Rational Truncation of an RNA Aptamer to Prostate-Specific Membrane Antigen Using Computational Structural Modeling

Abstract

RNA aptamers represent an emerging class of pharmaceuticals with great potential for targeted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface antigens with high affinity and specificity have been described. However, their clinical potential has yet to be realized. A significant obstacle to the clinical adoption of RNA aptamers is the high cost of manufacturing long RNA sequences through chemical synthesis. Therapeutic aptamers are often truncated postselection by using a trial-and-error process, which is time consuming and inefficient. Here, we used a "rational truncation" approach guided by RNA structural prediction and protein/RNA docking algorithms that enabled us to substantially truncateA9, an RNA aptamer to prostate-specific membrane antigen (PSMA),with great potential for targeted therapeutics. This truncated PSMA aptamer (A9L; 41mer) retains binding activity, functionality, and is amenable to large-scale chemical synthesis for future clinical applications. In addition, the modeled RNA tertiary structure and protein/RNA docking predictions revealed key nucleotides within the aptamer critical for binding to PSMA and inhibiting its enzymatic activity. Finally, this work highlights the utility of existing RNA structural prediction and protein docking techniques that may be generally applicable to developing RNA aptamers optimized for therapeutic use.

Improved Synthesis andIn VitroEvaluation of an Aptamer Ribosomal Toxin Conjugate

“…We chemically synthesized a 43 nucleotide minimized version of A9 (denoted here as A9.min) based on the minimal sequence reported by Archemix [14] and more recently reported by Rockey et al [15]. The same minimized sequence was identified from an analysis of a doped selection performed by our own group using a library based on the fulllength A9 ( [16] and unpublished results).…”

“…The same minimized sequence was identified from an analysis of a doped selection performed by our own group using a library based on the fulllength A9 ( [16] and unpublished results). Because the bulk of the reported characterization of this aptamer relied on the inhibition of the enzymatic activity of PSMA [14,15], we initially preformed a cell-based analysis by flow cytometry to confirm the specificity and determine the apparent binding constant for this molecule on PSMA-positive cells.…”

“…Although this minimized molecule has previously been shown to inhibit PSMA enzymatic activity with an IC50 of *10 nM [14,15], to inhibit PSMA cell migration with an IC50 of *200 nM [21] and even to localize to tumors following systemic administration to mice bearing PSMA-positive tumors [21], assays characterizing the direct cell binding capabilities of this molecule and its specificity for PSMA in the literature were lacking detail. Therefore, we performed a set of analyses using a fluorescently labeled aptamer (AF488-A9.min) to directly determine the cell binding characteristics of this molecule.…”

Diagnosis of prostate cancer using anti-PSMA aptamer A10-3.2-oriented lipid nanobubbles

“…Its molecular weight was significantly reduced, but it still has very good binding affinity to PSMA. 24,25 On the basis of previous studies on NBs carrying anti-PSMA monoclonal antibodies and nanobodies, this study aimed to couple the A10-3.2 aptamer as a ligand with lipid NBs and to study its imaging effect in prostate cancer. The purpose was not only to provide ultrasound molecular probes with better safety, small particle size, strong penetration and specificity for prostate cancer, but also to provide methods for relevant studies on targeted ultrasound NBs carrying aptamers.…”

A Theranostic “SMART” Aptamer for Targeted Therapy of Prostate Cancer

“…A9g is a truncated version of A9 that shows enhanced binding affinity to PSMA and, importantly, can inhibit its NAALADase/ glutamate carboxypeptidase II activity with an IC 50 of 10 nM (ref. 15). …”