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

Shiau Wei Liew; Dong Cao; Riley J. Petersen; Samuel E. Butcher; Scott G. Kennedy; Chun Kit Kwok

doi:10.1093/nar/gkaf137

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

Shiau Wei Liew, Dong Cao, Riley J. Petersen, Samuel E. Butcher, Scott G. Kennedy, Chun Kit Kwok^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

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Abstract

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. © The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.

Original language	English
Article number	gkaf137
Journal	Nucleic Acids Research
Volume	53
Issue number	5
Online published	8 Mar 2025
DOIs	https://doi.org/10.1093/nar/gkaf137
Publication status	Published - 24 Mar 2025

Funding

This work was supported by National Natural Science Foundation of China Project [32471343 and 32222089,]; Research Grants Council (RGC) of the Hong Kong Special Administrative Region (RFS2425-1S02, CityU 11100123, CityU 11100222, and CityU 11100421); Croucher Foundation Project (9509003); State Key Laboratory of Marine Pollution Seed Collaborative Research Fund (SCRF/0037, SCRF/0040, and SCRF0070); City University of Hong Kong projects (7030001, 9678302, and 6000827) to C.K.K., and the Hong Kong PhD Fellowship Scheme to S.W.L.; NIGMS Grant to S.G.K [R35 GM148206]; NIH grant to S.E.B [R35 GM118131]. This study made use of the National Magnetic Resonance Facility at Madison, an NIH Biomedical Technology Research Resource Center NIH R24GM141526. Helium recovery equipment, computers, and infrastructure for data archive were funded by the University of Wisconsin-Madison, NIH P41GM136463, R24GM141526 and by the United States National Science Foundation Mid-Scale Research Infrastructure Big Idea under grant no. 1946970. Funding to pay the Open Access publication charges for this article was provided by Research Grants Council of the Hong Kong SAR, China Projects [CityU 11100123]. Open Access made possible with partial support from the Open Access Publishing Fund of the City University of Hong Kong.

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This full text is made available under CC-BY 4.0. https://creativecommons.org/licenses/by/4.0/

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title = "A novel L-RNA aptamer to regulate the pUG fold RNA-induced gene expression in vivo",

abstract = "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. {\textcopyright} The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.",

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AU - Liew, Shiau Wei

AU - Cao, Dong

AU - Petersen, Riley J.

AU - Butcher, Samuel E.

AU - Kennedy, Scott G.

AU - Kwok, Chun Kit

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N2 - 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. © The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.

AB - 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. © The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

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RFS: Developing RNA Structure-Targeting Bi-/Multi-Functional Laptamer-Based Chimeras for Gene Regulation

GRF: Developing Novel Short Peptides to Target G-quadruplex Structure and Modulate Gene Function

GRF: Evolving New L-RNA Aptamers and Developing L-RNA aptamer-peptide Conjugates to Control G-quadruplex Structure-associated Gene Activity

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