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.