Probing RNA structure and interaction dynamics at the single molecule level.
RNA structures and their dynamic fluctuations lie at the heart of understanding key biological process such as transcription, splicing, translation and RNA decay. While conventional bulk assays have proven to identify and characterize key pathway intermediates, the generally dynamic nature of RNA structures renders the information obtained from time and ensemble averaging techniques necessarily lacking in critical details. Here we detail Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS), a method that readily monitors structural fluctuations of single RNA molecules through the repetitive interaction of fluorescent probes with an unlabeled, surface-immobilized RNA target of virtually any length and in any biological context. In addition, we demonstrate the broad applicability of SiM-KARTS by kinetically fingerprinting the binding of cognate tRNA ligand to single immobilized T-box riboswitch molecules. SiM-KARTS represents a valuable tool for probing biologically relevant structure and interaction features of potentially many diverse RNA metabolic pathways.
['Chauvier|Adrien|A|', 'Cabello-Villegas|Javier|J|', 'Walter|Nils G|NG|']
D001412:Bacillus subtilis D005456:Fluorescent Dyes D007091:Image Processing, Computer-Assisted D007700:Kinetics D008390:Markov Chains D008856:Microscopy, Fluorescence D009690:Nucleic Acid Conformation D009841:Oligonucleotides D015347:RNA Probes D012329:RNA, Bacterial D012343:RNA, Transfer D058928:Riboswitch D000072760:Single Molecule Imaging D012984:Software
RNA structure Kinetic analysis Riboswitch Conformational change Single-molecule fluorescence microscopy