TL;DR
The RModBlock strategy is showcased as a precise, efficient and versatile approach for manipulating RNA modifications, with broad applicability in basic and translational research and its therapeutic potential is highlighted.
Abstract RNA molecules carry a wide range of chemical modifications that play key roles in regulating their structure, stability, and function. These modifications are especially abundant in small and non-coding RNAs, such as transfer RNAs (tRNA), ribosomal RNA (rRNA), and related fragments, where they influence processes from translation to gene silencing. However, studying these modifications remains challenging due to the short length of these RNAs, their strong secondary structures, and the high density of chemical marks, all of which can interfere with standard sequencing workflows. This review summarizes current strategies for detecting and mapping RNA modifications in the small RNome. We outline the diversity of RNA classes and their characteristic modification patterns, and then discuss key methodological advances. These include demodification-based sequencing approaches for heavily modified RNAs, targeted chemical and enzymatic methods for site-specific mapping, and emerging direct RNA sequencing technologies that allow analysis of native molecules without prior conversion. We also highlight orthogonal validation techniques used to confirm modification identity and improve reliability. Together, these approaches provide complementary insights, but no single method is sufficient on its own. Careful experimental design and validation therefore remain essential for accurate and comprehensive analysis of RNA modifications.
FlexiAsCas12a joins the repertoire of Cas12a PAM variants, enabling access to an increasing number of target sequences by Cas12a nucleases, and expands the range of recognized PAM sequences by Cas12a variants to include NATN, NCCN and GTCN sequences.
A new chemically regulated circRNA translation system based on small ligand-induced stabilization of RNA-binding proteins (RBPs), engineered by incorporating RNA motifs into an internal ribosomal entry site and fusing RBPs with small ligand-stabilized conditional proteins is developed.
Chemically modified small interfering RNAs (siRNAs) are proving to be highly effective as therapeutic agents, but the extent and positioning of chemical modifications must be carefully tuned because the RNA interference (RNAi) machinery imposes intrinsic constraints on siRNA architecture. Here, using a SARS-CoV-2-targeting siRNA as a model, we have systematically investigated the positional and extent-dependent tolerance of 4'-thioRNA substitution within functional siRNAs. Single 4'-thioRNA substitution was broadly tolerated across multiple positions, and complete substitution of either the sense or antisense strand preserved robust RNAi activity. In contrast, full substitution of both strands resulted in a pronounced loss of activity. Physicochemical analysis suggested that this loss arises from interference with cleavage and removal of the sense strand during loading of the RNA-induced silencing complex (RISC). Sense-strand segmentation was therefore applied to fully 4'-thio-modified siRNA to mimic the post-cleavage state during RISC loading. This design partially restored RNAi activity, facilitating the development of fully 4'-thio-modified siRNAs with antiviral activity against SARS-CoV-2. Collectively, these findings expand the chemical space available for functional siRNA design.
The effectiveness of the SaCas9 modular base editors, the robustness of the platform’s modularity, and its feasibility for convenient screening of target-specific base editors are demonstrated.
Conditional activation of antisense oligonucleotides (ASOs) is a promising strategy for selective suppression of cancer cells without affecting normal cells. In this study, we developed a tripled-stranded ASO (tsASO) that is rendered inactive through complexation with two additional oligonucleotides. The key innovation is the use of partial overlap between the parent ASO and the biomarker sequence, combined with toehold-mediated strand displacement, enabling precise conditional activation. The tsASO effectively triggered RNase H-mediated degradation of DYNC1I2 and DARS1 RNAs exclusively in the presence of the ERBB2 sequence. In cell-free systems, the tsASO demonstrated high cleavage efficiency (up to 81%), comparable to the parent ASO efficiency, with minimal background activity in the absence of the biomarker sequence, validating the concept at the molecular level. However, in cells using lipid-based transfection, the tsASO exhibited nonspecific cytotoxicity that did not correlate with biomarker presence or target gene expression. Detailed analysis showed no clear support for known sequence-driven toxicity mechanisms (CpG/TLR9, G-quadruplexes) in the nonimmune cell lines, suggesting that the primary limitation is intracellular delivery rather than the tsASO design. Future work should focus on optimizing delivery platforms to achieve controlled cellular uptake and biomarker-dependent release, unlocking the therapeutic potential of this conditional gene silencing approach.