68. Development of Thioaptamers and X-aptamers Targeting Human Ovarian Cancer Cells

Abstract

Aptamers are oligonucleotide or peptide molecules that bind to a specific target with high affinity and specificity. A diverse range of modifications have been reported for either enhancing aptamers’ nuclease resistance or expanding their chemical functionalities. We created a combinatorial DNA aptamer library that had been modified with thio substitution of the phosphate backbone (thioaptamer, TA) at selected 5’dA positions for cell and tissue based selections. Based on morphological assessment of the tissue, we used an image-directed laser microdissection (LMD) technique to dissect Regions of Interest (ROIs) bound with thioaptamers, and further identified the targeted proteins. Using a Next Generation Sequencing (NGS) approach and alignments by our recently developed Aptaligner program, we are able to select tissue specific TAs in a more representative way from the TA libraries. We have successfully identified TAs that bind specifically to tumor endothelial cells and characterized the lead candidate TA, Endo28, as an Annexin A2-specific TA that allows for selective delivery to the tumor vasculature. Based on the primary sequences of several of the previously selected TAs and observed variations, we developed an X-aptamer (XA) library by conjugating drug-like ligands (X) to the DNA bases in a random fashion. We were able to select an XA with high affinity to CD44 hyaluronic acid binding domain (HABD) from a large combinatorial TA library modified with a drug (N-acetyl-2,3-dehydro-2- deoxyneuraminic acid, ADDA). The selected CD44 X-aptamers (amine form and ADDA form) show enhanced binding affinities with ADDA to CD44 over-expressed on human ovarian cancer IGROV cells. Using a systems biology approach that combines the use of 15 mm bead-based XA libraries with flow cytometry sorting and mass spectrometry, we collected beads binding to proteins labeled with different fluorescence dyes and identified their associated XA sequences by PCR and NGS. The differentially expressed proteins can be identified by an XA beads pulldown experiment to discover the potential biomarkers for ultimately TA or XA targeted nanoparticle delivery of drugs to tumors.