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.

The hybridization between two complementary strands of nucleic acid is the basis for a number of applications in DNA and RNA analysis, including in vivo RNA monitoring, microarrays, SNPs detection, and so on. The short oligonucleotide probes form Watson/Crick base pairs (A-T and G-C) with the analyzed nucleic acid. Molecular beacon (MB) probe is one of the most advantageous tools for nucleic acid analysis in real-time. A traditional MB probe consists of a DNA strand folded in hairpin motif with a fluorophore attached to the 5'end and a quencher attached to the 3' end. The loop segment is complementary to the analytes. Upon hybridization to a complementary single-stranded nucleic acid, MB probe switches to the elongated conformation, which separates the fluorophore from the quencher, resulting in high fluorescence signal. However, DNA or RNA folded in hairpin motifs are difficult to analyze by a conventional MB probes. Inefficient formation of the duplex between the secondary analyte and the MB probe results in low or undetectable fluorescent signal. In this project, we developed a tripartite probe consisting of one MB probe and two adaptor strands to genotype single nucleotide polymorphism (SNPs) in DNA hairpin motifs in real-time fluorescent assays. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to an MB probe. One adaptor strand hybridizes to the analyte and unwinds its secondary structure, and the other strand forms stable complex only with the fully complementary analyte sequence. The tri-component probe promises to simplify nucleic acid analysis at ambient temperatures in such application as in vivo RNA monitoring and isothermal detection of specific DNA/RNA targets.