A novel L-RNA aptamer to regulate the pUG fold RNA-induced gene expression <i>in vivo</i>

G-quadruplex (G4) is a guanine-rich secondary structure found in DNA and RNA involved in various biological roles. Recently, a non-canonical RNA G-quadruplex (rG4), known as poly(UG) (pUG) fold, was discovered in Caenorhabditis elegans. This unique structure was found to induce RNA interference (RNAi) upon recruitment of RNA-dependent RNA polymerase (RdRP), resulting in trans-generational gene silencing. Herein, we develop a novel L-RNA aptamer, L-apt3.1, that binds to the pUG fold. We uncover that L-apt3.1 consists of a parallel rG4 structural motif, and mutagenesis analysis illustrates that the rG4 motif in L-apt3.1 is essential for pUG fold recognition. We show that L-apt3.1 interacts strongly with pUG fold, and notably, it is the first reported aptamer that can bind to pUG fold in vitro. We also demonstrate that L-apt3.1 possesses great biostability in cellular environments and negligible toxicity in vivo. Furthermore, we report that L-apt3.1 can interact with pUG fold in vivo, and with a comparable performance to the G4 ligand, N-methyl mesoporphyrin, in inhibiting gene silencing in C. elegans. Overall, we demonstrate the development of pUG fold-targeting L-RNA aptamer for the first time, and show that this new aptamer tool can be applied to control pUG fold-mediated gene expression in vivo.