What are the strategies for minimizing MB false-positive signals in living cells?

Minimizing MB false-positive signals in living cells can be done by improving overall MB biostability. To do this, an MB may be synthesized with a backbone entirely partially modified with unnatural nucleotide analogs (e.g. LNA nucleotides, 2 Me/PS nucleotides). This way, there is improved bistability compared to an MB with a backbone composed of DNA or 2 Me nucleotides only. Another strategy is to minimize MB nuclear entry. An MB can be attached to either a nanoparticle, a tRNA molecule, or an siRNA-type element. Additionally, Dual-FRET MB can be used to minimize false-positive signals in living cells. In this method, there are 2 distinct MBs, one labeled with a donor fluorophore at the 3’ end and the other labeled with an acceptor fluorophore at the 5’ end. They are used to target adjacent regions on the same target RNA molecule. Shared-stem Mbs are used because the distance between the donor and acceptor fluorophores can be fixed to yield a stable FRET signal.