Tomasz Spiewla

Screening of 16 synthetic PAP/PAPS analogs by MST and ¹⁹ F NMR identified the 3′-phosphate as a major determinant of sulfotransferase recognition, while revealing broad tolerance toward other cofactor modifications.

PolyA tail regulates mRNA localization, stability, and translation. PolyA length affects the durability and translational activity of both endogenous and exogenously delivered mRNAs. However, long polyA stretches can undergo recombination during amplification in bacterial plasmids, impairing the production of in vitro transcribed (IVT) mRNA with long polyAs. PolyA tail segmentation with heteronucleotide spacers has recently emerged as a solution. Here, we developed segmented polyA patterns that stabilize the sequence during DNA amplification and enhance mRNA translation. We designed 15 novel genetically modified polyA variants, differing in the length, placement, and frequency of spacers, and the overall length (from ∼120 to ∼200 nucleotides). We evaluated their stability in DNA plasmids and homogeneity, translational activity, and durability in cell culture of the resulting mRNAs, comparing them to A ₉₀ tail and other known solutions, including those from existing mRNA vaccines. Selected sequences were validated in vivo . Surprisingly, we found that even frequent heteronucleotide insertions produce functional polyA tails. The most notable enhancements in protein production were observed for a segmented tail exceeding 200 nt in length [A ₃₀ (CA ₁₅ ) ₁₁ ; up to 6-fold compared to mRNA with A ₉₀ tails]. Our findings extend the scope of possible polyA modification strategies, offering new possibilities for advancing mRNA therapeutics.

Circularization is an important step for therapeutic messenger RNA (mRNA) enhancements. Current enzymatic and ribozymatic-based circularization methods face limitations including sequence constraints, purification challenges, and sub-optimal biological activity. Chemical strategies, while promising, have been restricted to short RNA sequences. Here, we report a method for chemically circularized in vitro transcribed RNAs of various lengths (chem-circRNAs; 35–4000 nt) with circularization efficiencies reaching up to 60%. This approach leverages a 5′ ethylenediamine modification and a periodate-oxidized 3′ end to drive intramolecular reductive amination. We demonstrate that this method is applicable to various sequences and modification compatible. We report the effective separation methods of chem-circRNAs from their linear precursors. We show that protein-coding chem-circRNAs are translationally active in cells and exhibit increased durability, like enzymatically circularized mRNAs. Furthermore, our method allows incorporation of functional modifications, including endocyclic N7-methylguanosine cap and N1-methylpseudouridine, enabling access to chemically defined translationally active circRNAs for therapeutic applications.

Despite the widespread use of mRNA vaccines against COVID-19, little is known about the metabolism of therapeutic RNAs. Here we use nanopore sequencing^1–3 to analyse individual therapeutic mRNA molecules, focusing on their poly(A) tails. We show that the Moderna mRNA-1273 vaccine⁴ has a poly(A) tail of around 100 nucleotides, followed by an mΨCmΨAG sequence. In cell lines, mRNA-1273 undergoes rapid degradation initiated by mΨCmΨAG removal, followed by CCR4–NOT-mediated deadenylation. However, in medically relevant preclinical models, particularly in macrophages, mRNA-1273 poly(A) tails are extended to up to 200 nucleotides by the TENT5A poly(A) polymerase^5–7, which is induced by the vaccine. Re-adenylation, which stabilizes target mRNAs, is consistently observed in synthetic mRNAs that encode proteins targeted to the endoplasmic reticulum, such as ovalbumin or antigens from Zika virus⁸ or the malaria parasite⁹. The extent of re-adenylation varies: the BioNTech–Pfizer BNT162b2 vaccine¹⁰ shows less potent re-adenylation than mRNA-1273, which correlates with a smaller proportion of membrane-associated BNT162b2. This highlights the crucial role of spatial accessibility to ER-resident TENT5A in determining re-adenylation efficiency. In vivo, TENT5A is expressed in immune cells that take up mRNA vaccine, and TENT5A deficiency reduces specific immunoglobulin production for mRNA vaccines after immunization in mice. Overall, our findings reveal a principle for enhancing the efficacy of therapeutic mRNAs, paving the way for improvement.

IFITs (interferon-induced proteins with tetratricopeptide repeats) are components of the innate immune response that bind to viral and cellular RNA targets to inhibit translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5′ ends. IFIT1 preferably binds RNAs modified with the m⁷G cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize noncanonical RNA 5′ ends, including hypermethylated and noncanonical RNA caps. Further insights into the structure-function relationship for IFIT1–RNA interactions are needed but require robust analytical methods. Here, we report a biophysical assay for quick, direct, in-solution affinity assessment of differently capped RNAs with IFIT1. The procedure, which relies on measuring microscale thermophoresis of fluorescently labeled protein as a function of increasing ligand concentration, is applicable to RNAs of various lengths and sequences without the need for their labeling or affinity tagging. Using the assay, we examined 13 canonically and noncanonically 5′-capped RNAs, revealing new binding preferences of IFIT1. The 5′ terminal m⁶A mark in the m⁷G cap had a protective function against IFIT1, which was additive with the effect observed for the 2′-Oposition (m⁶A_mcap-1). In contrast, an increased affinity for IFIT1 was observed for several noncanonical caps, including trimethylguanosine, unmethylated (G), and flavin-adenine dinucleotide caps. The results suggest new potential cellular targets of IFIT1 and may contribute to broadening the knowledge of the innate immune response mechanisms and the more effective design of chemically modified mRNAs.

