Visualization of the Delivery and Release of Small RNAs Using Genetic Code Expansion and Unnatural RNA-Binding Proteins.

Endogenously expressed noncoding RNAs are regulators of mRNA translation and affect diverse biological pathways spanning embryogenesis to cholesterol and fatty acid metabolism. Recently, microRNAs have become an important therapeutic target with strategies that employ oligonucleotides as both mimics and inhibitors of target microRNAs, successfully altering gene expression and cellular pathways in relevant contexts. However, delivery of these exogenous effectors remains a major challenge. Here, we present a method for evaluating noncoding RNA delivery using the viral suppressor of RNA silencing (VSRS) protein p19, optimized for cellular delivery of small RNAs. Using genetic code expansion technology, p-azidophenylalanine (AzF) was incorporated into a recombinant p19 protein and used to develop a fluorescence resonance energy transfer (FRET) sensor. AzF was used to attach FRET acceptor moieties using bioorthogonal chemistry. We show that this strategy not only gives rise to FRET signals that report on small RNA binding, but also allows for fluorescence quenching as well, convenient for measuring RNA release. We demonstrate the successful use of a modified version of the probe to track the delivery and release of small RNAs into mammalian cells. The results provide a basis for a further development of vehicles for small RNA delivery and release for intervening in noncoding RNA biology.

Neoantigen-specific T cell help outperforms non-specific help in multi-antigen DNA vaccination against cancer.

CD4+ T helper antigens are essential components of cancer vaccines, but the relevance of the source of these MHC class II-restricted antigens remains underexplored. To compare the effectiveness of tumor-specific versus tumor-unrelated helper antigens, we designed three DNA vaccines for the murine MC-38 colon carcinoma, encoding CD8+ T cell neoantigens alone (noHELP) or in combination with either "universal" helper antigens (uniHELP) or helper neoantigens (neoHELP). Both types of helped vaccines increased the frequency of vaccine-induced CD8+ T cells, and particularly uniHELP increased the fraction of KLRG1+ and PD-1low effector cells. However, when mice were subsequently injected with MC-38 cells, only neoHELP vaccination resulted in significantly better tumor control than noHELP. In contrast to uniHELP, neoHELP-induced tumor control was dependent on the presence of CD4+ T cells, while both vaccines relied on CD8+ T cells. In line with this, neoHELP variants containing wild-type counterparts of the CD4+ or CD8+ T cell neoantigens displayed reduced tumor control. These data indicate that optimal personalized cancer vaccines should include MHC class II-restricted neoantigens to elicit tumor-specific CD4+ T cell help.