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Compounds that bind specifically to double-stranded regions of RNA have potential as regulators of structure-based RNA function; however, sequence-selective recognition of double-stranded RNA is challenging. The modification of peptide nucleic acid (PNA) with unnatural nucleobases enables the formation of PNA–RNA triplexes. Herein, we demonstrate that a 9-mer PNA forms a sequence-specific PNA–RNA triplex with a dissociation constant of less than 1 nM at physiological pH. The triplex formed within the 5′ untranslated region of an mRNA reduces the protein expression levels both in vitro and in cells. A single triplet mismatch destabilizes the complex, and in this case, no translation suppression is observed. The triplex-forming PNAs are unique and potent compounds that hold promise as inhibitors of cellular functions that are controlled by double-stranded RNAs, such as RNA interference, RNA editing, and RNA localization mediated by protein–RNA interactions.

We determined hybridization rates of DNA probes bound to the surfaces of a human cell, and compared the rates with those determined in solution. The rates were slower on the cell surface than in solution-phase. The position of the nucleation site but not the location of the formed duplex relative to the cell surface adversely affected the hybridization kinetics.

mRNA forms various secondary and tertiary structures that affect gene expression. Although structures formed in the untranslated regions (UTRs) of mRNAs that inhibit translation have been characterized, stable mRNA structures in open reading frames (ORFs) may also cause translational halt or slow translation elongation. We previously established a method, termed a synchronized translation assay, that enables time course analysis of single turnover translation elongation. In this method, translation initiation, which is a rate determining step of the translation procedure, can be ignored because all ribosomes are synchronized on a specific position of mRNA before translation elongation is restarted from this position. In this paper, we used the synchronized translation assay to evaluate the effects of a G-quadruplex structure located at various positions within the mRNA ORF on translational halt.

RNA and protein are potential molecules that can be used to construct functional nanobiomaterials. Recent findings on riboswitches emphasize on the dominative function of RNAs in regulating protein functions through allosteric interactions between RNA and protein. In this study, we demonstrate a simple strategy to obtain RNAs that have a switching ability with respect to protein function in response to specific target molecules. RNA aptamers specific for small ligands and a trans-activation-responsive (TAR)-RNA were connected by random RNA sequences. RNAs that were allosterically bound to a trans-activator of transcription (Tat)-peptide in response to ligands were selected by repeated negative and positive selection in the absence and presence of the ligands, respectively. The selected RNAs interacted with artificially engineered Renilla Luciferase, in which the Tat-peptide was inserted within the Luciferase, in the presence of the specific ligand and triggered the “Lighting-UP” switch of the engineered Luciferase.

RNA interference (RNAi)-mediated silencing of specific genes represents a powerful tool for analyzing protein function. It also has profound biotechnological applications for cellular engineering and therapeutics. However, it is necessary to have a method that controls RNAi in response to artificially regulated stimulation. We designed a fluorescently labeled carrier protein to deliver short hairpin RNA (shRNA) with activity that could be regulated via photostimulation. We constructed a cell-permeable RNA-binding protein (RBP) by fusing the U1A RBP and a HIV-1 Tat peptide, which was labeled with an Alexa Fluor 546 fluorophore (TatU1A-Alexa). TatU1A-Alexa bound specifically to shRNA, which contains a U1A-binding sequence. The TatU1A-Alexa/shRNA complex was then internalized into cells via an endocytotic pathway and redistributed from endosomes to the cytosol by photostimulation, which induced RNAi-mediated gene silencing. This successive strategy was termed CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi).

Small interfering RNA (siRNA) causes sequence-specific gene silencing. In this study, siRNAs were induced into the cells by using a cell-penetrating peptide (CPP) and RNA-binding protein (RBP) as a safe and effective siRNA carrier (CPP-RBP). The diffusion of the siRNAs into the cytoplasm and RNAi mediated gene silencing were induced by photostimulation of Alexa546, which was connected to the CPP-RBP. Here, we investigated the effects of several CPP-RBPs for effective inducing the RNAi.

RNA interference (RNAi)-mediated silencing of specific genes represents a powerful tool for analyzing protein function. It also has profound biotechnological applications for cellular engineering and therapeutics. However, it is necessary to have a method that controls RNAi in response to artificially regulated stimulation. We designed a fluorescently labeled carrier protein to deliver short hairpin RNA (shRNA) with activity that could be regulated via photostimulation. We constructed a cell-permeable RNA-binding protein (RBP) by fusing the U1A RBP and a HIV-1 Tat peptide, which was labeled with an Alexa Fluor 546 fluorophore (TatU1A-Alexa). TatU1A-Alexa bound specifically to shRNA, which contains a U1A-binding sequence. The TatU1A-Alexa/shRNA complex was then internalized into cells via an endocytotic pathway and redistributed from endosomes to the cytosol by photostimulation, which induced RNAi-mediated gene silencing. This successive strategy was termed CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi).

In this study, we constructed fluorescently labelled protein carrier constructed from cell-penetrating peptide (CPP) and RNA binding domain (RBD) for intracellular shRNA or siRNA delivery. The protein carrier specifically bound to an RNA cargo containing short extended sequence tag for protein binding, and internalized into CHO cells together with the RNA. Although the internalized protein/RNA complexes showed cytoplasmic punctuate distributions, they widely spread into cytosol by photo irradiation. Moreover, redistributed RNA induced gene silencing only within the photo irradiated area.

Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detect specific nucleic acids using recombinant protein and oligonucleotides. A recombinant protein YRGnC-11ad, which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) was constructed and expressed in HeLa cells. Rev-peptide, which corresponds to amino acids 34-50 of the HIV-1 Rev protein, indicates disordered structure in solution but forms alpha-helical and elongated conformation upon binding to Rev response element RNA (RRE-RNA) and Rev-aptamer, respectively. We confirmed that YRGnC-11ad could specifically bind to RRE-RNA and Rev-aptamer in cell lysate, and fluorescent resonance energy transfer (FRET) signal was changed upon binding following the conformational change of Rev-peptide. To utilize this FRET signal change toward the detection of specific nucleic acids, we split the RRE-RNA sequence and connected to the complementary oligonucleotide for target nucleic acids. When each two oligonucleotides hybridized to an adjacent region of target nucleic acids correctly, a Rev-peptide binding site was reformed on the hybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase upon binding to the hybridized complex in cell lysate. These results suggest that the recombinant protein probe is available for specific nucleic acid detection.

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

The profiling of microRNAs (miRNAs) using microarray technology has been used in many recent biological and medical studies. In this study, arrays of peptide nucleic acids (PNAs) were prepared and ...

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