Development and Characterization of CD44-Targeted X-Aptamers with Enhanced Binding Affinity for Cancer Therapeutics

Area of Science:

• Biochemistry and Molecular Biology
• Cancer Research
• Biotechnology

Background:

• CD44 is a key cell surface molecule involved in cell adhesion, migration, and is a marker for cancer stem cells, driving tumor progression.
• Targeting CD44-positive cells, including cancer stem cells, is a promising strategy for cancer therapy and drug delivery.
• Existing aptamers (thioaptamers) provide a foundation for developing novel CD44-targeting agents.

Purpose of the Study:

• To develop and select high-affinity aptamers targeting the CD44 protein, specifically its hyaluronic acid binding domain (HABD).
• To investigate the impact of chemical modifications, such as ADDA conjugation, on aptamer binding affinity and specificity.
• To evaluate the efficacy of modified aptamers in targeting CD44-overexpressing cancer cells.

Main Methods:

• Construction of a bead-based X-aptamer (XA) library by conjugating drug-like ligands (X) to modified uridines on a thioaptamer backbone.
• Selection of high-affinity XAs targeting the CD44 HABD from the combinatorial library, including variants with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (ADDA).
• Assessment of binding affinity using CD44 protein and CD44-overexpressing human ovarian cancer IGROV cells; secondary structure prediction using MFold.

Main Results:

• A novel XA demonstrated up to a 23-fold enhancement in binding affinity to the CD44 protein.
• Selected CD44 X-aptamers, particularly the ADDA-modified forms, exhibited significantly improved binding to CD44-overexpressing IGROV cells.
• Specific motifs (X-aptamer motif 3 and 5) with ADDA modification showed superior binding, indicating the importance of ligand positioning for enhanced affinity and specificity.

Conclusions:

• Site-directed modification of aptamers, exemplified by ADDA conjugation, is a powerful strategy to enhance binding affinity and specificity for target molecules like CD44.
• The developed CD44 X-aptamers hold potential as targeted agents for cancer stem cells and for drug delivery applications.
• This approach provides a flexible and generalizable framework for creating high-performance aptamers against diverse molecular targets.