Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis. [1] tRNAs have a highly stable structure that can present a challenge for their detection using conventional techniques. [2] To enable signal amplification and lower detection limits, a split probe - split deoxyribozyme (sDz or BiDz) probe, which uses a double-labeled fluorogenic substrate as a reporter – has been introduced. In this project we developed an assay based on sDz probe to detect yeast tRNAPhe as a proof-of-principle highly structured target. An sDz probe was designed specific to tRNAphe that could efficiently unwind stable secondary and tertiary structure of the target RNA thereby providing an efficient tool for tRNA detection. [3]The efficiency of the developed sDz probe was compared with a currently used state-of-the-art hybridization probe – molecular beacon probe. The results obtained in the project further demonstrate the power of sDz probes for the detection of highly structured RNA analytes. The split probes show signal amplification capabilities in detection of structured analytes, which will benefit diagnostics, fundamental molecular biology research and therapeutic fields.