Efficient Protein Production via Internal Cap-Initiated Translation

In the rapidly evolving field of RNA therapeutics, circular messenger RNAs (circRNAs) have emerged as promising candidates due to their inherent stability and resistance to exonucleases. Unlike linear mRNAs, circRNAs lack free ends, which traditionally complicates the recruitment of the translational machinery required for protein synthesis. However, recent innovations have addressed this limitation through novel molecular designs that exploit internal initiation mechanisms, substantially enhancing the translational output of circRNAs. The latest breakthrough, as detailed by Fukuchi, Nakashima, Abe, and colleagues in Nature Biotechnology (2025), unveils two distinct strategies that leverage an internal cap-initiated approach, revolutionizing the efficiency with which circular mRNAs can produce functional proteins.

Traditional circRNAs employed in research and therapeutic settings commonly incorporate internal ribosome entry sites (IRES) to recruit ribosomal complexes and initiate translation. Although effective to some extent, IRES-dependent translation often suffers from lower efficiency and tissue-specific variability, which hampers wide therapeutic applicability. The innovation reported circumvents the dependency on IRES by integrally introducing an N^7-methylguanosine (m^7G) cap structure directly tethered to the circular RNA, forming what the authors term “cap-circ mRNA.” This groundbreaking molecular configuration provides a covalent attachment of the m^7G cap through a branched structure, enabling direct recruitment of eukaryotic initiation factors with high affinity, essentially mimicking the natural mechanism observed in linear capped mRNAs.

This covalently-attached cap does more than simply facilitate ribosome loading; it effectively transforms the circular RNA into a highly translatable molecule. Experimental evidence from the study demonstrates that cap-circ mRNAs outperform conventional IRES-containing circRNAs by a significant margin in protein production assays. Such an efficiency leap opens the door to practical therapeutic applications where protein yield is critical, including the development of vaccines, gene replacement therapies, and cancer immunotherapies. Notably, the authors highlight that this enhanced translation is not merely a laboratory artifact but translates effectively in vivo, particularly in murine models, suggesting robust physiological relevance.

Incorporation of the N^1-methylpseudouridine (m^1Ψ) modification further optimizes the cap-circ mRNA platform by reducing the immunostimulatory properties commonly associated with exogenous RNA molecules. RNA therapeutics often induce innate immune activation via pattern recognition receptors, which can limit therapeutic efficacy and safety. The introduction of m^1Ψ into cap-circ mRNAs diminishes recognition by these sensors, resulting in a much lower acute immunostimulatory response. This modification preserves potent translation while minimizing inflammatory side effects, a balance that is vital for clinical success, especially when repeated dosing regimens are required.

Complementing the covalent approach, the authors introduce a second, non-covalent strategy to enhance circRNA translation through hybridization with an m^7G cap-containing oligonucleotide. This innovative method involves annealing a synthetic oligonucleotide bearing an m^7G cap to the circular RNA’s complementary sequences, effectively grafting the cap structure onto the circRNA post-transcriptionally without permanent chemical modification. Remarkably, this technique enables a dramatic increase in translational efficiency—more than 50-fold compared to uncapped circular RNAs—highlighting the potency of cap-mediated initiation even when applied non-covalently.

The non-covalent cap attachment approach has exciting implications for modular RNA therapeutics. Because the cap oligonucleotide can be designed to target specific RNA sequences, this method allows selective activation of translation only in the presence of particular cellular RNAs, including endogenous capped mRNAs or long non-coding RNAs. This capability introduces a novel layer of cell-type or context-specific control over protein expression, paving the way for tailored therapies that minimize off-target effects and maximize therapeutic index.

A particularly compelling feature of this non-covalent system is its capability to support rolling circle-type translation. Rolling circle translation enables ribosomes to continuously translate repeated protein units from a single circular RNA molecule, theoretically generating large quantities of protein from minimal RNA input. The hybridized cap oligonucleotide facilitates this process by ensuring robust ribosomal recruitment and initiation, overcoming traditional barriers to efficient rolling circle translation. This could be transformative for the production of proteins that require long repetitive domains or for generating protein polymers with novel biophysical properties.

