Alternative probe chemistries for single-molecule analysis of long noncoding RNA

Single-molecule microscopy has been widely used to study the structure and dynamics of RNA, but extension to larger systems such as long noncoding RNA (lncRNA) has proven challenging. Methods such as single-molecule kinetic analysis of RNA transient structure (SiM-KARTS), where the binding of a short, complementary oligonucleotide probe is used to determine accessibility of a specific region of the RNA, are promising. However, adapting SiM-KARTS to systems as complex as lncRNA requires careful optimization of experimental variables that have not been thoroughly explored. In this work, SiM-KARTS, thermal denaturation experiments, and CD spectroscopy were used to analyze the binding behaviors of probes with alternative backbone chemistries, specifically DNA with locked nucleic acid (LNA) residues incorporated and morpholinos. A segment of lncRNA that enabled control over the accessibility of the target sequence was used as a model. We show that optimizing probe backbone chemistry can allow for a more precise distinction between different structures of the target RNA, and for fine-tuning of probe binding stability without the structural impacts that other variables such as ionic concentration may have. Specifically, we demonstrate that LNA probes exhibit a high degree of structural sensitivity in both their binding and unbinding kinetics. We further show that when binding and unbinding rates are considered holistically, LNA probes allow traces arising from different target RNA structures to be individually classified with a high degree of accuracy. These results provide design principles for the application of SiM-KARTS to target RNAs of increased complexity such as lncRNA.