Endogenous G-quadruplex-forming RNAs inhibit the activity of SARS-CoV-2 RNA polymerase.

Replication of RNA viruses is catalysed by virus-specific polymerases, which can be targets of therapeutic strategies. In this study, we used a selection strategy to identify endogenous RNAs from a transcriptome library derived from lung cells that interact with the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. Some of the selected RNAs weakened the activity of RdRp by forming G-quadruplexes. These results suggest that certain endogenous RNAs, which potentially form G-quadruplexes, can reduce the replication of viral RNAs.

Nearest-neighbor parameters for the prediction of RNA duplex stability in diverse in vitro and cellular-like crowding conditions.

RNA performs various spatiotemporal functions in living cells. As the solution environments significantly affect the stability of RNA duplexes, a stability prediction of the RNA duplexes in diverse crowded conditions is required to understand and modulate gene expression in heterogeneously crowded intracellular conditions. Herein, we determined the nearest-neighbor (NN) parameters for RNA duplex formation when subjected to crowding conditions with an ionic concentration relevant to that found in cells. Determination of the individual contributions of excluded volume effect and water activity to each of the NN parameters in crowded environments enabled prediction of the thermodynamic parameters and their melting temperatures for plenty of tested RNA duplex formation in vitro and in cell with significant accuracy. The parameters reported herein will help predicting RNA duplex stability in different crowded environments, which will lead to an improved understanding of the stability-function relationship for RNAs in various cellular organelles with different molecular environments.

Cladogenetic Orthogonal Light-Up Aptamers for Simultaneous Detection of Multiple Small Molecules in Cells.

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.

High-temperature adaptation of an OsNRT2.3 allele is thermoregulated by small RNAs.

Climate change negatively affects crop yield, which hinders efforts to reach agricultural sustainability and food security. Here, we show that a previously unidentified allele of the nitrate transporter gene OsNRT2.3 is required to maintain high yield and high nitrogen use efficiency under high temperatures. We demonstrate that this tolerance to high temperatures in rice accessions harboring the HTNE-2 (high temperature resistant and nitrogen efficient-2) alleles from enhanced translation of the OsNRT2.3b mRNA isoform and the decreased abundance of a unique small RNA (sNRT2.3-1) derived from the 5' untranslated region of OsNRT2.3. sNRT2.3-1 binds to the OsNRT2.3a mRNA in a temperature-dependent manner. Our findings reveal that allelic variation in the 5' untranslated region of OsNRT2.3 leads to an increase in OsNRT2.3b protein levels and higher yield during high-temperature stress. Our results also provide a breeding strategy to produce rice varieties with higher grain yield and lower N fertilizer input suitable for a sustainable agriculture that is resilient against climate change.

Applicability of the nearest-neighbour model for pseudoknot RNAs.

The validity of the nearest-neighbour (NN) model was verified in an RNA pseudoknot (PK) structure. The thermodynamic parameters of the second hairpin stem (S2) region, which separates the PK from a hairpin structure, were monitored using CD and UV melting. Different PKs with identical NN base pairs in the S2 region exhibited similar thermodynamic parameters, highlighting the validity of the NN model in this RNA tertiary structure motif.

Triple-Helical Binding of Peptide Nucleic Acid Inhibits Maturation of Endogenous MicroRNA-197.

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.

Transcriptome screening followed by integrated physicochemical and structural analyses for investigating RNA-mediated berberine activity.

Non-coding RNAs are regarded as promising targets for the discovery of innovative drugs due to their abundance in the genome and their involvement in many biological processes. Phytochemicals (PCs) are the primary source of ligand-based drugs due to their broad spectrum of biological activities. Since many PCs are heterocyclic and have chemical groups potentially involved in the interaction with nucleic acids, detailed interaction analysis between PCs and RNA is crucial to explore the effect of PCs on RNA functions. In this study, an integrated approach for investigating interactions between PCs and RNAs were demonstrated to verify the RNA-mediated PCs functions by using berberine (BRB) as a model PC. RNA screening of a transcriptome library followed by sequence refinement found minimal RNA motif consisting of a cytosine bulge with U-A and G-U neighbouring base pairs for interaction with BRB. NMR-based structure determination and physicochemical analyses using chemical analogues of BRB demonstrated the importance of electrostatic and stacking interactions for sequence selective interaction and RNA stabilization. The selective interaction with a relatively small RNA motif based on a chemical structure of a planer heterocyclic highlights the biological activities of various PCs mediated by the interactions with particular functional RNAs. In addition, the systematic and quantitative investigations demonstrated in this study could be useful for the development of therapeutic chemicals targeting functional RNAs, based on the PCs, in the future.

Effects of Modifying Thioflavin T at the N3-Position on Its G4 Binding and Fluorescence Emission.

We previously synthesized thioflavin T (ThT) with a hydroxyethyl group introduced at the N3-position (ThT-HE), which binds predominantly to the parallel G-quadruplex (G4) structure found in c-Myc and emits strong fluorescence. In this study, to investigate the effects of introduced substituents on G4 binding and fluorescence emission, a ThT derivative in which the hydroxyl group of ThT-HE was replaced with an amino group (ThT-AE) was synthesized for the first time. Furthermore, three other N3-modified ThT derivatives (ThT-OE2, ThT-SP, and ThT-OE11) having different substituent structures were synthesized by the N-acylation of the terminal amino group of ThT-AE, and their G4-binding and emission properties were investigated. The results showed that, although ThT-AE shows binding selectivity depending on the type of G4, its emission intensity is significantly decreased as compared to that of ThT-HE. However, ThT-OE11, which features an 11-unit oxyethylene chain attached to the terminal amino group of ThT-AE, regained about one-half of the emission intensity of ThT-HE while retaining selectivity for G4s. Accordingly, ThT-OE11 may be used as a key intermediate for synthesizing the conjugates of G4 binders and probes.

