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Recent successes in construction of light-up RNA aptamers allowed fluorescence-based live-cell imaging of RNAs. In addition, light-up aptamers have been converted into signaling aptamers that enable fluorometric detection of small chemicals. To date, only a single target chemical has been detected at a time in cells. In this study, we selected cladogenetic orthogonal light-up aptamers that output three different colors from the RNA library having the same ligand binding core. Two of the three functioned in mammalian cells. These two aptamers, which fluoresce blue and green upon binding of cognate fluorogen, were converted into signaling aptamers. Using these signaling aptamers in combination with a previously described light-up aptamer with red fluorescence, we demonstrated simultaneous detection of multiple chemicals in living cells. The cladogenetic orthogonal light-up aptamers developed in this study and the simple strategy for rational designing of the signaling aptamers will provide innovative advances in the field of RNA-based bioimaging.

Sequence specific recognition and functional inhibition of biomedically relevant double-helical RNAs is highly desirable but remains a formidable problem. The present study demonstrates that electroporation of a triplex-forming peptide nucleic acid (PNA), modified with 2-aminopyridine (M) nucleobases, inhibited maturation of endogenous microRNA-197 in SH-SY5Y cells, while having little effect on maturation of microRNA-155 or -27a. In vitro RNA binding and Dicer inhibition assays suggested that the observed biological activity was most likely due to a sequence-specific PNA-RNA triplex formation that inhibited the activity of endonucleases responsible for microRNA maturation. The present study is the first example of modulation of activity of endogenous noncoding RNA using M-modified triplex-forming PNA.

An RNA signaling aptamer is composed of two units: a sensing aptamer that binds the input target molecule and a working aptamer that binds the output target molecule to result in a detectable signal. A conformational change of the signaling aptamer that induces an allosteric interaction with the output target molecule in response to the input target molecule depends on a junction region, which connects the two aptamer units. Efficient and effective optimization of the junction region remains a technical challenge. In this study, we demonstrate a simple strategy for optimizing the junction region through functional RNA selection using RNA-capturing microsphere particles. From approximately 0.2 million sequence variants, a signaling aptamer that enabled intracellular detection of S -adenosyl methionine with a high signal-to-noise ratio, which is approximately 2-fold higher relative fluorescence increment compared to the previously reported signaling aptamer, was obtained after single round of selection. The technology demonstrated here can be used to select RNA sequences that carry out specific functions in response to particular stimuli.

The variance of sub-20 nm devices is a critical issue for large-scale integrated circuits. In this work, uniform sub-20 nm Si nanopillar (NP) arrays with a reduced diameter variance (to ±0.5 nm) and a cylindrical shape, which can be used for vertical gate-all-around metal-oxide-semiconductor field-effect transistors, were fabricated. For the fabrication process, an array of tapered Si NPs with a diameter of approximately 62.7 nm and a diameter variance of ±2.0 nm was initially fabricated by an argon fluoride lithography followed by dry etching. Then, the NPs were oxidized in a self-limiting region. After the oxide removal, a similar oxidation process was used again for the NPs. It is determined that by controlling oxidation in the self-limiting region, the diameter variance can be reduced in the height direction of Si NPs (as well as shape control) and between NPs, simultaneously with a controllable diameter decrease. This approach decreases the variance in size caused by conventional nanoprocessing and helps overcome the position-dependent variance for 300 mm φ wafers, which is caused by current semiconductor processing., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

Co-transcriptional RNA folding results in dynamic behavior of RNA transcripts. Extra stimuli can perturb the stability of the nascent RNA and alter its conformation and function. Here, an artificial RNA conformational switch, which consists of a 5' aptamer unit for sensing thiamine pyrophosphate (TPP) and a 3' working unit (TAR RNA) that binds Tat peptide connected by a switching sequence, was designed and cotranscriptionally functionalized. Co-transcriptional binding of TPP induced an RNA conformational change that allowed interaction of Tat peptide and the TAR RNA unit. Two conformational states, which are mutually exclusive depending on the TPP binding, did not show post-transcriptional interconversion. The irreversible RNA conformational switch demonstrated here, which kinetically coupled co-transcriptional RNA folding and molecular assembly in nonequilibrium biological systems, will enable efficient switching of RNA functions applicable for constructing intracellular molecular sensors.

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.

Conformational transitions of biomolecules in response to specific stimuli control many biological processes. In natural functional RNA switches, often called riboswitches, a particular RNA structure that has a suppressive or facilitative effect on gene expression transitions to an alternative structure with the opposite effect upon binding of a specific metabolite to the aptamer region. Stability of RNA secondary structure (-ΔG°) can be predicted based on thermodynamic parameters and is easily tuned by changes in nucleobases. We envisioned that tuning of a functional RNA switch that causes an allosteric interaction between an RNA and a peptide would be possible based on a predicted switching energy (ΔΔG°) that corresponds to the energy difference between the RNA secondary structure before (-ΔG°before) and after (-ΔG°after) the RNA conformational transition. We first selected functional RNA switches responsive to neomycin with predicted ΔΔG° values ranging from 5.6 to 12.2 kcal mol(-1). We then demonstrated a simple strategy to rationally convert the functional RNA switch to switches responsive to natural metabolites thiamine pyrophosphate, S-adenosyl methionine, and adenine based on the predicted ΔΔG° values. The ΔΔG° values of the designed RNA switches proportionally correlated with interaction energy (ΔG°interaction) between the RNA and peptide, and we were able to tune the sensitivity of the RNA switches for the trigger molecule. The strategy demonstrated here will be generally applicable for construction of functional RNA switches and biosensors in which mechanisms are based on conformational transition of nucleic acids.

