Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

Single‐molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real‐time single‐molecule measurement of the structure‐changing dynamics of nucleic acids.     

Specific Binding of a d-RNA G-Quadruplex Structure with an l-RNA Aptamer

G-quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4-binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l -RNA aptamer that targets a d -RNA G-quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l -RNA aptamer, Ap3-7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA-rG4–RHAU53 binding. Our approach and findings open a new door in further developing G4-specific tools for diverse applications.     

Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL)

Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH₄Cl/OsO₄-based conversion of 6-thioguanosine (6sG) into A′, where A′ constitutes a 6-hydrazino purine derivative. A′ retains the Watson–Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.     

Bundling mRNA Strands to Prepare Nano‐Assemblies with Enhanced Stability Towards RNase for In Vivo Delivery

Ribonuclease (RNase)‐mediated degradation of messenger RNA (mRNA) poses a huge obstruction to in vivo mRNA delivery. Herein, we propose a novel strategy to protect mRNA by structuring mRNA to prevent RNase attack through steric hinderance. Bundling of mRNA strands through hybridization of RNA oligonucleotide linkers allowed the preparation of mRNA nano‐assemblies (R‐NAs) comprised of 7.7 mRNA strands on average, mostly below 100 nm in diameter. R‐NA formation boosted RNase stability by around 100‐fold compared to naïve mRNA and preserved translational activity, allowing protein production. A mechanistic analysis suggests that an endogenous mRNA unwinding mechanism triggered by 5′‐cap‐dependent translation may induce selective R‐NA dissociation intracellularly, leading to smooth translation. R‐NAs showed efficient mRNA transfection in mouse brain, demonstrating the feasibility for in vivo administration.     

The Mechanism of Nonenzymatic Template Copying with Imidazole‐Activated Nucleotides

The emergence of the replication of RNA oligonucleotides was a critical step in the origin of life. An important model for the study of nonenzymatic template copying, which would be a key part of any such pathway, involves the reaction of ribonucleoside‐5′‐phosphorimidazolides with an RNA primer/template complex. The mechanism by which the primer becomes extended by one nucleotide was assumed to be a classical in‐line nucleophilic‐substitution reaction in which the 3′‐hydroxyl of the primer attacks the phosphate of the incoming activated monomer with displacement of the imidazole leaving group. Surprisingly, this simple model has turned out to be incorrect, and the dominant pathway has now been shown to involve the reaction of two activated nucleotides with each other to form a 5′–5′‐imidazolium bridged dinucleotide intermediate. Here we review the discovery of this unexpected intermediate, and the chemical, kinetic, and structural evidence for its role in template copying chemistry.