveringsreagens og det samme plasmid blandet med poly-L-lysin.
[This apstract has been rewritten with the help of AI based on the project's original abstract]
Keywords
DNA Origami ; pTagRFP-C ; Gene Delivery ; Transfection ; HeLa Cells ; aPCR ; Asymmetric PCR ; PCR ; AGE ; Poly-L-Lysine ; Cholesterol ; oxDNA ; caDNAno ; Maya ; vHelix ; AFM