Traditional protein-targeting therapeutics by small molecules can target ~3000 proteins, representing <2% of the human genome. In comparison, ~80% of human genome is transcribed into RNAs, which are mostly targetable by oligonucleotides. Thus, RNA-targeting therapeutics enable a much bigger spectrum of drug targets. With nearly 20 FDA-approved oligonucleotide drugs and numerous ongoing clinical trials, RNA-targeting therapeutics by oligonucleotides have significantly addressed unmet medical needs. However, challenges persist in gene expression enhancement and comprehensive dual-RNA modulation. Here, I set out to address these two challenges. Firstly, I identified two ASOs designed to target intron 1 of FXN pre-mRNA, leading to a ~2-fold increase in FXN mRNA levels in multiple cell models. Despite rigorous controls such as two RNA quantification assays and normalization by multiple housekeeping genes, the FXN activation by these two ASOs wasn’t driven by direct binding to the FXN pre-mRNA or a mutual off-target transcript. Surprisingly, it’s found dependent on guanosine-rich motifs in the full PS backbone, suggesting non-base-paired off-target effects. Secondly, we developed siRNASO, an oligonucleotide scaffold integrating the functionalities of siRNA and ASO into a single molecule. siRNASOs demonstrated potent single RNA silencing and comprehensive dual-RNA modulation, including RNA silencing, splicing modulation, and RNA editing in vitro and in vivo. Notably, siRNASO exhibits excellent tolerability in the mouse CNS, suggesting its potential as a therapeutic platform for CNS disorders. Overall, the research in this thesis will serve as a valuable foundation for future research and therapeutic applications, significantly contributing to the advancement of RNA-targeting therapeutics.