Date of Award

Summer 8-11-2016

Degree Type

Dissertation

Degree Name

PhD. Chemistry

Department

Chemistry and Biochemistry

Advisor

David Sabatino, Ph.D.

Committee Member

Cecilia Marzabadi, Ph.D.

Committee Member

Wyatt Murphy, Ph.D.

Keywords

siRNA Nanostructures, RNA Nanotechnology, Chaperones, Glucose Regulated Protiens

Abstract

Over the past two decades, advances in RNA structural biology have improved our understanding of the structures and folding properties of naturally occurring RNAs. RNA sequences and structures participate in many specific biological functions, such as those performed by messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), micro RNA (miRNA), short-interfering RNA (siRNA), small nuclear RNA (snRNA) and many others. The noncoding RNAs, such as siRNA, do not express proteins but have been utilized in a wide range of applications, including RNA interference (RNAi) and the regulation of mRNA expression. These important biological functions have been implemented in gene therapy and for screening malignant gene targets. In spite of their therapeutic potential, naturally occurring siRNAs are limited by poor pharmacological properties which has hindered their translation into the clinic. However, recent studies have highlighted the fruitful applications of modified siRNAs, including the use of siRNA nanostructures in cancer detection and treatment. In this thesis, the prerequisite conditions for forming stable RNA hybrid assemblies are described in Chapter 2. These conditions are critically important for the generation of stable higher-order RNA nanostructures. Inspired by the widespread biological function of self-assembled RNA hybrids, linear RNA templates and two complementary strands were self-assembled in order the determine the requirements for efficient RNA hybridization into stable three-component systems (3CS). In this study the RNA sequence composition and length were found to impact hybridization and self-assembly. Moreover, buffer conditions were also evaluated in order to explore the influence of ionic strength and metal cation composition on stable RNA hybridization. The complementary RNAs were annealed in buffer and analyzed by native PAGE, thermal denaturation and CD spectroscopy. The data supported the stable 3CS self-assembly on a thirty nucleotide (30nt) RNA template and with complementary 15nt and 23 nt RNA sequences in Tris buffer. These conditions were shown to favor the self-assembly of higher-order RNA structures, such as the siRNA nanostructures in Chapter 3.

The genetically encoded, self-assembled siRNA nanostructures targeting the Glucose Regulated Proteins (GRP) were developed for applications in siRNA screening of these important oncologic targets and for potentiating cancer gene therapy. In our RNAi nanotechnology approach, linear, V- and Y-shape RNA templates were synthesized by semi-automated solid phase RNA synthesis with the use of a ribouridine branchpoint synthon which was used generate the V- and Y-shape RNA templates. The RNA templates were then hybridized in Tris buffer with their complementary strands, in stoichiometric ratios which favored hybridization and self-assembly into genetically encoded spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes. The siRNA self-assembly was confirmed by native PAGE while TEM imaging validated the sizes and shapes of the siRNA nanostructures. Moreover, thermal denaturation and CD spectroscopy were used to ascertain the prerequisite siRNA hybrids for their RNAi applications. In a 24 sample siRNA screen conducted within the AN3CA endometrial cancer cells known to overexpress tumorigenic GRP78 activity, the self-assembled siRNAs targeting multiple sites of GRP78 mRNA demonstrated more potent and long-lasting anticancer activity relative to their linear controls. Extending the scope of our RNAi screening approach, the self-assembled siRNA hybrids (5 nM) that targeted GRP-75, 78 and 95 were tested within endometrial (AN3CA), cervical (HeLa) and breast (MDA-MB-231) cancer cell lines with respect to the control non-cancerous lung (MRC5) cell line. The results indicated that the non-cancerous MRC5 lung cell line which displayed normal glucose regulated chaperone levels was found to tolerate siRNA treatment and demonstrated less toxicity relative to the cancer cells that were found to be addicted to glucose regulated chaperome. Therefore, the GRP targeting siRNAs were found to elicit more potent anti-cancer activity due to an overexpression and strong dependence of GRP activity in cancer. The serum stability of the self-assembled siRNAs was also investigated relative to the linear siRNA control. The data analyzed on a denaturing PAGE indicated a quick (< 4 h) degradation profile of the linear siRNA hybrid while the siRNA nanostructures were disassembled into their native RNA templates with no further degradation observed over the course of a 48 h fetal bovine serum (FBS) treatment. Thus, the RNA templates have been proposed to contribute to the prolonged (72 h) RNAi effect observed within the cancer cells. In sum, these remarkable self-assembled siRNA nanostructures may thus encompass a new class of potent siRNAs that may be useful in screening important oncogene targets while improving siRNA therapeutic efficacy and specificity in cancer.

 
 

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