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Synthesis and characterization of covalently-linked dendrimer bioconjugates and the non-covalent self-assembly of streptavidin-based megamers

This work details the attachment of dendrimers to proteins, peptides and single stranded DNA (ssDNA). Dendrimers based on melamine satisfy many of the synthetic demands in the field of bioconjugate chemistry including: monodispersity, synthetic flexibility and scalability. The solution-phase syntheses of both ssDNA-dendrimer and peptide-dendrimer bioconjugates is described, and thorough characterization by matrix-assisted laser desorption ionization/ time-of-flight (MALDI-TOF) mass spectrometry, UV-vis spectroscopy, fluorescence spectroscopy, and polyacrylamide gel electrophoresis is discussed.
Non-covalent DNA-dendrimer complexes have been shown to facilitate antisense gene delivery, but are vulnerable to dissociation and subsequent enzymatic degradation within the cell. In an effort to prepare biocompatible antisense agents capable of effectively shielding ssDNA from intracellular nuclease digestion, disulfide-linked ssDNA-dendrimers were prepared and rigorously characterized to rule out the possibility of an electrostatic-based interaction.
Hybridization assays were performed to determine if the covalently-attached dendrimer affected the ability of the attached ssDNA strand to anneal with a complementary sequence to form double-stranded DNA (dsDNA)-dendrimers. Results indicate that ssDNA-dendrimer conjugates readily anneal to complementary ssDNA strands either in solution or attached to gold surfaces. Nuclease digestions of conjugates in solution suggested that enzymatic manipulation of dsDNA-dendrimers is possible, offering promise for DNA-based computation and other fields of DNA-nanotechnology.
Much larger bioconjugates consisting of dendrimers, proteins and peptides were prepared with the goal of obtaining molecular weights sufficient for enhanced permeability and retention (EPR) in tumors. While the dendrimer provides the advantages of a purely synthetic route for drug delivery, the protein portion of the bioconjugate provides a monodisperse, macromolecular scaffold for the non-covalent self-assembly of the dendrimers. The strategy presented herein is based on the strong interaction between biotin and the 60 kD tetrameric protein streptavidin. Each monomer of streptavidin is capable of binding 1 biotin molecule, thus when biotin functionalized peptide-dendrimers are added to streptavidin they bind to form a cluster of dendrimers, or a megamer.
The biotinylated peptides that link the dendrimers to the streptavidin core provide a way to actively target specific cell types for drug delivery. Megamer formation through the addition of tetrameric streptavidin was successful as indicated by MALDI-TOF, UV-vis titration and gel electrophoresis assays.

Identiferoai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/1319
Date17 February 2005
CreatorsMcLean, Megan Elizabeth
ContributorsSimanek, Eric
PublisherTexas A&M University
Source SetsTexas A and M University
Languageen_US
Detected LanguageEnglish
TypeBook, Thesis, Electronic Dissertation, text
Format4826754 bytes, electronic, application/pdf, born digital

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