Inosine Substitutions in RNA Activate Latent G-Quadruplexes

Abstract

It is well-accepted that gene expression is heavily influenced by RNA structure. For instance, stem-loops and Gquadruplexes (rG4s) are dynamic motifs in mRNAs that influence gene expression. Adenosine-to-inosine (A-to-I) editing is a common chemical modification of RNA which introduces a nucleobase that is iso-structural with guanine, thereby changing RNA base-pairing properties. Here, we provide biophysical, chemical, and biological evidence that A-to-I exchange can activate latent rG4s by filling incomplete G-quartets with inosine. We demonstrate the formation of inosine-containing rG4s (GIquadruplexes) in vitro and verify their activity in cells. GIquadruplexes adopt parallel topologies, stabilized by potassium ions. They exhibit moderately reduced thermal stability compared to conventional G-quadruplexes. To study inosine-induced structural changes in a naturally occurring RNA, we use a synthetic approach that enables site-specific inosine incorporation in long RNAs. In summary, RNA GI-quadruplexes are a previously unrecognized structural motif that may contribute to the regulation of gene expression in vivo.

Visualization of Deep Tissue G-quadruplexes with a Novel Large Stokes-Shifted Red Fluorescent Benzothiazole Derivative

“…Significantly, ThT-NA /G4 complexes showed excellent photostability (Figures S11 and S12), suggesting their promising potential for live-cell G4 imaging. An intracellular G4 structure shows dynamic changes, and the underlying G4 structure may affect various physiological functions. , The initial tests were constructed using fixed HepG2 cells and imaged with confocal laser scanning microscopy (CLSM) to determine the feasibility. When the cells were stained with ThT-NA , bright red fluorescence signals were observed in the cytoplasm and nucleus.…”

“…G-quadruplex (G4) is a noncanonical nucleic acid secondary structure formed by Hoogsteen-type hydrogen bonding in guanine-rich sequences. 1,2 Many studies have discovered the implication of G4 structures in regulating fundamental cellular functions, 3 such as telomere maintenance, 4 replication, 5 transcriptions, 6 and translation. 7,8 Recent studies have established clear connections between G-quadruplexes and human diseases, including cancer and neurodegenerative diseases.…”

“…G-quadruplex (G4) is a noncanonical nucleic acid secondary structure formed by Hoogsteen-type hydrogen bonding in guanine-rich sequences. , Many studies have discovered the implication of G4 structures in regulating fundamental cellular functions, such as telomere maintenance, replication, transcriptions, and translation. , Recent studies have established clear connections between G-quadruplexes and human diseases, including cancer and neurodegenerative diseases. , Notably, some human diseases related RNA viruses (like hepatitis C virus and coronavirus) also had been demonstrated to fold spontaneously to form the G4 structure . Consequently, detecting G4 structures in situ is critical for further elaboration of G4 structure–function interactions and their potential applications in diagnosing and treating these diseases. , Circular dichroism (CD) and nuclear magnetic resonance are widely used to characterize G4 structures in vitro .…”

Inosine‐Based Supramolecular Hydrogel for Highly Efficient PD‐L1 Blockade Therapy via Mediating CD8+ T Cells

“…[ 32 ] Due to the lack of the 2‐amino group, inosine removes one hydrogen bond from the G‐quartets, thereby destabilizing them in the presence of K + . [ 33 ] In the present study, we presume that the first release of inosine in IPBisoG hydrogel is attributed to the insufficient hydrogen bonds; this needs to be further explored in the future. With the release of inosine, the structure of the gel was destroyed, and aPDL1 was subsequently released.…”

Oxidative Damage Induces a Vacancy G-Quadruplex That Binds Guanine Metabolites: Solution Structure of a cGMP Fill-in Vacancy G-Quadruplex in the Oxidized BLM Gene Promoter

“…The above data suggest that cGMP-fill-in oxidized BLM -vG4s may be formed in cells because they are stable at body temperature and can function as gene regulators. Notably, the T m value of the cGMP-fill-in oxidized vG4 (46 °C) is significantly lower than the native BLM -G4 (>90°C) (Figures c, c, and S1a); hence, the oxidation-induced vG4 may provide a tunable switch for gene regulation by interaction with cellular guanine metabolites. , Thus, it is of great interest to solve the solution structure of a cGMP-fill-in oxidized vG4 for further molecular insights.…”