Circularization is at the frontier of therapeutic messenger RNA (mRNA) enhancements. Currently available enzymatic and ribozymatic methods for generating circular RNAs (circRNAs) face several challenges related to sequence limitations, purification, and sub-optimal biological activity. The chemical circularization of synthetic RNA fragments potentially overcomes these limitations but is applicable only to extremely short sequences. Here, we report a novel approach for accessing circular RNAs based on the chemical circularization of in vitro transcribed RNA. We efficiently accessed chemically circularized RNAs (chem-circRNAs) by making in vitro transcribed precursor RNAs modified at the 5′ end with an ethylenediamine moiety, which undergoes an intramolecular reaction with the periodate-oxidized RNA 3′ end under reductive amination conditions. We demonstrate that this method is modification-compatible and applicable to various sequences. Additionally, we report methods for the effective separation of chem-circRNAs from their linear precursors. Using this approach, we prepared multiple chemically-obtained circular RNAs (chem-circRNAs; 35–1500 nt long) with circularization efficiencies reaching up to 60%. We show that protein-coding chem-circRNAs are translationally active in living cells and exhibit increased durability, similar to enzymatically circularized mRNAs. We also demonstrate that this approach enables unprecedented access to chemically modified circRNAs, such as circ-mRNAs incorporating a functional endocyclic N7-methylguanosine cap or modified with N1-methylpseudouridine within the RNA body. Notably, circRNAs containing an endocyclic cap structure engage in the most efficient, cap-dependent mechanism of translation. Our approach makes chemically-modified circularized full-length protein-coding RNAs easily accessible, thereby opening new avenues for the design, modification, and functionalization of circular mRNAs.

Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA‐based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5′‐end, called 5′‐cap. Depending on the 5′‐terminal sequence and its methylation pattern, different proteins may be involved in the translation initiation and regulation, but a deeper understanding of these mechanisms requires specialized molecular tools to identify natural binders of mRNA 5′‐end variants. Here, a series of 8 new synthetic 5′‐cap analogs that allow the preparation of RNA molecules with photoreactive tags using a standard in vitro transcription reaction are reported. Two photoreactive tags and four different modification sites are selected to minimize potential interference with cap‐protein contacts and to provide complementary properties regarding crosslinking chemistry and molecular interactions. The tailored modification strategy allows for the generation of specific crosslinks with model cap‐binding proteins, such as eIF4E and Dcp2. The usefulness of the photoreactive cap analogs is also demonstrated for identifying the cap‐binding subunit in a multi‐protein complex, which makes them perfect candidates for further development of photoaffinity labeling probes to study more complex mRNA‐related processes.

IFIT proteins (interferon-induced proteins with tetratricopeptide repeats) are key components of the innate immune response that bind to viral and cellular RNA targets to inhibit viral translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5’ ends. IFIT1 preferably binds RNAs modified with the 7-methylguanosine (m ⁷ G) cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize non-canonical RNA 5’ ends, including hypermethylated and non-canonical RNA caps. Deciphering the structure-function relationship for IFIT1-RNA interaction may improve understanding of cellular selection of IFIT targets and guide the design of exogenously delivered therapeutic RNAs, but requires high-throughput and robust analytical methods. Here, we report a biophysical assay for quick, direct, in-solution affinity assessment of differently capped RNAs with IFIT1. The procedure, which relies on measuring microscale thermophoresis (MST) of fluorescently labelled protein as a function of increasing ligand concentration, is applicable to various RNA lengths and sequences without the need for labelling or affinity tagging. Using the assay, we examined thirteen canonically and non-canonically 5’-capped RNAs, revealing new binding preferences of IFIT1. The 5’ terminal m ⁶ A mark in the m ⁷ G cap had a protective function against IFIT1, which was additive with the effect observed for the 2’-O position (m ⁶ A _m cap-1). In contrast, an increased affinity for IFIT1 was observed for several non-canonical caps, including trimethylguanosine (TMG), unmethylated (G), and flavin-adenine dinucleotide (FAD) caps. The results suggest new potential cellular targets of IFIT1 and may contribute to broadening the knowledge on the mechanisms of the innate immune response as well as the more effective design of chemically modified mRNAs.