All ETDs from UAB

Advisory Committee Chair

Eugenia Kharlampieva

Advisory Committee Members

Maaike Everts

David E Graves

Aaron L Lucius

David A Schneider

Document Type

Dissertation

Date of Award

2017

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

Nucleic acids are known to play numerous important roles in the cell. Recently, interest in the delivery of DNA to treat disease, also known as gene therapy, has increased with new non-viral encapsulation strategies becoming available. Unfortunately, challenges with sufficient delivery of the DNA still plague these systems causing the need for improved DNA delivery vehicles. To overcome DNA delivery barriers, two-component poly(methacrylic acid) (PMAA)/ poly(N-vinylpyrrolidone) (PVPON) microcapsules were developed using Layer-by-Layer (LbL) technology. G-quadruplex forming deoxyoligonucleotides of the human telomeric sequence were used to model the delivery of the thrombin binding aptamer (TBA), a DNA therapeutic that folds into a G-quadruplex structure. Because the structure of the nucleic acid therapeutic dictates their mechanism of action, the structural stability of G-quadruplex sequences of the human telomere were explored, first, before these structures were encapsulated within PMAA/PVPON microcapsules. Release of the DNA from the microcapsules was achieved using a controlled therapeutic ultrasound technique. A summary of these exper-iments is described below. In Chapter 3, a library of G-quadruplex forming deoxyoligonucleotides was built by mutating either one, two, three, or four adenines at a time to a thymine in the loop se-quence regions of the structure. The unfolding properties and energetics associated with each sequence were examined using differential scanning calorimetry (DSC) and circular dichroism spectropolarimetry (CD). The melting profiles for the wild-type human telo-meric G-quadruplex sequence were biphasic in nature, indicating the presence of an un-folding intermediate. As the number of modified loops increased, the melting profiles were fit by a single monophasic curve. DSC and circular dichroism (CD) spectra were collected to determine how the mutations influenced the stability and secondary struc-tures of these G-quadruplex forming deoxyoligonucleotides. It was found that all se-quences could fold into G-quadruplex structures and varied by the magnitude of their peaks at 245 and 295 nm. CD spectra were also collected as a function of temperature and then analyzed by singular value decomposition to further probe the biphasic melting profiles observed by DSC. Results from deconvolution of CD data are consistent with the presence of intermediates during the unfolding for the wild-type G-quadruplex as well as the loop modified G-quadruplexes. In Chapter 4, the encapsulation and release of G-quadruplex forming deoxyoligo-nucleotides of the human telomeric sequence by two-component PMAA/PVPON multi-layer hydrogel capsules is described. LbL deposition was used to coat PMAA and PVPON layers onto CaCO3 inorganic microparticles that were co-precipitated with the G-quadruplex structures. The PMAA molecules within the multilayer were crosslinked with cystamine and dissolution of the CaCO3 cores with ethylenediaminetetraacetic acid (EDTA) yielded hollow microcapsules loaded with G-quadruplex forming deoxyoligonu-cleotides. The microcapsules were characterized using a variety of techniques including FTIR, Zeta-potential measurements as a function of pH, and scanning electron microsco-py. These studies demonstrate that these capsules are highly efficient at releasing their DNA cargo, unharmed, by treatment with ultrasound to mechanically burst the capsules.

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