RNA has emerged beyond its traditional biological role to become a premier biorecognition element in biosensor design. While single-target RNA sensors demonstrate high specificity, multi-pocket variants, such as the tetrahydrofolate (THF) riboswitch, enable complex signal integration and cooperative allosteric regulation. Intriguingly, the THF riboswitch binds both folinic acid and purine derivatives with high affinity, yet exhibits different signal transduction outputs. The mechanistic basis for this decoupling of molecular recognition from sensor-proper folding remains a critical hurdle. Herein, we established a comprehensive multi-modal analytical platform, including engineered fluorescent sensors, position-specific RNA labeling, stopped-flow kinetics, microscale thermophoresis (MST), and single-molecule FRET, to decipher this decoupling mechanism. Our investigations support that the functional activation of the THF riboswitch is dictated decisively by transient binding kinetics (target residence time) rather than overall affinity alone. Furthermore, we demonstrate that concentration-dependent heterotropic cooperativity among distinct ligands functions as an allosteric amplifier, broadening the dynamic range and lowering the detection threshold of the sensor. By elucidating these intricate kinetic principles, this work is valuable for engineering next-generation, logic-gated RNA aptasensors for multiplexed diagnostics.

The synthesis of large RNA with precise modifications at specific positions is in high demand for both basic research and therapeutic applications, but efficient methods are limited. Engineered DNA polymerases have recently emerged as attractive tools for RNA labelling, offering distinct advantages over conventional RNA polymerases. Here, through semi-rational designs, we engineered a DNA polymerase variant and used it to precisely incorporate a diverse range of modifications, including base modifications, 2 '-ribose modifications and backbone modifications, into desired positions within RNA. We achieved efficiencies exceeding 85% in the majority of modification cases, demonstrating success in introducing 2 '-O-methyl, phosphorothioate, N4-acetylcytidine and a fluorophore to specific sites in eGFP and Firefly luciferase messenger RNA. Our mRNA products with N4-acetylcytidine, 2 '-O-methyl and/or phosphorothioate have demonstrated the ability to enhance stability and affect protein production. This method presents a promising tool for the comprehensive functionalization of RNA, enabling the introduction of plentiful modifications irrespective of RNA lengths and sequences.

Cyclic diadenosine monophosphate (C-di-AMP) synthases are key enzymes for synthesizing c-di-AMP, a potent activator of the stimulator of interferon genes (STING) immune pathway. However, characterizing these enzymes has been hampered by the lack of effective sensors. While c-di-AMP riboswitches, as natural aptamers, hold the potential as RNA biosensors, their poorly comprehended structural dynamics and inherent "OFF" genetic output pose substantial challenges. To address these limitations, we synthesized over 10 fluorophore-labeled samples to probe the conformational changes of the riboswitch at the single-molecule level. By integrating these dynamic findings with steady-state fluorescence titration, mutagenesis, in vivo assays, and strand displacement strategy, we transformed the natural aptamer into a c-di-AMP biosensor. This engineered biosensor reversed its genetic output from "OFF" to "ON" upon c-di-AMP binding, exhibiting a 50-fold improvement in the c-di-AMP detection limit. Leveraging this refined biosensor, we developed a robust strategy for high-throughput in vivo evolution of c-di-AMP synthases.

Riboswitches represent a class of non-coding RNA that possess the unique ability to specifically bind ligands and, in response, regulate gene expression. A recent report unveiled a type of riboswitch, known as the guanidine-IV riboswitch, which responds to guanidine levels to regulate downstream genetic transcription. However, the precise molecular mechanism through which the riboswitch senses its target ligand and undergoes conformational changes remain elusive. This gap in understanding has impeded the potential applications of this riboswitch. To bridge this knowledge gap, our study investigated the conformational dynamics of the guanidine-IV riboswitch RNA upon ligand binding. We employed single-molecule fluorescence resonance energy transfer (smFRET) to dissect the behaviors of the aptamer, terminator, and full-length riboswitch. Our findings indicated that the aptamer portion exhibited higher sensitivity to guanidine compared to the terminator and full-length constructs. Additionally, we utilized Position-specific Labelling of RNA (PLOR) combined with smFRET to observe, at the single-nucleotide and single-molecule level, the structural transitions experienced by the guanidine-IV riboswitch during transcription. Notably, we discovered that the influence of guanidine on the riboswitch RNA’s conformations was significantly reduced after the transcription of 88 nucleotides. Furthermore, we proposed a folding model for the guanidine-IV riboswitch in the absence and presence of guanidine, thereby providing insights into its ligand-response mechanism.

