To aid the design of our probes, we first sought to determine whether both palmi,c glyceride chains (di-C16:0 PI5P 1) are required for binding of PI5P to hUHRF1. It has previously been shown that di-C16:0 chains are required for hUHRF1 binding, as di-C16:0 PI5P 1, di-C16:0 PI3P 2 and di-C16:0 PE 3, all bind UHRF1, while di-C8:0 PI5P 4, di-C14:0 PE 5, di-C18:0 PE 6 do not, revealing the selec,vity of hUHRF1 lipid binding (Figure ). However, the ability of UHRF1 to dis,nguish between molecules containing a single C16:0 lipid, in addi,on to another lipid chain of varying lengths, has not been determined. To inves,gate this, we synthesized two PI5P deriva,ves, 7 and 8 (Figure ), containing a mixture of C16:0 and C8:0 famy acid chains. The synthe,c details can be found in the supplementary informa,on (Scheme S1). Microscale thermophoresis (MST) analysis of biding to hUHRF1 confirmed that the di-C16:0 chains were required for protein affinity, as neither C16:0, C8:0 PI5P 7, nor C8:0, C16:0 PI5P 8 showed significant binding to hUHRF1 (Supplementary Figure ). These data confirmed that the development of a func,onalized PI5P probe for hUHRF1 requires minimizing any changes to the structure of the C16 chains. The design of the probe 9 (Figure ), therefore, incorporates