Novel Biomolecular Carriers for Gene Delivery in Mammalian Cells. Investigation of DNA origami as a Potential Gene Delivery System
Author
Larsen, Anne-Kathrine Kure
Term
4. term
Education
Publication year
2020
Submitted on
2020-06-03
Pages
107
Abstract
Dette projekt undersøgte DNA-origami—at folde DNA-strenge til bestemte former—som en mulig ny metode til at levere gener og få dem til at udtrykkes i pattedyrsceller. Vi brugte et plasmid med Cas9 (et genredigeringsenzym) og EGFP (et grønt fluorescerende mærkeprotein) som udgangspunkt for at skabe et DNA-origami-skelet. For at forberede de nødvendige DNA-stykker forsøgte vi at amplificere en region i pCas9_GFP-plasmidet med PCR, en standardmetode til at kopiere DNA. Skabelonen modstod dog fuld denaturering, sandsynligvis fordi kyllinge β-aktin-promotoren og et kimært intron er GC-rige (høj andel af G og C), og fordi DNA’et kan danne en hårnålsstruktur, hvilket tilsammen hæmmede fuldlængde-amplifikation. Vi skiftede derfor til lentiCas9-EGFP-plasmidet og lykkedes med at fremstille den nødvendige dobbeltstrengede målregion. Vi modellerede to mulige DNA-origami-designs, og simuleringer indikerede, at begge er stabile under fysiologiske betingelser (salt og temperatur som i kroppen). For at overvåge foldning udførte vi indledende FRET-målinger (Förster-resonansenergioverførsel) med annealerede oligonukleotid-konstruktioner (korte, parrede DNA-strenge) og et model-origami. Vi så et FRET-signal for de annealerede oligos, men der kræves flere forsøg for klart at konkludere noget om denaturerede oligos og egentlige DNA-origami-strukturer.
This project examined DNA origami—folding DNA strands into designed shapes—as a possible new way to deliver genes and drive their expression in mammalian cells. We used a plasmid carrying Cas9 (a gene-editing enzyme) and EGFP (a green fluorescent marker) as the basis for a DNA origami scaffold. To prepare the needed DNA pieces, we tried to amplify a region of the pCas9_GFP plasmid by PCR, a standard method for copying DNA. However, the template resisted full denaturation, likely because the chicken β-actin promoter and a chimeric intron are GC-rich (high in G and C bases), and because the DNA can form a hairpin structure, both of which hindered full-length amplification. We therefore switched to the lentiCas9-EGFP plasmid and successfully produced the required double-stranded target region. We designed two candidate DNA origami structures, and simulations suggested that both are stable under physiological conditions (salt and temperature similar to the body). To monitor folding, we carried out initial FRET measurements (Förster resonance energy transfer) using annealed oligonucleotide constructs (short paired DNA strands) and a model origami. We detected a FRET signal for the annealed oligos, but further experiments are needed to draw clear conclusions for denatured oligos and full DNA origami structures.
[This abstract was generated with the help of AI]
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