摘要TMPRSS2 plays a crucial role in facilitating the entry of both the influenza virus and the SARS-CoV-2 coronavirus into host cells.Recent studies have identified a guanine-rich sequence in the proximal promoter region of the TMPRSS2 gene,which can form G-quadruplex structures(TMPRSS2-G4s)that are potential targets for small molecules to inhibit TMPRSS2 expression.However,the structural details of the major TMPRSS2-G4 and its complex with small mo-lecules remain unknown,hindering the development of antiviral drugs targeting TMPRSS2-G-quadruplexes(G4s).This study reports the first high-resolution nuclear magnetic resonance(NMR)solution structure of the major TMPRSS2-G4,which consists of a three-tetrad core par-allel-stranded G4.Both 3'and 5'flanking regions form well-defined capping structures stabil-ized by multiple hydrogen bonds.Importantly,we found that berberine,an antiviral alkaloid,strongly binds to the major TMPRSS2-G4 and determined its binding complex structure with TMPRSS2-G4 at a 2∶1 binding stoichiometry.Each berberine molecule recruits an adjacent flanking residue,forming a coplanar structure superimposed on two outer G-tetrads.Moreover,we demonstrated that the major TMPRSS2-G4 can stably form within a longer deoxyribonucleic acid(DNA)context and be targeted by small molecules to inhibit DNA poly-merase activity.Overall,this study provides structural insights into the recognition mechan-ism of small molecules by the major TMPRSS2-G4 and may facilitate the development of novel antiviral therapeutics targeting TMPRSS2-G4.