Aug 2026· Analytical Chemistry· Vol 98 32, pp.
23819-23826
· 0 citations· 27 references
Medicine
TL;DR
A new chemically regulated circRNA translation system based on small ligand-induced stabilization of RNA-binding proteins (RBPs), engineered by incorporating RNA motifs into an internal ribosomal entry site and fusing RBPs with small ligand-stabilized conditional proteins is developed.
Abstract
Circular RNAs (circRNAs) have emerged as a promising platform for RNA-based therapeutics, ascribed to their excellent stability and prolonged protein translation capacity. However, modulation of circRNA translation remains a great challenge. Here, we develop a new chemically regulated circRNA translation system based on small ligand-induced stabilization of RNA-binding proteins (RBPs). The system is engineered by incorporating RNA motifs into an internal ribosomal entry site and fusing RBPs with small ligand-stabilized conditional proteins. We show that this design allows small ligand-modulated translation of circRNAs with a wide dynamic range and dose dependency. It is demonstrated that this strategy is modular and versatile to different RBPs and small-ligand-stabilized conditional proteins. We further employ this strategy to control the translation of functional proteins, including a guanine nucleotide exchange factor for modulating signaling pathways and a bispecific engager for regulating T cell activities. This system is highly modular and versatile, highlighting its great potential for basic research and therapeutic applications.
RNA-binding proteins (RBPs) are central regulators of post-transcriptional gene expression, recognizing RNAs through sequence, structure, and chemical modifications. Post-transcriptional RNA modifications, including m6A, m1A, m5C, m7G, and pseudouridine (Ψ), form the epitranscriptome, a dynamic regulatory layer that modulates RNA stability, localization, and translation. These modifications are interpreted by specialized “reader” RBPs that translate epitranscriptomic marks into functional outcomes. Dysregulation of RNA modifications or their associated reader RBPs has been increasingly linked to the development of cancers, neurological disorders, and other diseases, highlighting their potential for therapeutic manipulation. This review summarizes key RNA modifications and regulating RBPs with a specific emphasis on how dysregulation can lead to cancers. We further discuss current approaches for investigating and manipulating reader RBP–RNA interactions, highlighting how these methods enable new opportunities for therapeutic discovery.
G. Vega-Hernández, Amanda L. Garner· RSC Chemical Biology· 0 citations
Viruses encode diverse regulatory elements, but their breadth and mechanisms remain poorly defined. To address this gap, we performed massively parallel reporter assays spanning ∼200,000 genomic segments from 297 vertebrate-infecting viral genera. We identified numerous viral elements that enhance RNA stability and translation through TENT4-mediated mixed tailing, distributed across 19 genera and grouped into six distinct subclasses, indicating extensive convergent evolution. We also found diverse TENT4-independent elements acting through alternative pathways. One such element, Pt1 from Potamipivirus, stabilizes linear mRNA to levels comparable to circular RNA, suggesting its potential for RNA therapeutics. Pt1 directly recruits canonical poly(A) polymerases (PAPγ/α)-previously thought to function exclusively in transcription-coupled nuclear pre-mRNA processing-to drive cytoplasmic polyadenylation. Together, these findings chart the rich landscape of viral regulation, extend the scope of poly(A)-tail biology, and establish the virome as a valuable source for uncovering host RNA regulatory mechanisms.
Jenny J Seo, Chemin Lee, Dongbin Lim et al.· Cell· 0 citations
ABSTRACT RNA‐binding proteins (RBPs) are central regulators of post‑transcriptional gene expression, controlling RNA stability, localization, translation, and alternative splicing. Their functions arise not only from intrinsic RNA‐binding domains but also from dynamic interactions with noncoding RNAs, metabolites, cofactors, and other RBPs. Here, we summarize the structural diversity and core biological activities of canonical and noncanonical RBPs, and delineate how competitive and cooperative regulatory networks dictate RBP function in disease, with an emphasis on cancer. Competitive mechanisms, including lncRNA‐mediated sequestration, antagonistic crosstalk between miRNAs and RBPs, and competition among RBPs for shared substrates, can redirect RNA fate. In contrast, cooperative mechanisms assemble multimolecular ribonucleoprotein complexes that reinforce oncogenic or tumor‐suppressive programs. Dysregulation of these networks promotes proliferation, metastasis, immune evasion, and therapy resistance. We also review emerging therapeutic strategies that target RBP‐centered regulatory circuits, including antisense oligonucleotides, small molecules, protein degraders, and natural products, and we evaluate representative preclinical studies and clinical trials. By integrating mechanistic principles with translational evidence, this review provides a network‐based framework for exploiting RBPs as therapeutic vulnerabilities and for advancing next‐generation precision oncology.
