Dissertations, Theses, and Capstone Projects

Date of Degree

6-2023

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Prabodhika Mallikaratchy

Committee Members

Jill Bargonetti-Chavarria

Nicolas Biais

Morgan Huse

Julio Gallego-Delgado

Subject Categories

Biochemistry | Cancer Biology | Cell Biology | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides | Therapeutics

Keywords

Aptamer, Nucleic Acid Ligands, SELEX, Cell-SELEX, LIGS, CD19, CD20, Cancer Biomarkers, B-cell Antigens

Abstract

Nucleic acid ligands called aptamers are single-stranded DNA or RNA molecules which fold into functional three-dimensional structures to facilitate their target binding with high affinity and specificity. The method used to generate aptamers is an in vitro process called Systematic Evolution of Ligands by Exponential enrichment (SELEX). A variant of SELEX, cell-SELEX has been used to select aptamers against cell-surface proteins in their native state. We recently introduced a novel method called Ligand-Guided Selection (LIGS) to identify aptamers against cell-surface markers. Herein, we expanded LIGS method into a multiplexing platform to partition multiple aptamers against B-cell-specific antigens, CD19 and CD20, expressed in a single B lymphoma cell population. To identify specific aptamers against CD19 and CD20, the evolved cell-SELEX and LIGS libraries were sequenced using the Illumina high-throughput (HT) DNA sequencing platform and analyzed using FASTAptamer and Galaxy. Sequences from primary families were selected based on defined fold-enrichment ratio values, resulting in 18 hit sequences for CD19 and 13 hit sequences for CD20. The specificity of the candidate hit sequences was screened using CD19- and CD20-positive cells or CD19- or CD20-negative cell lines. The best two aptamers towards each antigen (CD19 or CD20) were characterized using biochemical analysis. This study establishes a cell-based multiplexing platform termed multi-target LIGS to partition highly specific synthetic ligands (aptamers) against clinically relevant biomarkers expressed in their natural state.

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