Prion Delivery via Peptide-Nucleic Acid Co-assembly: the Inside-out Virus Open Access

Chan, Mandy (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/xs55mc11m?locale=en
Published

Abstract

Protein-nucleic acid interaction drives the chemical evolution of life as well as the pathogenesis of neurodegenerative diseases. In order to better understand this interaction between these two important biopolymers, we aimed to develop an inside-out virus that reverses the normal viral architecture and functions of protein and nucleic acid. Such a virus would deliver information stored in proteins using nucleic acids as a vehicle for cellular uptake. Here we provide the preliminary results of the construction of a fluorescent peptide-DNA co-assembly and its cellular internalization performance. Fluorophore-tagged, prion-like amyloid-β peptide and single-stranded DNA were co-assembled to achieve a PBS-stable, heterogeneous system with nanotubes and fibers. Dilution of the tagged-DNA as an initial attempt to resolve the heterogeneity yielded interesting results as maturing ribbons were replaced by bundled fibers. Flow cytometry on assembled peptide-treated non-small lung cancer cell lines showed that peptide fibers did not associate with cells. Meanwhile, confocal micrographs demonstrated the stability, co-localization of components, and preferential attachment of the co-assemblies to cells.

Table of Contents

 Introduction. 1

Results and Discussion. 4

Fluorophore-tagged peptide/nucleic acid co-assembly. 4

Figure 1. 5

Figure 2. 5

Figure 3. 5

Figure 4.  6

Figure 5. 7

Cellular delivery of Rho-peptide measured by flow cytometry. 8

Figure 6. 9

Figure 7.. 10

Confocal microscopy of the interaction of co-assembly with cellular environment. 10

Figure 8. 11

Figure 9. 13

Conclusion. 14

Materials and Methods. 16

Peptide synthesis. 16

Rhodamine-tagged peptide preparation. 16

Desalting and disaggregation. 17

Transmission electron microcopy (TEM) 18

Fourier Transform Infrared (FT-IR) Spectroscopy. 19

Sonication, and resuspension of assembly in PBS. 19

Cell incubation and flow Cytometry. 19

Cell fixing and confocal microscopy. 20

References. 21

 

 

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