Probing transcript RNA G-quadruplexes using an engineered RHAU-RNase HI enzyme.

G-quadruplexes (G4s) in functional RNAs regulate telomere maintenance, mRNA processing, and disease pathways, but their detection in long transcripts is limited by biophysical methods. Herein, we engineered RHAU-RNase HI, fusing the G4-binding RHAU domain (aa 53-192) with RNase HI (Bacillus halodurans), to probe RNA G4s via enhanced RNA-DNA hybrid cleavage with antisense oligonucleotides (ASOs). The results showed RHAU-RNase HI fusion protein selectively binds G4-containing R1 RNA while RNase HI alone showed no detectable binding. Both RHAU-RNase HI and RNase HI enzymes exhibited comparably low cleavage efficiencies toward G4-free RNA substrate (R2) under standardized condition. In contrast, RHAU-RNase HI exhibited approximately 4-fold greater cleavage activity than RNase HI toward the G4-containing RNA substrate (R1). In addition, RHAU-RNase HI exhibited approximately 8-fold higher cleavage efficiency at the G4-proximal site than the G4-distal site. Collectively, our results show that RHAU-RNase HI recognizes G4 structures and enhances the cleavage of RNA-DNA hybrids adjacent to G4 motifs. These findings highlight RHAU-RNase HI as a promising biochemical tool for G4 structure mapping in complex RNA sequences.