Biology subjects

Spiewla, T.

Publications and source records attributed to Spiewla, T..

5 recordsLinked to original sources

bioRxiv2025

PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

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 A90 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 [A30(CA15)11; up to 6-fold compared to mRNA with A90 tails]. Our findings extend the scope of possible polyA modification strategies, offering new possibilities for advancing mRNA therapeutics.

molecular biology↗

bioRxiv2024

Chemical circularization of in vitro transcribed RNA opens new avenues for circular mRNA design

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.

biochemistry↗

bioRxiv2024

An MST-based assay reveals new binding preferences of IFIT1 for canonically and non-canonically capped RNAs

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 (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 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 m6A mark in the m7G cap had a protective function against IFIT1, which was additive with the effect observed for the 2-O position (m6Am 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.

biochemistry↗

bioRxiv2023

Trinucleotide mRNA cap analog N6-benzylated at the site of posttranscriptional m6Am mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo

Eukaryotic mRNAs undergo co-transcriptional 5-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am - a common epitranscriptomic mark unique to the mRNA 5-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analog of the m6Am-cap - m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as an RP-HPLC purification handle, allowing separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic setting as anti-cancer vaccines. The biochemical characterization suggests several phenomena underlying the biological properties of AvantCap: (i) increased competitiveness of the mRNA 5-end for eIF4E protein by reducing its propensity for unspecific interactions, (ii) direct involvement of eIF3 in alternative translation initiation, (iii) subtle differences in mRNA impurity profiles, or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of the m6Am in mRNA.

biochemistry↗

bioRxiv2022

SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response

Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Modernas mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails. We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (m{Psi}), is terminated by a long poly(A) tail (~100 nucleotides) followed by an m{Psi}Cm{Psi}AG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of m{Psi}Cm{Psi}AG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after m{Psi}Cm{Psi}AG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after m{Psi}Cm{Psi}AG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination. Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.

immunology↗