In-stem molecular beacon targeted to a 5′-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity

Abstract

Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5′- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.

Interrogation of highly structured RNA with multicomponent deoxyribozyme probes at ambient temperatures

“…By increasing the assay's time and/or temperature, it is possible to further improve the LOD. The 70-min LOD for tRNA Phe is about two orders of magnitude lower than the previously reported LOD determined for the in-stem MBP targeting a similar region of the unmodified tRNA transcript (Miyoshi et al 2019).…”

“…S6). Indeed, previous works required tRNA concentrations in the high nM to µM range to trigger the target dependent response (Li et al 2017;Miyoshi et al 2019).…”

“…The loops are involved in tertiary interactions (Shi and Moore 2000), which add to the great thermal stability of the L-shaped native tRNA molecule and resistance to unfolding, and further disfavors tRNA interactions with complementary probe sequences. Even though MBPs that can detect specific tRNA sequences have been previously reported (Li et al 2012;Miyoshi et al 2019), it required designing and testing multiple expensive MBPs to find the one that achieves moderate performance. The optimized MBPs exhibited detection limits of ∼23-27 nM with nonmodified tRNA transcripts as targets, which are known to be less thermodynamically stable than mature tRNA (Serebrov et al 1998).…”

Photocontrolled apoptosis induction using precursor miR-664a and an RNA carrier-conjugated with photosensitizer

“…The resulting template DNA was precipitated with 2-propanol. T7 RNA polymerase was prepared as described previously 21 . The transcription reaction was carried out at 37 °C for 4 h in a reaction mixture containing 40 mM Tris–HCl (pH 8.0), 24 mM MgCl 2 , 5 mM dithiothreitol, 10 mM guanosine monophosphate, 2 mM of each NTP, 1.8 U/mL inorganic pyrophosphatase (Sigma, St. Louis, MO, USA), 26.2 µg/mL purified T7 RNA polymerase, and 10 µg/mL DNA template.…”

FRET probe for detecting two mutations in one EGFR mRNA

“…In recent years, many methods for RNA detection and quantification have emerged, [6][7][8] including polymerase chain reaction (PCR)based methods 9,10 that use cell extracts; however, these methods cannot be applied to living cells. Methods applicable to living cells include those using fluorescent probes, such as fluorescent proteins, [11][12][13] RNA aptamers, 14,15 and molecular beacons, [16][17][18] which bind to target RNAs. These detection methods are widely used in biological research and medical diagnosis, 19,20 but further methodological considerations are required to cope with different cases of medical diagnosis.…”