Dissertation

Aptamer Engineering for Enhanced Delivery of Functional Proteins for Cancer Therapy

Pan, Xiaoshu

2020

Overview

Nucleic acid aptamers, also known as “chemical antibodies”, generated from cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX), can be used to recognize specific biomarkers on the cell membrane with high affinity and specificity. Nucleic acid aptamers can be coupled to proteins in order to target those proteins to specific cell types. In comparison to other targeting strategies, aptamers have many advantages such as a small size and low immunogenicity, and they can be easily modified chemically.This dissertation investigated whether conjugating DNA aptamer to a variety of functional proteins could be used to significantly enhanced the targeted delivery of functional proteins in cancer therapy. Firstly, a bispecific circular aptamer (bc-apt) composed of a cell-specific aptamer and an anti-His tag aptamer, showed good binding properties to specific cells as well as to the polyhistidine tag on functional proteins. By noncovalently and stably tethering His-proteins with specific biomarkers on cancer cells, bc-apt was demonstrated to enhance the intracellular delivery of His-EGFP for the cell imaging and His-RNase A for a better therapeutic outcome. However, it was also found that the binding performance of bc-apt was affected by protein size and the surface charge of the proteins. To tackle the unmet need of targeted delivery of the larger proteins such as the gene editing protein Cas9, aptamer-Cas9 bioconjugates were therefore constructed using the amine-reactive homo-bifunctional crosslinker disuccinimidyl suberate (DSS) and then demonstrated to specifically and efficiently bind with and enter the targeted cells. DSS crosslinking reagents were shown to efficiently conjugate Cas9 with DNA aptamers with different DNA to protein ratios, however, the resulting aptamer-Cas9 bioconjugates showed a low level of undesired bioactivity in both cell-free and in vitro conditions compared to wild-type Cas9. In order to prevent the adverse effects of bioconjugation, we proposed that the bifunctional cleavable linker AzMMMan, which was reported to be hydrolyzed in a mild acidic environment like endosomes, is adequate to construct reversible aptamer-Cas9 bioconjugates for the desired targeted gene editing in cancer therapy.Consequently, all results in the dissertation showed that therapeutic proteins were efficiently conjugated with DNA aptamers via chemical crosslinkers or aptamer-His tag interaction, and the resulting DNA-protein complexes in both strategies were promising in enhancing the targeted delivery of various functional proteins for cancer therapy.

• Publisher: ProQuest Dissertations & Theses
• Subject: Chemistry
• ISBN: 9798357544681