KARR-seq is a method that uses N3-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA-RNA interactions and higher-order RNA structures within cells, independent of local protein binding. This technique allows for the detection of widespread intermolecular RNA-RNA interactions with high sensitivity and accuracy. Using KARR-seq, researchers identified that translation represses mRNA compaction under native and stress conditions. They also determined the higher-order RNA structures of respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) and identified RNA-RNA interactions between the viruses and host RNAs that potentially regulate viral replication. RNA functions are primarily determined by their higher-order structures, which include the assembly and organization of multiple RNA secondary structural elements and three-dimensional (3D) RNA conformations mediated by other biomacromolecules. However, these structures are difficult to determine due to their highly dynamic nature, especially when responding to perturbations. High-throughput sequencing-based approaches have been applied to reveal RNA-RNA interactions across the transcriptome. Psoralen-based methods, such as PARIS, RAP-RNA, LIGR-seq, SPLASH and COMRADES, directly capture RNA duplexes and were applied to study the impact of pairwise RNA interactions on RNA metabolism. These methods predominantly capture base-pairing interactions but may miss the distance information between spatially proximal single-stranded RNA fragments. Protein-mediated approaches, including CLASH, RPL, MARIO and RIPPLiT, complement psoralen crosslinking by revealing physical distances between RNAs, but they usually exhibit limited sensitivity for transcripts with modest expression levels and mostly capture RNAs bound by particular proteins. RIC-seq improved the sensitivity by performing protein-mediated proximity ligation in cells and incorporating biotinylated nucleotides during the ligation step. However, because the local protein concentration and the strength of protein-RNA association are spatially heterogeneous, RNA regions with weak protein-RNA engagement could be underrepresented. Therefore, it is desirable to develop technologies that do not rely solely on protein-RNA crosslinking to study higher-order RNA structures and RNA-RNA interactions. KARR-seq accurately reveals RNA tertiary structures and identifies intermolecular RNA-RNA interactions. Using KARR-seq, researchers showed that cytoplasmic mRNA has less compact structures than their nuclear counterparts, with translation resolving higher-order RNA structures under native and stress conditions. They detected RNA-RNA interactions that affect pre-rRNA processing. Furthermore, they mapped the tertiary structures of RSV and VSV RNAs and detected hundreds of interactions between viral and host RNAs. Host mRNAs that interact with RSV and VSV enrich different molecular pathways. The blockage of RNA-RNA interactions between RSV RNA and host mRNAs represses RSV replication. KARR-seq thus enables precise and sensitive mapping ofKARR-seq is a method that uses N3-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA-RNA interactions and higher-order RNA structures within cells, independent of local protein binding. This technique allows for the detection of widespread intermolecular RNA-RNA interactions with high sensitivity and accuracy. Using KARR-seq, researchers identified that translation represses mRNA compaction under native and stress conditions. They also determined the higher-order RNA structures of respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) and identified RNA-RNA interactions between the viruses and host RNAs that potentially regulate viral replication. RNA functions are primarily determined by their higher-order structures, which include the assembly and organization of multiple RNA secondary structural elements and three-dimensional (3D) RNA conformations mediated by other biomacromolecules. However, these structures are difficult to determine due to their highly dynamic nature, especially when responding to perturbations. High-throughput sequencing-based approaches have been applied to reveal RNA-RNA interactions across the transcriptome. Psoralen-based methods, such as PARIS, RAP-RNA, LIGR-seq, SPLASH and COMRADES, directly capture RNA duplexes and were applied to study the impact of pairwise RNA interactions on RNA metabolism. These methods predominantly capture base-pairing interactions but may miss the distance information between spatially proximal single-stranded RNA fragments. Protein-mediated approaches, including CLASH, RPL, MARIO and RIPPLiT, complement psoralen crosslinking by revealing physical distances between RNAs, but they usually exhibit limited sensitivity for transcripts with modest expression levels and mostly capture RNAs bound by particular proteins. RIC-seq improved the sensitivity by performing protein-mediated proximity ligation in cells and incorporating biotinylated nucleotides during the ligation step. However, because the local protein concentration and the strength of protein-RNA association are spatially heterogeneous, RNA regions with weak protein-RNA engagement could be underrepresented. Therefore, it is desirable to develop technologies that do not rely solely on protein-RNA crosslinking to study higher-order RNA structures and RNA-RNA interactions. KARR-seq accurately reveals RNA tertiary structures and identifies intermolecular RNA-RNA interactions. Using KARR-seq, researchers showed that cytoplasmic mRNA has less compact structures than their nuclear counterparts, with translation resolving higher-order RNA structures under native and stress conditions. They detected RNA-RNA interactions that affect pre-rRNA processing. Furthermore, they mapped the tertiary structures of RSV and VSV RNAs and detected hundreds of interactions between viral and host RNAs. Host mRNAs that interact with RSV and VSV enrich different molecular pathways. The blockage of RNA-RNA interactions between RSV RNA and host mRNAs represses RSV replication. KARR-seq thus enables precise and sensitive mapping of