Ribonuclease P (RNase P) plays a vital role in the maturation of tRNA across bacteria, archaea, and eukaryotes. However, how RNase P assembles various components to achieve specific cleavage of precursor tRNA (pre-tRNA) in different organisms remains elusive. In this study, we employed single- molecule fluorescence resonance energy transfer to probe the dynamics of RNase P from E. coli ( Escherichia coli) and Mja ( Methanocaldococcus jannaschii) during pre-tRNA cleavage by incorporating five Cy3-Cy5 pairs into pre-tRNA and RNase P. Our results revealed significant differences in the assembly and catalytic mechanisms of RNase P between E. coli and Mja at both the RNA and protein levels. Specifically, the RNA of E. coli RNase P (EcoRPR) can adopt an active conformation that is capable of binding and cleaving pre-tRNA with high specificity independently. The addition of the protein component of E. coli RNase P (RnpA) enhances and accelerates pre-tRNA cleavage efficiency by increasing and stabilizing the active conformation. In contrast, Mja RPR is unable to form the catalytically active conformation on its own, and at least four proteins are required to induce the correct folding of Mja RPR. Mutation experiments suggest that the functional deficiency of Mja RPR arises from the absence of the second structural layer, and proper intermolecular assembly is essential for Mja RNase P to be functional over a broad temperature range. We propose models to illustrate the distinct catalytic patterns and RNA-protein interactions of RNase P in these two organisms.

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.

Parkinson's disease (PD) diagnosis based on magnetic resonance imaging (MRI) is still challenging clinically. Quantitative susceptibility maps (QSM) can potentially provide underlying pathophysiological information by detecting the iron distribution in deep gray matter (DGM) nuclei. We hypothesized that deep learning (DL) could be used to automatically segment all DGM nuclei and use relevant features for a better differentiation between PD and healthy controls (HC). In this study, we proposed a DL-based pipeline for automatic PD diagnosis based on QSM and T1-weighted (T1W) images. This consists of (1) a convolutional neural network model integrated with multiple attention mechanisms which simultaneously segments caudate nucleus, globus pallidus, putamen, red nucleus, and substantia nigra from QSM and T1W images, and (2) an SE-ResNeXt50 model with an anatomical attention mechanism, which uses QSM data and the segmented nuclei to distinguish PD from HC. The mean dice values for segmentation of the five DGM nuclei are all >0.83 in the internal testing cohort, suggesting that the model could segment brain nuclei accurately. The proposed PD diagnosis model achieved area under the the receiver operating characteristic curve (AUCs) of 0.901 and 0.845 on independent internal and external testing cohorts, respectively. Gradient-weighted class activation mapping (Grad-CAM) heatmaps were used to identify contributing nuclei for PD diagnosis on patient level. In conclusion, the proposed approach can potentially be used as an automatic, explainable pipeline for PD diagnosis in a clinical setting.

Growing RNAs fold differently as they are transcribed, which modulates their finally adopted structures. Riboswitches regulate gene expression by structural change, which are sensitive to co-transcriptionally structural biology. Here we develop a strategy to track the structural change of RNAs during transcription at single-nucleotide and single-molecule resolution and use it to monitor individual transcripts of the SAM-VI riboswitch (riboSAM) as transcription proceeds, observing co-existence of five states in riboSAM. We report a bifurcated helix in one newly identified state from NMR and single-molecule FRET (smFRET) results, and its presence directs the translation inhibition in our cellular translation experiments. A model is proposed to illustrate the distinct switch patterns and gene-regulatory outcome of riboSAM when SAM is present or absent. Our strategy enables the precise mapping of RNAs' conformational landscape during transcription, and may combine with detection methods other than smFRET for structural studies of RNAs in general.

BACKGROUND:Cogan's syndrome (CS) is a rare autoimmune disorder characterized by non-syphilitic interstitial keratitis (IK) and Menière-like cochlear vestibular symptoms, which may also have systemic effects. Corticosteroids are first-line treatment. DMARDs and biologics have been used to treat ocular and systemic symptoms of CS.CASE PRESENTATION:This is a case of a 35-year-old female who reported hearing loss, eye redness and photophobia. Her condition progressed to a sudden sensorineural hearing loss, tinnitus, and constant vertigo accompanied by cephalea. CS was diagnosed after excluding other diseases. The patient still developed bilateral sensorineural hearing loss after receiving hormone, methotrexate, cyclophosphamide, and a variety of biological agents. Joint symptoms were relieved after treatment with a JAK inhibitor (tofacitinib), and hearing did not deteriorate further.CONCLUSIONS:CS should be involved in the differential diagnosis of keratitis. Early identification and intervention of this autoimmune disease can minimize disability and irreversible damage.

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.

