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Gene expression involves concurrent and consecutive events of unidirectional nature, such as transcription occurring from 5' to 3' end and translation from N to C terminus. Recent functional studies have shown the importance of kinetically coupled nucleic acid folding events that influence gene expression processes. For example, mRNA conformational dynamics during transcription and translation regulate gene expression and subsequent protein functionalization. The structure, stability, and kinetic properties of nucleic acids are sensitive to the intracellular molecular environment and can be regulated by using artificially developed molecules. Here, we review our current understanding of how mRNA conformational dynamics affect the consecutive and concurrent processes involved in gene expression and discuss how novel pharmaceutical agents designed to influence RNA conformational dynamics, could be developed to treat various diseases.

mRNAs encodes not only information that determines amino acid sequences but also additional layers of information that regulate the translational processes. Notably, translational halt at specific position caused by rare codons or stable RNA structures is one of the potential factors regulating the protein expressions and structures. In this study, a quadruplex-forming potential (QFP) sequence derived from an open reading frame of human estrogen receptor α (hERα) mRNA was revealed to form parallel G-quadruplex and halt the translation elongation in vitro. Moreover, when the full-length hERα and variants containing synonymous mutations in the QFP sequence were expressed in cells, translation products cleaved at specific site were observed in quantities dependent on the thermodynamic stability of the G-quadruplexes. These results suggest that the G-quadruplex formation in the coding region of the hERα mRNA impacts folding and proteolysis of hERα protein by slowing down or temporarily stalling the translation elongation.

Cotranscriptional folding of an RNA transcript enables formation of metastable RNA structures. Thermodynamic and kinetic properties of RNA G-quadruplex formation have previously been investigated using purified guanine-rich oligonucleotides. Here, we describe a method for analysis of cotranscriptional dynamics of the G-quadruplex formation based on real-time monitoring of the fluorescence of G-quadruplex ligands. For RNA sequences with the potential to form mutually exclusive hairpin or G-quadruplex structures, the efficiency of G-quadruplex formation during transcription depended on position of the hairpin forming sequence. The real-time monitoring enabled evaluation of environmental effects on RNA dynamics, as we demonstrated facilitation of post-transcriptional G-quadruplex formation under molecular crowding conditions. The strategy demonstrated here provides folding insights into the G-quadruplex during transcription that should be involved in gene regulation.

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.

The interaction between proteins and ligands was evaluated by T7 RNA polymerase transcription with a DNA-modified ligand. The principle of this method is suppression of T7 RNA polymerase transcription by binding of a protein to small ligand modified by conjugation with a T7 RNA polymerase promoter. To demonstrate proof of principle, biotin or antifolate methotrexate was modified by covalent attachment of a T7 RNA promoter. Using these T7 RNA promoter-modified ligands, T7 RNA polymerase transcriptions were performed in the presence or absence of an anti-biotin antibody or recombinant human dihydrofolate reductase, respectively. Transcription was suppressed in the presence of each binding protein plus its modified ligand, but not in the absence of the binding protein.

In this study we describe the spatial regulation of RNA interference (RNAi) using an RNA-carrier protein labeled with a fluorescent dye and a light source to trigger the RNAi. We demonstrate photo-dependent gene silencing using several dyes with different excitation wavelengths. Additionally, we use light from a halogen lamp and a photomask to produce photopatterned RNAi, and laser light to trigger single-cell RNAi on cell culture plates.

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as 'bridge-builder' to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA.

HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

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.

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 ...