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To investigate RNA degradation in live cells, detection methods that do not require RNA extraction from cells are necessary. In this study, we examined the utility of fluorescence lifetime measurements using a probe attached to the end of an RNA molecule for detecting RNA degradation. We optimized a short fluorescein-labeled RNA sequence whose fluorescence lifetime varied significantly before and after degradation. The selected HHG-fluorescein sequence (H = U, C, or A) is a promising RNA labeling unit (fluorescence lifetime probe) for live cell imaging of RNA degradation.

Many cellular events are thought to be controlled by the temporal upregulation of multiple RNAs; the timing of the upregulation of these RNAs is not always the same. In this study, we first show that our light-directed intracellular RNA delivery method induced high concentrations of RNA in a short period. This effect was beneficial for the temporal control of cellular events by functional RNAs. Next, we stimulated the short-term upregulation of two different RNAs at different time points. Cytosolic delivery of a first RNA was induced by red light; thereafter, cytosolic delivery of a second RNA was induced by near-infrared light. The time difference between the introduction of the first and second RNA can be short (0.5-4 h) or long (>8 h). This strategy shows the potential for future applications of the deliberate control of time-dependent RNA concentration to guide various cellular functions by multiple RNAs.

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) may potentially treat a wide variety of diseases through RNA interference-mediated silencing of specific genes. MicroRNAs (miRNAs) are endogenous regulatory RNA molecules that also modulate gene expression, and thus potential therapeutic approaches using miRNAs have attracted attention. For clinical application of these small RNAs, efficient and safe RNA delivery to target tissues and cells is necessary. Current challenges to RNA delivery are the penetration of negatively charged RNAs through the cell membrane and specific delivery of RNA into target cells. Photocontrolled intracellular RNA delivery is a promising strategy with high target specificity. This strategy includes photodependent endosomal escape of RNA or photodependent release of RNAs from carrier particles. In this chapter, photocontrolled intracellular RNA delivery methods employing gold or silver nanoparticles, upconversion nanoparticles, proteins, or polymers are discussed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

[Display omitted] Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore–PNA–quencher (fPq) and a quencher–DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA–DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis. [ABSTRACT FROM AUTHOR]

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNALeu or Phe-tRNAPhe as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNALeu or Phe-tRNAPhe, is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched β-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys. [ABSTRACT FROM AUTHOR]

[Display omitted] Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics. [ABSTRACT FROM AUTHOR]