RNA G-quadruplex structure targeting and imaging: recent advances and future directions

Abstract

RNA guanine (G)-quadruplexes (rG4s) are noncanonical structures formed by G-rich RNA sequences and have been demonstrated to play critical roles in various biological events, including translation, transcription, RNA processing, and other cellular functions. In contrast to DNA G-quadruplexes (dG4s), research on rG4s has been relatively limited until recently. Recent advances in targeting and imaging of rG4s have opened new avenues for understanding their functional significance and therapeutic potential. In this review, we summarize the innovative platforms and tools being developed to target rG4s, highlight the novel and important imaging probes that have been generated and applied for rG4 structure visualization in different biological contexts, and discuss the challenges and perspectives for further advancing these technologies and toolsets to facilitate rG4 targeting and imaging with greater precision and resolution across the Tree of Life. These scientific developments and breakthroughs will enable the discovery of new biological insights regarding rG4s and help decipher their molecular mechanisms and implications for health and disease.

Keywords: G-quadruplex structure; RNA; imaging; nucleic acids; targeting.

FIGURE 1.

Structural diversities and biological functions of RNA G-quadruplexes. (A) The chemical structure of a G-quartet and the representative topologies of intermolecular and intramolecular G-quadruplex structures. (B) Representative three-dimensional structure of RNA G-quadruplexes. Protein Data Bank (PDB ID): 2KBP, 8X0S, and 6K84. (C) Representative biological roles of RNA G-quadruplexes in both coding mRNAs and noncoding RNAs (Dumas et al. 2021; Caterino and Paeschke 2022; Kharel et al. 2023; Sahayasheela and Sugiyama 2024; Shukla and Datta 2024).

FIGURE 2.

Diverse tools, including small molecules, peptides, aptamers, antibodies, and derived combinatorial tools, are used to target rG4s and regulate rG4-mediated gene expression. (A) Representative small molecules with different scaffolds for rG4 targeting, such as cationic porphyrins (TmPyP4), quinoline derivatives (cPDS), polyaromatic compounds (RGB-1), and guanine-clamp analogs (PhpC). (B) De novo peptides specifically target G4 structures selected by G4-mRNA display-seq. An mRNA library was linked to puromycin linker and translated in vitro to create the mRNA-peptide fusion library, followed by reverse-transcription and screening against an rG4 target to isolate functional sequences. The tandem and cyclic version of peptide showed enhanced binding to rG4 and downregulated rG4-mediated gene expression. (C) L-RNA aptamers bind to rG4, leading to disruption of rG4–protein interaction and inhibition of gene expression. (D) Antibody BG4 binding to rG4 for rG4 imaging in cells via confocal microscopy. Icon adapted from “confocal-scanning-laser-microscope-CSLM” by DBCLS (https://togotv.dbcls.jp/en/pics.html) is licensed under CC-BY 4.0 Unported (https://creativecommons.org/licenses/by/4.0/). (E) Antisense oligonucleotides composed of γPNA oligomers potently invade and destabilize rG4, resulted in formation of γPNA–RNA duplex. (F) Dual binding of an rG4 ligand cPDS and an antibody BG4 to TERRA rG4 in a two-stage manner. cPDS initially binds competitively to TERRA rG4, followed by a slow conformational rearrangement of ternary complex with BG4. (G) A bifunctional guanine-RHAU23 peptide conjugate (GRPC) for G-vacancy-bearing G-quadruplexes (GVBQs) targeting. The guanine group fills into the G-vacancy in a GVBQ, facilitating the binding of RHAU23 to the GVBQs.

FIGURE 3.

Principle of rG4 imaging techniques. (A) Fluorescent small molecules that light up upon binding to rG4 structures. (B) Immunofluorescent staining of rG4 structure using immuno-tag G4 binding ligand. (C) Selective rG4 imaging using an antisense conjugated rG4 fluorescent ligand, or proximity-induced rolling circle amplification (RCA) triggered by click reaction between antisense and rG4-binding ligand of close proximity. (D) Fluorogenic bifunctional aptamer (FLAP) by connecting rG4 targeting aptamer and fluorogenic aptamer to image rG4 structure in the presence of its corresponding fluorescent ligand.