The unbiased simulations offer insights into the initial stages of the RNA transfection, in particular the fusion of LNP and ensosomal membrane. The LNP diffuses inside the vesicle and approaches the vesicle membrane, separated by a thin layer of water (Figure ). The lipid surfaces come close enough that individual lipids can interact. Negatively charged PAPS interacts with the positively charged IL, and one of them is pulled out of the lipid layer, lying with its tail on the lipid surface (lipid handshake, Figure ). If the lipid adopts an extended conformation and embeds its other tail into the lipid vesicle (in the case of IL) or LNP (in the case of PAPS), fusion begins (Figure ). Once the two lipid compartments are connected by this single lipid, fusion is initiated and progresses rapidly (Figure ). LNP fusion with the lipid vesicle induces deformation of the vesicle and mixing of LNP and vesicle lipids. Initially, the intact lipid vesicle maintained a spherical shape, but the fusion process disrupted this geometry, leading to elongations and other deformations of the vesicle (Figure ). Cholesterol from both the LNP and vesicle intermingled and became evenly distributed throughout the final structure. DSPC diffused into the inner leaflet of the vesicle, while a small fraction of ILs flipped to the outer leaflet. However, the majority of ILs remained in contact with RNA (Figure ). In the final configuration, the RNA was predominantly surrounded by cholesterol (originating from both the vesicle and the LNP) and some residual DSPC. Notably, very few other vesicle lipids, apart from cholesterol, interacted with RNA by the end of the simulation. The RNA-rich compartment was encased in a lipid monolayer primarily composed of vesicle lipids (Figure ). Although the induced deformations and RNA-lipid clustering might impact the stability of the resulting particle, we did not observe any RNA translocation outside the vesicle. This phenomenon has been reported for dsDNA in lipoplexes , but for RNA in LNPs, it remains an open question.