Synthetic 5′ caps & mRNA engineering

Messenger RNAs (mRNAs) have recently entered the public stage as highly versatile therapeutic modalities. We aim to advance messenger RNA (mRNA) by a) identifying and developing new ways of controlling mRNA activities and their therapeutic potential at the molecular level and b) preparing and studying novel synthetic 5′-capped mRNAs for trigger- or cell-type specific control

Messenger RNAs (mRNAs) have recently entered the public stage as highly versatile therapeutic modalities (1). Prominent examples are the mRNA-based vaccines by Moderna and BioNTech/Pfizer against infection by SARS-Cov2. In addition to antiviral vaccines, mRNA holds enormous potential toward a broad spectrum of applications including cancer immunotherapies, protein-supplement therapies, genome editing, and cell re-programming (2). Clinically proven examples include mRNA-based vaccines containing modified nucleotides and 5′ cap structures, allowing potential vaccines against newly sequenced pathogens to be designed and synthesized rapidly, then advanced through the regulatory process in months rather than years.

Light-induced mRNA translation

We have devised a technique, termed FlashCap, that controls the translation of any mRNA by light (3). The translation initiation starts on the 5′ end of the mRNA, which is naturally protected against decay by a 5′ cap (4). The production of mRNAs, including the addition of synthetic caps, is well established in hundreds of research groups and in a considerable number of companies, even for large quantities (up to 50 litres in vaccine production). FlashCaps add to the natural 5′ cap of the mRNA additional synthetic photocaging groups that completely prevent the translation of the mRNA. These synthetic photocaging groups can be removed by irradiation with light, which leads to the release of the natural 5′ cap structure and enables the immediate translation of the mRNA (Figure 1). In simple terms, we developed light-activation of mRNA expression. FlashCaps are non-toxic and compatible with the established production methods for mRNA. In addition, there is no indication to date that they would have undesired effects on cells. The released mRNA is indistinguishable from the natural mRNA. FlashCaps are therefore the first applicable solution to make mRNA studies controllable by light, without requiring new production steps and without introducing artefacts into measurements.

Figure 1: Concept of FlashCaps for light-induced translation. A single photo-cleavable group at the cap 0 impairs binding to eIF4E. FlashCaps are compatible with routine protocols for transcription and transfection. Following light-induced deprotection, the native mRNA with a 5′ cap 0 is released and translated.

This approach is highly interdisciplinary, ranging from synthetic chemistry to cell and developmental biology. Of note, FlashCaps can be directly used in transcription reactions and can be applied by any researcher in the life and medical sciences for any mRNA of interest to conveniently analyze protein function with a spatio-temporal resolution.

A significant advancement in our research is the Medronate-FlashCap, which features a methylene group between the β and γ phosphates (Figure 2). This modification not only strengthens its resistance to degradation by the decapping enzyme DcpS but also increases stability within cell lysate. When these caps are incorporated into mRNAs, they produce translationally muted mRNAs that can be activated to produce proteins upon brief light exposure. This capability is crucial for targeted gene expression studies and the refinement of mRNA-based therapeutic strategies.