The two cap-dependent translation strategies outlined by Fukuchi and colleagues collectively address the long-standing challenge of poor translational efficiency in circRNAs, expanding their utility beyond niche applications. By integrating structural RNA chemistry, enzymology, and translational biology, these innovations significantly reduce the molecular size of therapeutic constructs while boosting their output, which is particularly relevant for delivery platforms with constrained payload capacities such as lipid nanoparticles or viral vectors.

Beyond therapeutic protein production, these advances also open up new avenues in synthetic biology and molecular engineering, where precise control over protein synthesis kinetics and quantity is essential. The ability to modulate circRNA translation dynamically through cap modifications or oligonucleotide hybridization offers researchers a powerful toolkit to engineer bespoke protein expression profiles in diverse cellular environments.

It is also noteworthy that both strategies favor a cleaner immunological profile compared to traditional mRNA therapeutics. The reduced immune activation observed with cap-circ mRNAs modified with m^1Ψ aligns well with the growing emphasis on tolerability and patient safety in RNA drug development. Moreover, the non-covalent hybridization method offers flexibility in fine-tuning immune responses by altering oligonucleotide design, length, or chemistry, enabling next-generation RNA therapeutics to better integrate into complex biological systems.

In concert, these internal cap-initiated mechanisms demonstrate a paradigm shift in circRNA design, moving from passive reliance on secondary structure elements like IRES toward active chemical and hybridization-based modulators of the translational landscape. Such a transition mirrors advances seen historically in linear mRNA therapeutics, where optimization of the 5’ cap and nucleotide modifications propelled the field forward. The translation of these principles into circular RNA forms is a logical but challenging step that has now been convincingly realized.

Future investigations are expected to expand upon these findings by exploring diverse cap architectures, alternative nucleotide modifications, and the interplay between circRNA topology and cellular translation factors. Additionally, comprehensive in vivo studies will be critical to elucidate biodistribution, stability, and long-term expression patterns in clinically relevant models. The foundational work presented herein lays the groundwork for these pursuits and underscores the clinical potential of next-generation circRNA platforms.

Considering the rapid growth of RNA therapeutics, particularly following the success of mRNA vaccines, innovations such as these internal cap-initiated translation systems could extend the reach and versatility of RNA-based interventions. By combining enhanced translation efficiency, minimized molecular size, and refined immunological profiles, these circRNA technologies position themselves as strong contenders for diverse therapeutic applications, including chronic disease treatment, personalized medicine, and beyond.

As the RNA therapeutics landscape becomes more sophisticated, tools that enable precise, efficient protein synthesis with minimal immunogenicity will be invaluable. The discoveries reported by Fukuchi and colleagues represent a significant leap forward, marrying fundamental RNA biochemistry with translational medicine imperatives. The future of circular mRNA is bright, promising more effective, durable, and adaptable treatments that leverage the unique advantages of RNA circularity, all while overcoming prior translational bottlenecks.

Subject of Research: Molecular innovations to enhance translation efficiency of circular mRNA therapeutics through internal cap-initiated mechanisms.

Article Title: Internal cap-initiated translation for efficient protein production from circular mRNA.

Article References:
Fukuchi, K., Nakashima, Y., Abe, N. et al. Internal cap-initiated translation for efficient protein production from circular mRNA. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02561-8

Image Credits: AI Generated

Tags: cap-circ mRNA innovationcircular messenger RNAsefficient protein productionenhancing translational efficiencyexonuclease resistance in RNAsinternal cap-initiated translationmolecular design breakthroughsN^7-methylguanosine cap structureribosome entry sitesRNA therapeuticstherapeutic applications of circRNAstranslational machinery recruitment