Carrier PNA for shRNA delivery into cells

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as 'bridgebuilder' 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

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it might also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched towards the C-termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur prior to assembly. Using high-throughput imaging of protein homomers in vivo in E. coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization

Conformational Dynamics of the RNA G-Quadruplex and its Effect on Translation Efficiency

During translation, intracellular mRNA folds co-transcriptionally and must refold following the passage of ribosome. The mRNAs can be entrapped in metastable structures during these folding events. In the present study, we evaluated the conformational dynamics of the kinetically favored, metastable, and hairpin-like structure, which disturbs the thermodynamically favored G-quadruplex structure, and its effect on co-transcriptional translation in prokaryotic cells. We found that nascent mRNA forms a metastable hairpin-like structure during co-transcriptional folding instead of the G-quadruplex structure. When the translation progressed co-transcriptionally before the metastable hairpin-like structure transition to the G-quadruplex, function of the G-quadruplex as a roadblock of the ribosome was sequestered. This suggested that kinetically formed RNA structures had a dominant effect on gene expression in prokaryotes. The results of this study indicate that it is critical to consider the conformational dynamics of RNA-folding to understand the contributions of the mRNA structures in controlling gene expression

Selection of RNAs for Constructing “Lighting-UP” Biomolecular Switches in Response to Specific Small Molecules

<div><p>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 <i>Renilla</i> 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.</p> </div

Nucleic Acids Chemistry and Engineering: Special Issue on Nucleic Acid Conjugates for Biotechnological Applications

Nucleic acids not only store genetic information in their primary sequence but also exhibit biological functions through the formation of their unique structures [...

Detection of target molecules by the Lighting-UP switch.

<p>(A, B) Relative luminescence intensities (LI) of the engineered Luciferase in buffer containing 20 mM phosphate (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 100 ng/ µL tRNA, and 0.005% Tween 20 at 37°C. An aliquot (0.04 µL) of the <i>in vitro</i> translation product was mixed with varying concentrations of (A) th5N-G6 or (B) tc5N-G6 in the absence (blue) or presence of 3 mM theophylline (red) or 100 µM tetracycline (green). tRNA was added at 100 ng/ µL to improve the signal to noise ratio. (C) Theophylline (red) or caffeine (purple) at the indicated concentrations was mixed with the <i>in vitro</i> translation product in the presence of 25 nM th5N-G6. (D) Tetracycline (green) or doxycycline (orange) at indicated concentrations was mixed with the <i>in vitro</i> translation product in the presence of 50 nM tc5N-G6. (E) Chemical structures of target molecules and analogs.</p

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction

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 (−Δ<i>G</i>°) 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 (ΔΔ<i>G</i>°) that corresponds to the energy difference between the RNA secondary structure before (−Δ<i>G</i>°_before) and after (−Δ<i>G</i>°_after) the RNA conformational transition. We first selected functional RNA switches responsive to neomycin with predicted ΔΔ<i>G</i>° 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, <i>S</i>-adenosyl methionine, and adenine based on the predicted ΔΔ<i>G</i>° values. The ΔΔ<i>G</i>° values of the designed RNA switches proportionally correlated with interaction energy (Δ<i>G</i>°_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

The interaction of initial RNA libraries and the Tat-peptide.

<p>(A, B) Fluorescence intensities (FI) of the TMR-Tat at 590 nm mixed with G0 RNA libraries in a buffer containing 20 mM phosphate (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 20 ng/ µL tRNA, and 0.005% (v/v) Tween 20 at 37°C. TMR-Tat was mixed with varying concentrations of (A) th5N-G0 and (B) tc5N-G0 in the absence (blue) or presence of 3 mM theophylline (red) or 100 µM tetracycline (green).</p

Unusual −1 Ribosomal Frameshift Caused by Stable RNA G‑Quadruplex in Open Reading Frame

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