Tertiary structures formed by mRNAs impact the efficiency of the translation reaction. Ribosomal frameshift is a well-characterized recoding process that occurs during translation elongation. Pseudoknot and stem-loop structures may stimulate frameshifting by causing a translational halt at a slippery sequence. In this study, we evaluated the efficiency of an unusual -1 frameshift caused by a noncanonical RNA G-quadruplex structure in mammalian cells. The reporter gene construct consisting of a fluorescent protein and Luciferase enabled evaluation of apparent and absolute values of the -1 frameshift efficiency and revealed significant increase of the efficiency by G-quadrupex forming potential sequence. In addition, berberine, a small molecule that binds to and stabilizes G-quadruplex structures, further increased the frameshift efficiency. These results indicate that the stable G-quadruplex structure stimulates the unusual -1 frameshift and has a potential to regulate the frameshift with its ligand.

Nucleic acid aptamers are receptors of single-stranded oligonucleotides that specifically bind to their targets. Significant interest is currently focused on development of small molecule aptamers owing to their applications in biosensing, diagnostics, and therapeutics involving low molecular weight biomarkers and drugs. Despite great potential for their diverse applications, relatively few aptamers that bind to small molecules have been reported, and methodologies to enhance and broaden their functions by expanding chemical repertories have barely been examined. Here we describe construction of a modified DNA library that includes (E)-5-(2-(N-(2-(N(6)-adeninyl)ethyl))carbamylvinyl)-uracil bases and discovery of high-affinity camptothecin-binding DNA aptamers using a systematic evolution of ligands by the exponential enrichment method. Our results are the first to demonstrate the superior efficacy of base modification on affinity enhancement and the usefulness of unnatural nucleic acid libraries for development of small molecule aptamers.

Monopolar spindle 1 (Mps1) is an attractive cancer drug target due to the important role that it plays in centrosome duplication, the spindle assembly checkpoint, and the maintenance of chromosomal stability. A design based on JNK inhibitors with an aminopyridine scaffold and subsequent modifications identified diaminopyridine 9 with an IC50 of 37 nM. The X-ray structure of 9 revealed that the Cys604 carbonyl group of the hinge region flips to form a hydrogen bond with the aniline NH group in 9. Further optimization of 9 led to 12 with improved cellular activity, suitable pharmacokinetic profiles, and good in vivo efficacy in the mouse A549 xenograft model. Moreover, 12 displayed excellent selectivity over 95 kinases, indicating the contribution of its unusual flipped-peptide conformation to its selectivity.

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.

Bioactive small molecules such as metabolites and drugs play important roles in regulating biological functions. A technique for visualizing such small molecules is very useful to understand their molecular mechanisms. In this study, an RNA-protein conjugate, which consists of an RRE-RNA sensor protein (EYFP-Rev-ECFP) and an altered RRE-RNA, was constructed to detect bioactive small molecules by fluorescent resonance energy transfer (FRET). We designed a theophylline-aptamer-inserted RRE-RNA (Theo-RRE) to detect theophylline as a model target molecule. Theo-RRE formed an RNA-protein conjugate with EYFP-Rev-ECFP in the presence of theophylline. As a result, theophylline was specifically detected down to 10 microM by the FRET increase in distinction from theophylline analogue, caffeine, in cell lysates.

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.

Intermolecular enzyme complementation assay is a useful method for detecting protein-protein interactions. Specifically, bioluminescent signals produced from reconstructed split luciferase fragments are powerful tools for in vivo analysis because the bioluminescent signals have been visualized both in cultured cells and living animals. However, they are limited for detection and evaluation of biological events relevant to intermolecular protein-protein interactions. In this study, we constructed an intramolecular luciferase complementation probe for detecting target biomolecules other than protein-protein interactions. It consists of peptide-inserted firefly luciferase (PI-FLuc) containing a short peptide between internally divided firefly luciferase. The inserted short peptide triggers FLuc complementation or discomplementation and subsequent reactivation or inactivation of FLuc activity through its induced fit conformational changes. We chose RNA binding arginine rich motif (ARM) peptides, Rev and/or Tat, for model peptide insertion, and expressed constructed PI-FLuc probe variants using a wheat germ cell-free protein synthesis system. They showed FLuc activity changes, reactivation, or inactivation after binding to their specific RNA targets. Furthermore, to expand the versatility of the PI-FLuc RNA detection system, we designed split-RNA probes built to reform the ARM peptide binding site in the presence of arbitrarily selected target-RNA. As a result, the target RNA was homogeneously detected by FLuc luminescent signals mediated by a cooperative function of the PI-FLuc and split-RNA probe sets.

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.