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition.

The stability of Watson-Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na+ or K+ in the presence or absence of Mg2+). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°37 and Tm values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg2+, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR-Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.

Nearest-neighbor parameters for predicting DNA duplex stability in diverse molecular crowding conditions.

The intracellular environment is crowded and heterogeneous. Although the thermodynamic stability of nucleic acid duplexes is predictable in dilute solutions, methods of predicting such stability under specific intracellular conditions are not yet available. We recently showed that the nearest-neighbor model for self-complementary DNA is valid under molecular crowding condition of 40% polyethylene glycol with an average molecular weight of 200 (PEG 200) in 100 mM NaCl. Here, we determined nearest-neighbor parameters for DNA duplex formation under the same crowding condition to predict the thermodynamics of DNA duplexes in the intracellular environment. Preferential hydration of the nucleotides was found to be the key factor for nearest-neighbor parameters in the crowding condition. The determined parameters were shown to predict the thermodynamic parameters (∆H°, ∆S°, and ∆G°37) and melting temperatures (Tm) of the DNA duplexes in the crowding condition with significant accuracy. Moreover, we proposed a general method for predicting the stability of short DNA duplexes in different cosolutes based on the relationship between duplex stability and the water activity of the cosolute solution. The method described herein would be valuable for investigating biological processes that occur under specific intracellular crowded conditions and for the application of DNA-based biotechnologies in crowded environments.

Signaling Aptamer Optimization through Selection Using RNA-Capturing Microsphere Particles.

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.

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.

RNA-Capturing Microsphere Particles (R-CAMPs) for Optimization of Functional Aptamers.

RNA aptamers are useful building blocks for constructing functional nucleic acid-based nanoarchitectures. The abilities of aptamers to recognize specific ligands have also been utilized for various biotechnological applications. Solution conditions, which can differ depending on the application, impact the affinity of the aptamers, and thus it is important to optimize the aptamers for the solution conditions to be employed. To simplify the aptamer optimization process, an efficient method that enables re-selection of an aptamer from a partially randomized library is developed. The process relies on RNA-capturing microsphere particles (R-CAMPs): each particle displays different clones of identical DNA and RNA sequences. Using a fluorescence-activated cell sorter, the R-CAMPs that are linked to functional aptamers are sorted. It is demonstrated that after a single round of reselection, several functional aptamers, including the wild-type, are selected from a library of 16 384 sequences. The selection using R-CAMPs is further performed under the solution containing high concentration of ethylene glycol, suggesting applicability in various conditions to optimize an aptamer for a particular application. As any type of RNA clone can be displayed on the microspheres, the technology demonstrated here will be useful for the selection of RNAs based on diverse functions.

Validation of the nearest-neighbor model for Watson-Crick self-complementary DNA duplexes in molecular crowding condition.

Recent advancement in nucleic acid techniques inside cells demands the knowledge of the stability of nucleic acid structures in molecular crowding. The nearest-neighbor model has been successfully used to predict thermodynamic parameters for the formation of nucleic acid duplexes, with significant accuracy in a dilute solution. However, knowledge about the applicability of the model in molecular crowding is still limited. To determine and predict the stabilities of DNA duplexes in a cell-like crowded environment, we systematically investigated the validity of the nearest-neighbor model for Watson-Crick self-complementary DNA duplexes in molecular crowding. The thermodynamic parameters for the duplex formation were measured in the presence of 40 wt% poly(ethylene glycol)200 for different self-complementary DNA oligonucleotides consisting of identical nearest-neighbors in a physiological buffer containing 0.1 M NaCl. The thermodynamic parameters as well as the melting temperatures (Tm) obtained from the UV melting studies revealed similar values for the oligonucleotides having identical nearest-neighbors, suggesting the validity of the nearest-neighbor model in the crowding condition. Linear relationships between the measured ΔG°37 and Tm in crowding condition and those predicted in dilute solutions allowed us to predict ΔG°37, Tm and nearest-neighbor parameters in molecular crowding using existing parameters in the dilute condition, which provides useful information about the thermostability of the self-complementary DNA duplexes in molecular crowding.

Co-Transcriptional Molecular Assembly Results in a Kinetically Controlled Irreversible RNA Conformational Switch.

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.

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit.

In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg2+ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg2+ -containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg2+ solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.

Pursuing origins of (poly)ethylene glycol-induced G-quadruplex structural modulations.

Molecular crowding conditions provided by high concentration of cosolutes are utilized for characterization of biomolecules in cell-mimicking environment and development of drug-delivery systems. In this context, (poly)ethylene glycols are often used for studying non-canonical DNA structures termed G-quadruplexes, which came into focus by emerging structural biology findings and new therapeutic drug design approaches. Recently, several reports were made arguing against using (poly)ethylene glycols in role of molecular crowding agents due to their direct impact on DNA G-quadruplex stability and topology. However, the available data on structural details underlying DNA interaction is very scarce and thus limits in-depth comprehension. Herein, structural and thermodynamic analyses were strategically combined to assess G-quadruplex-cosolute interactions and address previously reported variances regarding the driving forces of G-rich DNA structural transformations under molecular crowding conditions. With the use of complementary (CD, NMR and UV) spectroscopic methods and model approach we characterized DNA G-quadruplex in the presence of the smallest and one of the largest typically used (poly)ethylene glycols. Dehydration effect is the key contributor to ethylene-glycol-induced increased stability of the G-quadruplex, which is in the case of the large cosolute mainly guided by the subtle direct interactions between PEG 8000 and the outer G-quartet regions.

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins.

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can 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 toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia 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 mRNA in Gene Expression as New Pharmaceutical Target.

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.