Date of Degree

6-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Prabodhika Mallikaratchy

Committee Members

Mark Pezzano

Kevin H. Gardner

Jimmie E. Fata

Daniel Heller

Subject Categories

Biochemistry | Bioinformatics | Cancer Biology

Keywords

Aptamers, cell-SELEX, CD3 Receptor

Abstract

Oligonucleotide aptamers (nucleic acid-based affinity reagents) are an emerging class of synthetic molecules that display high affinity and specificity towards their targets. Aptamer molecules for a target of interest are obtained using a combinatorial chemistry-based method termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX is an in vitro selection process in which a random oligonucleotide library is subjected to repeated cycles of target incubation, separation, and amplification until target-specific evolved sequences become prevalent in the library. Typically, SELEX is used against target molecules such as small molecules and proteins, in their purified state. However, aptamers selected against purified cell surface proteins show limited scope, due to aptamer’s inability to recognize the cell surface protein expressed in its natural environment. To address this issue, a modified SELEX method, named cell-SELEX, was developed, which uses the whole live cells as targets. Cell-SELEX enabled the identification of aptamers against cell surface proteins in their native environment without the need for target purification. A major limitation of cell-SELEX, however, is that the targeting epitope on the cell surface cannot be predefined. In this dissertation, we developed a novel variant of the SELEX method named ligand-guided selection (LIGS) to successfully identify specific aptamers against predetermined cell surface proteins in their native, functional state. This method is designed to uniquely exploit the selection step, which is the core of the SELEX process.

In chapter 2, the LIGS method is introduced as a new biochemical-screening platform that employs the binding of naturally occurring, stronger and highly specific secondary molecular entity to its target as a partition step, to identify highly specific artificial nucleic acid ligands. Here, we used an antibody (Ab) that binds to the membrane-bound Immunoglobulin M (mIgM) to selectively elute aptamers that are specific for mIgM from a SELEX pool that is partially enriched toward mIgM expressing Ramos cells. Three aptamers that were selected showed high specificity toward Ramos cells. Furthermore, our results show that the aptamers identified by LIGS could be outcompeted by mIgM Ab, demonstrating that LIGS can be successfully applied to select aptamers from a partially evolved cell-SELEX library, against predetermined receptor proteins using a cognate ligand.

In Chapter 3, we explored the applicability of LIGS towards a multi-domain receptor complex, using an anti-CD3ε mAb against the cluster of differentiation 3 (CD3ε), as the guiding ligand against one of the domains of the T-cell Receptor (TCR) complex expressed on Jurkat.E6 cells. We discovered three specific aptamers against TCR complex expressed on an immortalized line of human T lymphocyte cells. These findings demonstrate that specific aptamers can be identified utilizing an antibody against a single domain of a multidomain protein complex in their endogenous state with neither post- nor pre-SELEX protein manipulation.

Chapter 4 outlines, the systematic truncation of an aptamer named R1, which was selected against mIgM in the study concluded in chapter one, to design shorter variants with enhanced affinity. Importantly, herein, we succeeded to show that the specificity of the most optimized variant of R1 aptamer (R1.2) is similar to that of the anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, the results demonstrated that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature indicating that low-affinity aptamers generated in LIGS could be enhanced by post-SELEX modifications without compromising their specificity.

Since the aptamers obtained from initial LIGS experiments possessed only moderate binding affinities to their target receptors, we developed a comprehensive version of ligand-guided selection (LIGS), by optimizing LIGS to identify higher affinity aptamers with high specificity. In addition, we expanded the LIGS method by performing specific aptamer elution at 25 °C, utilizing multiple monoclonal antibodies (mAbs) against cultured cells and primary cells obtained from human donors expressing the same receptor. Eluted LIGS libraries were subjected to Illumina high-throughput (HT) DNA sequencing and were analyzed by bioinformatics tools to discover five DNA aptamers with apparent affinities ranging from 3.06 ± 0.485 nM to 325 ± 62.7 nM against the target, T-cell receptor-cluster of differentiation epsilon (TCR-CD3ε) expressed on human T-cells. The specificity of the aptamers was validated utilizing multiple strategies, including competitive binding analysis and a double-knockout Jurkat cell line generated by CRISPR technology. The cross-competition experiments using labeled and unlabeled aptamers revealed that all five aptamers compete for the same binding site. Collectively, the data presented in Chapter 5, introduce a modified LIGS strategy as a universal platform to identify highly specific multiple aptamers toward multi-component receptor proteins in their native state without changing the cell-surface landscape. These aptamers can be used to develop therapeutic agents, especially for cancer immunotherapy.

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