Published on Angewandte Chemie (27 October 2024)
Author(s): Dr. Danyang Ji, Dr. Bo Wang, Dr. Kwok Wai Lo, Dr. Chi Man Tsang, Dr. Chun Kit Kwok
Abstract
L-RNA aptamers have been developed to target G-quadruplexes (G4s) and regulate G4-mediated gene expression. However, the aptamer selection process is laborious and challenging, and aptamer identification is subject to high failure rates. By analyzing the previously reported G4-binding L-RNA aptamers, we found that the stem-loop (SL) structure is favored by G4 binding. Herein, we present a robust and effective G4-SLSELEX-Seq platform specifically for G4 targets by introducing a pre-defined stem-loop structure library during the SELEX process. Using G4-SLSELEX-Seq, we identified an L-RNA aptamer, L-Apt1-12, for the Epstein–Barr nuclear antigen 1 (EBNA1) RNA G4 (rG4) in just three selection rounds. L-Apt1-12 maintained the stem-loop structure initially introduced, and possessed a unique G-triplex motif that is important for the strong binding affinity and specificity to EBNA1 rG4. L-Apt1-12 effectively downregulated endogenous EBNA1 protein expression in human cancer cells and showed selective toxicity towards Epstein–Barr virus (EBV)-positive cancer cells, highlighting its potential for targeted therapy against EBV-associated cancers. Furthermore, we demonstrated the robustness and generality of G4-SLSELEX-Seq by selecting L-RNA aptamers for the amyloid precursor protein (APP) rG4 and the hepatitis C virus subtype 1a (HCV-1a) rG4, obtaining high-affinity aptamers in three selection rounds. These findings demonstrated G4-SLSELEX-Seq as a robust and efficient platform for the selection of rG4-targeting L-RNA aptamers.
Graphical Abstract
We combine mirror-image SELEX with a pre-structured library for the first time, using a pre-defined stem-loop (SL) structure library to develop the G4-SLSELEX-Seq platform. G4-SLSELEX-Seq can serve as a general L-RNA aptamer SELEX platform for RNA G-quadruplex (rG4) targets. L-Apt1-12 for the EBNA1 rG4 is selected in three rounds (within a week). It can effectively inhibit EBNA1 mRNA translation in Epstein–Barr virus (EBV)-positive cancer cells.
Read more: https://onlinelibrary.wiley.com/doi/full/10.1002/anie.202417247