Single-Molecule Analysis of the Improved Variants of the G-Quadruplex Recognition Protein G4P

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al. synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity toward G4s than the previously published BG4 antibody. To get insight into G4P- G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein's affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.

Automated summary

As many as 700,000 unique sequences in the human genome can fold into G-quadruplexes (G4s), which play important roles in various cellular processes. A small protein called G4P has been synthesized and shown to bind G4 structures with better selectivity than previous methods. By studying its interaction with different G4s, the researchers found that increasing the number of recognition motifs in G4P enhances its affinity for certain G4s and its ability to interact with sequences containing multiple G4s.