Although numerous studies have shown that MBs can be used to visualize endogenous RNA in living cells, the susceptibility of MBs to nonspecific interactions and/or nuclease degradation and the resulting false-positive signals has limited their use to the study of highly overexpressed RNA. This has led to the evaluation of alternative nucleic acid chemistries that are intended to confer increased resistance to nuclease degradation and less susceptibility to nonspecific interactions. In this study, we systematically analyzed the subcellular trafficking, false-positive signal generation, and functionality of MBs with 2Me and 2MePS modifications. Interestingly, it was found that 2Me MBs, which are perhaps the most widely utilized MB composition, were highly susceptible to nonspecific interactions and/or nuclease degradation and emitted a false-positive signal that increased with time. These false-positive signals, however, could be completely eliminated simply by attaching the MBs to NeutrAvidin, which prevented their translocation to the nucleus; at least when microinjection was used to deliver the MB-NeutrAvidin conjugates into living cells. Unfortunately, the absence of false-positive signals did not carryover to when microporation was used to deliver the MB-NeutrAvidin conjugates. In addition, false-positive signals could not be eliminated when 2MePS MBs were utilized regardless of whether or not they were conjugated to NeutrAvidin. Therefore, if highly sensitive measurements of RNA expression are required, 2Me MB-NeutrAvidin conjugates seem to provide the best option. This was the only strategy tested that did not result in any detectable false-positive signals. Of course, one concern with incorporating NeutrAvidin into the MB design is that it could impede the free diffusion of the MBs in the cytoplasm, thereby reducing hybridization kinetics. Alternatively, NeutrAvidin could potentially reduce the accessibility of MBs to any RNAs associated with cytosolic proteins. These possible shortcomings must be taken into consideration when using MB-NeutrAvidin conjugates.
When a more high-throughput approach to studying gene expression is required, then 2Me MB-NeutrAvidin conjugates no longer provide a suitable option for highly sensitive measurements of RNA due to the high level of nonspecific MB opening observed following microporation. Even more problematic was that the extent of false-positives increased with time. The 2MePS MBs also opened nonspecifically following microporation, but to a much lower extent and the false-positive signal did not vary with time, making it easier to account for. NeutrAvidin could also be conjugated to 2MePS MBs to reduce nuclear sequestration, although the addition of NeutrAvidin did not seem to result in any significant improvement in reducing the false-positives.
It should be noted that although the results that were presented here were for MEF-3T3 cells, similar findings were also obtained with HeLa and MCF-7 cells (data not shown). Therefore, we believe the behavior observed is true in general and not specific for MEF-3T3 cells.
Overall, these results suggest that the optimal MB backbone may be dependent on the mechanism of delivery. Further, we demonstrate that oligonucleotide modifications can have a profound impact on the fate of MBs in the intracellular environment, highlighting the need to continue evaluating alternative MB designs.