Crystallographic analysis of G-clamp-RNA complex assisted by large scale RNA-binding profile.

We present the X-ray crystal structure of a complex between a G-clamp and an internal loop motif of pre-mir-125a, selected from high affinity RNAs identified in a large-scale RNA-binding profile. This X-ray crystal structure reveals that the G-clamp interacts with three distinct guanine bases, forming robust bonds through hydrogen bonding and stacking interactions.

Visualizing liquid-liquid phase transitions.

Liquid-liquid phase condensation governs a wide range of protein-protein and protein-RNA interactions in vivo and drives the formation of membrane-less compartments such as the nucleolus and stress granules. We have a broad overview of the importance of multivalency and protein disorder in driving liquid-liquid phase transitions. However, the large and complex nature of key proteins and RNA components involved in forming condensates such as stress granules has inhibited a detailed understanding of how condensates form and the structural interactions that take place within them.

Large-scale analysis of small molecule-RNA interactions using multiplexed RNA structure libraries.

The large-scale analysis of small-molecule binding to diverse RNA structures is key to understanding the required interaction properties and selectivity for developing RNA-binding molecules toward RNA-targeted therapies. Here, we report a new system for performing the large-scale analysis of small molecule-RNA interactions using a multiplexed pull-down assay with RNA structure libraries. The system profiled the RNA-binding landscapes of G-clamp and thiazole orange derivatives, which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively.

Large-Scale Analysis of RNA-Protein Interactions for Functional RNA Motif Discovery Using FOREST.

RNA transcripts can form a variety of higher-order structures. We developed a large-scale affinity analysis system, FOREST (Folded RNA Element Profiling with Structure Library), to investigate the function of these RNA structures on transcriptome-wide scale. Here we describe a protocol to analyze RNA-protein interactions using FOREST .

RNA structure-wide discovery of functional interactions with multiplexed RNA motif library.

Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets.