Pre‐Defined Stem‐Loop Structure Library for the Discovery of L‐RNA Aptamers that Target RNA G‐Quadruplexes

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.