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Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.

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.

Pancreatic cancer is one of the cancers with very poor prognosis; there is an urgent need to identify novel biomarkers to improve its clinical outcomes. Circulating tumor DNA (ctDNA) from liquid biopsy has arisen as a promising biomarker for cancer detection and surveillance. However, it is known that the ctDNA detection rate in resected pancreatic cancer is low compared with other types of cancer. In this study, we collected paired tumor and plasma samples from 145 pancreatic cancer patients. Plasma samples were collected from 71 patients of treatment-naive status and from 74 patients after neoadjuvant therapy (NAT). Genomic profiling of tumor DNA and plasma samples was conducted using targeted next-generation sequencing (NGS). Somatic mutations were detected in 85% (123/145) of tumors. ctDNA was detected in 39% (28/71) and 31% (23/74) of treatment-naive and after-NAT groups, respectively, without referring to the information of tumor profiles. With a tumor-informed approach (TIA), ctDNA detection rate improved to 56% (40/71) and 36% (27/74) in treatment-naive and after-NAT groups, respectively, with the detection rate significantly improved (p = 0.0165) among the treatment-naive group compared to the after-NAT group. Cases who had detectable plasma ctDNA concordant to the corresponding tumor showed significantly shorter recurrence-free survival (RFS) (p = 0.0010). We demonstrated that TIA improves ctDNA detection rate in pancreatic cancer, and that ctDNA could be a potential prognostic biomarker for recurrence risk prediction

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.