Search Results (5)

Precisely fluorescently labeling specific nucleotide sites of RNA is critical for gaining insights into the structure and function of RNA through multiple fluorescence detection techniques. The position-selective labeling of RNA (PLOR) method provides a promising strategy to achieve this, wherein the fluorophore-modified NTPs can be co-transcriptionally introduced to specific sites of nascent RNA by using T7 RNA polymerase (T7 RNAP). However, due to steric hindrance limitations, the efficiency of T7 RNAP in recognizing and incorporating large fluorophore-modified NTPs into RNA is far from satisfactory. To overcome this challenge, in this work, we developed an efficient PLOR variant (ePLOR) for the site-specific fluorescent labeling of RNA by integrating PLOR with a post-transcriptional SPAAC (strain-promoted azido-alkyne cycloaddition) click chemistry reaction. The efficiency of the SPAAC reaction occurring on RNA is nearly 100%. Consequently, ePLOR enables the precise fluorescent labeling of designated sites across various structural regions of SAM-VI riboswitch and adenine riboswitch RNA, with labeling and synthesis efficiencies that are 2–2.5 times higher than those of PLOR. The strategy developed in this work can be used for the efficient synthesis of a broader spectrum of long-strand RNAs with site-specific fluorescent labeling and greatly facilitate the detection of the structure and function of these RNAs. Full article

T7 RNA polymerase is the most widely used enzyme in RNA synthesis, and it is also used for RNA labeling in position-selective labeling of RNA (PLOR). PLOR is a liquid–solid hybrid phase method that has been developed to introduce labels to specific positions of RNA. Here, we applied PLOR as a single-round transcription method to quantify the terminated and read-through products in transcription for the first time. Various factors, including pausing strategies, Mg²⁺, ligand and the NTP concentration at the transcriptional termination of adenine riboswitch RNA have been characterized. This helps to understand transcription termination, which is one of the least understood processes in transcription. Additionally, our strategy can potentially be used to study the co-transcription behavior of general RNA, especially when continuous transcription is not desired. Full article

Riboswitches are natural biosensors that can regulate gene expression by sensing small molecules. Knowledge of the structural dynamics of riboswitches is crucial to elucidate their regulatory mechanism and develop RNA biosensors. In this work, we incorporated the fluorophore, Cy3, and its quencher, TQ3, into a full-length adenine riboswitch RNA and its isolated aptamer domain to monitor the dynamics of the RNAs in vitro and in cell. The adenine riboswitch was sensitive to Mg²⁺ concentrations and could be used as a biosensor to measure cellular Mg²⁺ concentrations. Additionally, the TQ3/Cy3-labeled adenine riboswitch yielded a Mg²⁺ concentration that was similar to that measured using a commercial assay kit. Furthermore, the fluorescence response to the adenine of the TQ3/Cy3-labeled riboswitch RNA was applied to determine the proportions of multiple RNA conformational changes in cells. The strategy developed in this work can be used to probe the dynamics of other RNAs in cells and may facilitate the developments of RNA biosensors, drugs and engineering. Full article

Riboswitches are important model systems for the development of approaches to search for RNA-targeting therapeutics. A principal challenge in finding compounds that target riboswitches is that the effector ligand is typically almost completely encapsulated by the RNA, which severely limits the chemical space that can be explored. Efforts to find compounds that bind the guanine/adenine class of riboswitches with a high affinity have in part focused on purines modified at the C6 and C2 positions. These studies have revealed compounds that have low to sub-micromolar affinity and, in a few cases, have antimicrobial activity. To further understand how these compounds interact with the guanine riboswitch, we have performed an integrated structural and functional analysis of representative guanine derivatives with modifications at the C8, C6 and C2 positions. Our data indicate that while modifications of guanine at the C6 position are generally unfavorable, modifications at the C8 and C2 positions yield compounds that rival guanine with respect to binding affinity. Surprisingly, C2-modified guanines such as N2-acetylguanine completely disrupt a key Watson–Crick pairing interaction between the ligand and RNA. These compounds, which also modulate transcriptional termination as efficiently as guanine, open up a significant new chemical space of guanine modifications in the search for antimicrobial agents that target purine riboswitches. Full article

Riboswitches are naturally occurring RNA aptamers that control the expression of essential bacterial genes by binding to specific small molecules. The binding with both high affinity and specificity induces conformational changes. Thus, riboswitches were proposed as a possible molecular target for developing antibiotics and chemical tools. The adenine riboswitch can bind not only to purine analogues but also to pyrimidine analogues. Here, long molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) computational methodologies were carried out to show the differences in the binding model and the conformational changes upon five ligands binding. The binding free energies of the guanine riboswitch aptamer with C74U mutation complexes were compared to the binding free energies of the adenine riboswitch (AR) aptamer complexes. The calculated results are in agreement with the experimental data. The differences for the same ligand binding to two different aptamers are related to the electrostatic contribution. Binding dynamical analysis suggests a flexible binding pocket for the pyrimidine ligand in comparison with the purine ligand. The 18 μs of MD simulations in total indicate that both ligand-unbound and ligand-bound aptamers transfer their conformation between open and closed states. The ligand binding obviously affects the conformational change. The conformational states of the aptamer are associated with the distance between the mass center of two key nucleotides (U51 and A52) and the mass center of the other two key nucleotides (C74 and C75). The results suggest that the dynamical character of the binding pocket would affect its biofunction. To design new ligands of the adenine riboswitch, it is recommended to consider the binding affinities of the ligand and the conformational change of the ligand binding pocket. Full article