Ling Li, Xiu-Li Yan, Qing Ji et al.· MedComm· 0 citations
Abstract Synthetic riboswitches are efficient tools to render gene expression inducible by small molecules. Here, we present a novel riboswitch platform based on RNA triplexes stabilizing non-polyadenylated transcripts while still allowing translation of the respective messenger RNA. By incorporating a tetracycline aptamer, we rendered triplex formation dependent upon the presence of the ligand. The new riboswitch platform was able to induce gene expression up to 25-fold as a standalone switch. Combination of the triplex system with an aptazyme placed into the 5′-UTR achieved a 42-fold induction of gene expression. Further, we were able to exchange the triplex, the aptamer, the maturation platform cleaving off the poly-A tail of the switch platform, and the regulated transgene, demonstrating a high degree of modularity. The new riboswitch platform is designed to function independently of nuclear processes such as transcription and RNA processing, potentially expanding the application of these synthetic riboswitches to strictly cytoplasmic therapeutic RNA vectors.
Pierre Gommeringer, Elisabeth Müller, Sıla Köse et al.· Nucleic Acids Research· 1 citation
The topology of RNA therapeutics is emerging as a critical design dimension in precision oncology. Unlike linear mRNA, circular RNA (circRNA) lacks free ends, conferring exceptional resistance to exonuclease degradation and enabling sustained protein expression for days to weeks. Beyond their use as engineered therapeutics, endogenous circRNAs exhibit cancer-associated expression patterns and persistence in biofluids, supporting complementary roles in tumor biology and as candidate biomarkers for diagnosis and longitudinal disease monitoring. This review argues that circular topology should be viewed as an active pharmacologic variable, not merely a stability enhancement. We dissect recent advances in cap-independent translation initiation, including IRES elements and m6A-driven mechanisms, rolling-circle translation for multi-epitope vaccine design, and programmable stability circuits that integrate tumor-microenvironment cues such as miRNA signatures. Delivery innovations are equally transformative: antibody-guided lipid nanoparticles and engineered extracellular vesicles enable increasingly selective RNA delivery, while local depot formulations and organ-selective systemic routes expand therapeutic reach. Safety considerations are re-evaluated as double-edged tools-innate immunogenicity can serve as a self-adjuvant for cancer vaccines, whereas back-splice-junction neoantigens offer both vaccine opportunities and tolerance risks. Recent advances in scarless circularization, topology-sensitive purification, dsRNA depletion, and lyophilized formulations have begun to address key manufacturing bottlenecks, although clinical-scale recovery and process scalability remain insufficiently characterized. Key applications include circRNA cancer vaccines, transient CAR-T/NK cell engineering, tumor-suppressor replacement, and circRNA-encoded bispecific T-cell engagers. The field now requires real-time pharmacokinetic tracking, reproducible and scalable manufacturing, validated liquid-biopsy assays, and indication-specific regulatory pathways to translate circRNA from bench to bedside.
Amr A. El-Sehrawy, H. Al-Ameer, J. Rizaev et al.· Biotechnology and Bioenginee...· 0 citations
mRNA is a new medical modality as vaccine and raises hopes to become a protein replacement agent. Despite notable advances in manufacturing, stabilization and modification, its short half-life remains a major limitation. Circular RNA bears potential to solve this issue, as degradation of mRNA proceeds primarily from the 5' and 3' ends. However, circular RNAs normally lack a 5' cap and are less efficiently translated. We present a strategy to circularize mRNA covalently via click chemistry by modifying the 5' cap and the 3' poly(A) tail with bioorthogonal functional groups. The resulting Cyclic Click Cap-containing mRNA (CyCliCap-mRNA) is translationally muted by a photocleavable protecting group. Upon irradiation by light, the photocleavable protecting group is removed and the respective linear Cap0-mRNA released. We show that CyCliCap-mRNA can be activated for efficient translation at late timepoints (24-48 h post transfection) in HeLa cells, which is not possible with the respective linear mRNA. The light-mediated opening releases linear mRNA with a native Cap0 and thus overcomes the problem of inefficient translation of circular RNAs.
Bayram Terzi, Greta Charlotte Dahm, Kristoffer Heintz et al.· Angewandte Chemie· 0 citations