We hypothesized that partially-PS-modified probes may exhibit an optimal balance of nuclease resistance while avoiding excess nonspecific binding. To test this, MBs were synthesized with different numbers and distributions of PS linkages. In a variety of cell types including HEK293, HeLa, Jurkat, and primary BJ cells, we observed a general trend of MB performance relative to the degree of PS modification (Figure 2). For example, 2Me MBs and 2Me/PSSTEM MBs, which have a fully PS-modified stem, were both highly susceptible to nonspecific opening . Incorporating PS in the loop domain significantly improved MB stability, as 2Me/PS10-LOOP MBs exhibited lower FPSs than 2Me/PSSTEM MBs, despite having the same total number (10) of PS modifications. Consistent with this observation, 2Me/PSLOOP MBs, which have a fully-PS-modified loop domain and a phosphodiester stem, exhibited even lower FPSs than 2Me/PS10-LOOP MBs . Only 1%−3% of the 2Me/PSLOOP MBs opened nonspecifically within 10 h after delivery into several different cell types . FPSs generated by 2Me/PSLOOP MBs were lower than those generated by 2Me/PSFULL MBs, suggesting that when the loop domain is highly PS-modified, stem domain modification is disadvantageous, as the additional PS groups can induce nonspecific binding while offering no additional increase in nuclease resistance. Overall, these findings demonstrate the feasibility of reducing the number of PS modifications in the MB backbone to reduce nonspecific binding while maintaining nuclease resistance.Figure 2Nonspecific opening of non-PS-modified and PS-modified MBs in living cells over timeFollowing microporation of 5 µM MBs that have no endogenous RNA targets into HeLa, HEK293, Jurkat, or primary BJ cells, the extent of MB opening was quantified over the course of 10 h . The MBs tested include 2Me (●), 2Me/PSSTEM (○), 2Me/PS10-LOOP (▴), 2Me/PSALT (△), 2Me/PSLOOP (■), and 2Me/PSFULL (□). Data are presented as mean ± S.E. from at least 30 cells. B. Representative images of MBs in HeLa cells, acquired at 10 h post microporation. The inset shows an expanded segment of the image. Arrows point to bright spots indicative of nonspecific binding in the nucleus (Scale bar, 10 µm). PS, phosphorothioate; MB, molecular beacon; 2Me, 2′-O-methyl. The graphs and images are reproduced with permission from Elsevier .