Phospha,dylinositol 5-phosphate (PI5P) is one of seven essen,al membrane phospholipids (phospha,dylinositols/PIs) and is thought to play a crucial role in cellular signalling, cell prolifera,on, the DNA damage repair response, and gene transcrip,on. In comparison to other members of the PI family, PI5P exists in low concentra,ons, and is usually found in the membranes of cellular compartments including in the cell nucleus, 2 the Golgi apparatus, 3 in lysosomes, and in mitochondria. Despite PI5P being discovered over 25 years ago, the metabolism and func,on of PI5P are s,ll poorly understood. Chemical probes capable of revealing the cellular PI5P interactome are scarce, and so any addi,onal probes have the poten,al to greatly enhance our understanding of the biological func,ons of PI5P. Phosphoinosi,des are also known to interact with soluble nuclear proteins in a membrane-independent manner. For example, an X-ray crystal structure of PIP3 bound to the nuclear protein SF-1 (steroidogenic factor-1) reveals that the PIP3 acyl groups are bound within the protein, rather than to the membrane. The charged inositol headgroup is solvent exposed, crea,ng an enhanced binding surface for co-factors, that increases the ac,vity of SF-1. PI5P is thought to be par,cularly important in controlling nuclear processes in a similar manner. It has been reported to modulate ING2-mediated p53 acetyla,on and apoptosis, 9 inhibit Cul3 E3 ligase ac,vity, 10 and alter TAF3-mediated transcrip,on. Of par,cular interest to us, is the role that PI5P plays as an allosteric ac,vator of hUHRF1 [ubiqui,n-like with plant homeodomain (PHD) and really interes,ng new gene (RING) finger domains 1]. hUHRF1 is a fundamental epigene,c regulator that protects the genome by regula,ng DNA methyla,on levels, recognizing and binding damaged DNA, and recrui,ng DNA repair factors. 15 hUHRF1 comprises five domains connected via flexible linker regions (Figure ). These five domains are: the ubiqui,n-like domain (UBL), which directs E3 ligase ac,vity of hUHRF1 towards the N-terminal tail of histone H3 in chroma,n; the Tandem Tudor Domain (TTD), which recognizes methyla,on at lysine 9 (K9Me) of the N-terminal tail of histone H3; the plant homeodomain (PHD), which recognizes the unmodified very N-terminal residues of histone H3 (unmodified arginine 3, H3RMe0); the SET-and RING-associated (SRA) domain, which binds hemimethylated DNA; and the RING domain, which catalyzes ubiqui,na,on of lysine 18 (H3K18) and/or lysine 23 (H3K23) on histone H3 (Figure ). Recently, we have shown that PI5P interacts simultaneously with two distant flexible linker regions of hUHRF1 (Linkers 2 and 4) connec,ng dis,nct domains of the protein. This interac,on enhances the binding of hUHRF1 to H3K9Me3, however, the exact mechanis,c details of how PI5P regulates the func,on of hUHRF1 are yet to be fully explored. To enable further understanding of the PI5P interactome, and probe how PI5P interacts with hUHRF1, we aimed to develop a phospha,dylinositol 5-phosphate-based photoaffinity probe. To achieve this goal, we designed a probe that incorporates both a photoaffinity component, for protein crosslinking, and an alkyne click tag, to enable visualiza,on or enable affinity purifica,on schemes (pull-down) (Figure ). As we have previously shown that even small changes in the lipid chain conforma,on of PI5P can abolish binding to UHRF1, we considered which photoac,vatable func,onal groups and click tag to employ, and where they should be placed to minimize disrup,ons of the PI5P-protein interac,on. Based on these considera,ons, we selected a diazirine-based photo crosslinker, as it is the smallest light ac,vatable group, and an alkyne as the click tag. The alkyne was incorporated at the end of the acyl chain, without altering the length of the chain, to minimally perturb protein binding.