Sebastian Golojuch

Chemically modified nucleic acids such as antisense oligonucleotides, small interfering RNA and CRISPR guide RNA have emerged as transformative modalities in modern medicine, providing new strategies for the treatment of genetic and metabolic disorders, viral infections and cancer. However, their application is limited by challenges related to intracellular delivery and targeting, advances in which would benefit from a broader range of methods for chemical functionalization. Here we describe a streamlined approach using inexpensive, bench-stable alcohols or nucleosides that enables site-selective modification at the 5′ and 3′ termini and at internal sites of synthetic oligonucleotides. Our strategy offers a conceptually straightforward solution to this problem through the in situ generation of phosphoramidites on the solid-supported oligonucleotide. The methodology displays high coupling efficiency, high functional-group compatibility with azides, amines and thiols, and efficient recovery of excess alcohols/monomers, thus offering a robust platform for advancing and diversifying therapeutic oligonucleotide modification.

While cap-dependent translation remains the primary focus in mRNA-based therapeutics, cap-independent translation holds promise for targeting diseases ranging from cancer to neurodegeneration. However, cap-independently translated mRNAs are unstable, produce less protein than capped mRNAs, and current methods for their improvement are imperfect. Here, we propose the use of in vitro transcription priming with azido-modified dinucleotide primer and post-transcriptional modification utilising click chemistry to improve the properties of cap-independently translated mRNAs. Our results demonstrate a significant enhancement in mRNA stability and protein output without eliciting immunogenicity. Moreover, we show how the mRNA 5′-end modification strategy can be used to investigate transfection and cap-independent translation processes in cells overcoming burdens associated with previous methods. Together, our findings support cap-independent translation as a viable alternative to the established cap-dependent process and provide tools for further exploration and enhancement of this modality.

SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 μM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay