To reduce nonspecific signals, MBs have been conjugated to macromolecules that are either too big to traverse the nuclear pores, such as quantum dots  and pegylated NeutrAvidins , or are quickly exported to the cytoplasm, such as tRNAs  and small interfering RNA (siRNA)-like molecules . Alternatively, MBs have been synthesized with degradation-resistant oligonucleotides containing locked nucleic acids (LNA) or modified internucleotide linkages (such as phosphorothioate, PS) , , . By incorporating PS linkages throughout the loop domain of a 2′-O-methyl (2Me) MB backbone, we have recently developed an MB architecture called the 2Me/PSLOOP MB, based on the latter approach . The 2Me/PSLOOP MB exhibits a marginal level of FPSs in various cell types and can be used for imaging single RNA transcripts in living cells . Here we highlight the work undertaken to develop 2Me/PSLOOP MBs and explore their capabilities for single-molecule RNA imaging. We envision that the use of 2Me/PSLOOP MBs to study RNAs in living cells can further our knowledge of the role of RNAs in health and disease.