1,N6-Ethenoadenosine is a fluorescent etheno-bridged adenosine analog widely used as a probe for nucleotide-binding proteins. It serves as an efficient fluorescent substrate and allosteric inhibitor of phosphofructokinase, with binding kinetics exhibiting fast-phase catalytic site association followed by a slow-phase conformational transition from the R to T state. The analog also forms stable DNA lesions, inducing frameshift mutations during translesion synthesis with human DNA polymerase η while being poorly incised by RNase H2, highlighting its utility in studying DNA damage tolerance and repair mechanisms.