Currently, debate over the primary causes of MB FPSs in living cells remains unresolved. Our findings that MBs with different PS modifications exhibit large differences in the degree of nonspecific opening can help explain why MBs open nonspecifically in cells. For example, single-stranded endonucleases appear to be the primary cause of MB nonspecific opening, as levels of FPSs are inversely correlated with the extent of PS modifications in the single-stranded loop domain . Exonucleases appear to have little impact on MB degradation, as PS modifications in the stem have little effect on MB stability . Presumably, the fluorophore and the quencher sterically block access by 5′- and 3′-exonucleases. The tendency of highly-PS-modified MBs to aggregate and emit FPSs is consistent with previous studies showing that PS-modified ODNs are prone to nonspecific binding to cellular proteins , , , , , . Accordingly, the higher nonspecific signals emitted by 2Me/PSFULL than 2Me/PSLOOP MBs suggest that any nuclease resistance gained by PS modifications in the stem domain is offset by increased nonspecific binding due to greater number of PS modifications.