Figure 3. DNA probe competition game to study encounter preference.

a, Schematic showing DNA probe competition game results among diacyllipid, cholesterol and tocopherol anchors on the Ramos cell membrane. Initially, 150 nM cholesterol, 300 nM diacyllipid and 400 nM tocopherol conjugates were incubated with 5 × 10⁵ Ramos cells ml⁻¹. b, Fluorescence microscopy study of the TCC competition game on the Ramos cell membrane. In this game, BHQ-2 quencher-labelled cholesterol (cholesterol-S1-BHQ-2/B) was given two possible destinations, TMR-labelled cholesterol (red, cholesterol-S2/W-TMR) or Quasar 670-labelled tocopherol (green, tocopherol-S2/W-Quasar 670) anchor sites. The quenching of TMR fluorescence (‘winning’ site) was apparently faster than that of Quasar 670 fluorescence (‘losing’ site), indicating that cholesterol–cholesterol encounters are preferred than those of cholesterol– tocopherol. Scale bar, 50 µm. c, Route selection was between Dabcyl-labelled anchor (red bar) and non-labelled anchor (blue bar). In the divergent stacked bar chart, all the bar length represents 100%. The left-shifted bar indicates a preferred X–Y encounter, while right-shifted bar shows that a Y–Z encounter was preferred. Taking C–T–L as an example, in this game, dye-labelled probe strand initially conjugated with tocopherol anchor T is given two possible destinations, quencher-labelled cholesterol C or unlabelled diacyllipid L anchor sites. Here, the divergent stacked bar chart indicates 54% tocopherol anchor will encounter preferably with a cholesterol anchor (C–T route), while 46% encounters with a diacyllipid anchor (T–L route). Original flow cytometry data are shown in Supplementary Fig. 8 for three repeated measurements. The relative route choice was calculated based on equation (10) in the Methods. Gibbs free energy changes (ΔG) were calculated based on the corresponding competition results.