Development of the binding molecules for the RNA higher-order structures based on the guanine-recognition by the G-clamp

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

“…1b). [31][32][33] G-clamp was used to validate our system because it binds strongly to a wide range of RNAs. Conversely, the TO derivatives, TO-PRO-1 and TO-PRO-3, are known as uorescent light-up probes for imaging and uorescent indicator displacement (FID) assays (Fig.…”

“…These results corresponded to the fact that G-clamp mostly recognizes G base in the ssRNA regions. 32 Next, to validate our screening platform for RNA structures, we selected 17 sequences from the higha nity (top 100), intermediate-a nity (101-1000), and low-a nity (1001-1824) groups and measured their apparent dissociation constants (K Dapp ) by uorescence titration (Figure S4). The RNA motifs with three base pairs of a common stem (5′-AGC-motif-GCU-3′) were used to measure K Dapp .…”

Development of a selective ligand for G–G mismatches of CGG repeat RNA inducing the RNA structural conversion from the G-quadruplex into a hairpin-like structure

“…The synthesis of the G-clamp-dimer ligand is summarized in Scheme 1. The carboxylic acid intermediate 3 was prepared according to previously reported methods 18,22 and subjected to a condensation reaction with the mono-Boc protected tris(2aminoethyl)amine linker 4 to afford compound 5. Finally, the Cbz-and Boc-protecting groups were simultaneously removed using thioanisole and TFA followed by purification using reverse phase HPLC to obtain the pure compound 2.…”

“…1A) consisting of 1,3-diazaphenoxazine (G-clamp) units linked with 3,3′-diamino- N -methyldipropylamine, which formed hydrogen bonds with unpaired or mispaired guanines in the stem-loop RNA. 18 The 1 : 1 binding mode with a nanomolar dissociation constant ( K d ) was confirmed for the target guanines, whereas there was no binding to the guanine paired with the cytosine base in the stem region. In addition, the ligand's fluorescence increased in response to the binding with the guanine base in the RNA.…”

“…Our G-clamp-dimer takes a closed form in aqueous buffered solution, which causes self-quenching due to the intra-molecular stacking interaction. 18 The self-quenched form is opened through RNA binding, resulting in a light-up response based on the recovery of the 480 nm fluorescence emission from the self-quenching state (Fig. S3A †).…”