Mg 2+ is well-known to facilitate the structural folding of RNA. However, the thermodynamic and dynamic roles of Mg 2+ in RNA folding remain elusive. Here, we exploit smFRET (single-molecule fluorescence resonance energy transfer) and ITC (isothermal titration calorimetry) to study the mechanism of Mg 2+ in facilitating the folding of TPP (thiamine pyrophosphate) riboswitch RNA. The results of smFRET identify that the presence of Mg 2+ compacts the RNA, but also enlarge the dynamical dispersity among individual RNA molecules, resulting in a large gain of entropy. The compact yet flexible conformations triggered by Mg 2+ may help the riboswitch to recognize its specific ligand and further fold. This is supported by the ITC experiments, in which the Mg 2+ induced RNA folding is driven by entropy ( ΔS ) instead of enthalpy ( ΔH ). Our results complement the understanding of Mg 2+ -induced RNA folding. The strategy developed in this work can be used to model other RNAs’ folding at different conditions.

Labeling RNA molecules at specific positions is critical for RNA research and applications. Such methods are in high demand but still a challenge, especially those that enable native co-synthesis rather than post-synthesis labeling of long RNAs. The method we developed in this work meets these requirements, in which a leader RNA is extended on the hybrid solid-liquid phase by an engineered transcriptional complex following the pause-restart mode. A custom-designed short oligonucleotide is used to functionalize the engineered complex. This remarkable co-transcriptional labeling method incorporates labels into RNAs in high yields with great flexibility. We demonstrate the method by successfully introducing natural modifications, a fluorescent nucleotide analogue and a donor-acceptor fluorophore pair to specific sites located at an internal loop, a pseudoknot, a junction, a helix, and the middle of consecutive identical nucleotides of various RNAs. This newly developed method overcomes efficiency and position-choosing constraints that have hampered routine strategies to label RNAs beyond 200 nucleotides (nt).

RNAs adopt various conformations to perform different functions in cells. Incapable of acquiring intermediates, the key initiations of ligand recognition in the adenine riboswitch have not been characterized. In this work, stopped-flow fluorescence was used to track structural switches in the full-length adenine riboswitch in real time. We used PLOR (position-selective labeling of RNA) to incorporate fluorophores into desired positions in the RNA. The switching sequence P1 responded to adenine more rapidly than helix P4 and the binding pocket, followed by stabilization of the binding pocket, P4, and annealing of P1. Moreover, a transient intermediate consisting of an unwound P1 was detected during adenine binding. These events were observed in both the WT riboswitch and a functional mutant. The findings provide insight into the conformational changes of the riboswitch RNA triggered by a ligand.

Ligand binding and temperature play important roles in riboswitch RNAs' structures and functions. However, most studies focused on studying structural dynamics or gene-regulation function of riboswitches from the aspect of ligand, instead of temperature. Here we combined NMR, ITC, stopped-flow and in vivo assays to investigate the ligand-triggered switch of adenine riboswitch from 10 to 45°C. Our results demonstrated that at single-nucleotide resolution, structural regions sensed ligand and temperature diversely. Temperature had opposite effects on ligand-binding and gene-regulation of adenine riboswitch. Compared with higher temperature, the RNA bound with its cognate ligand obviously stronger, while its regulatory capacity was weakened at lower temperature. In addition, application of specific-labelled RNAs to the stopped-flow experiments identified the real-time folding of the specific positions upon ligand addition at different temperatures. The kissing loop and internal loop at the riboswitch responded to ligand and temperature differently. The distinct thermo-dynamics of adenine riboswitch exposed here may contribute to the fields of RNA sensors and drug design.

Modifications of short RNAs at specific sites can be achieved commercially by solid-phase chemical synthesis method. However, labelling long RNAs is still challenging for the routine methods. Position-selective Labelling of RNA (PLOR) is a hybrid phase transcription method that allows to label RNAs at desired sites with great flexibility and decent efficiency. In principle, PLOR is a promising method for synthesis of long modified RNAs that are unable to be generated by solid-phase chemical synthesis and other methods. However, as a recently developed method, PLOR has been only applied to label a 71nt and a 104nt RNA, and the limited sequence applications of PLOR may hinder its potential usages. To extend PLOR to more RNAs, we tested the PLOR performances for various RNA sequences. Considering that the controlled transcriptional pauses at the initiation stage in PLOR may lead to different preferences on RNA sequences from in vitro transcription method, we here focused on identifying the effects of the 5MODIFIER LETTER PRIME-end and initiated lengths of RNA on PLOR. In addition, our work demonstrated that PLOR efficiencies also varied with linker sizes of DNA templates. This work can facilitate PLOR to be the choice of synthesizing long modified RNAs for more users in the near future.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and ranks as the fifth leading cause of visual impairment, but an understanding of DR development has been hampered by the lack of an efficient metabolomic tool. Herein, vanadium core-shell nanorods are developed for metabolic fingerprinting to probe molecular variation in DR. First, a series of vanadium core-shells are constructed with different elemental composition and structural parameters, using silica nanorods to support vanadium oxide. The plasma metabolic fingerprints (MFs) are extracted by the optimized vanadium core-shell nanorod-assisted laser desorption/ionization mass spectrometry, by analyzing 500 nL of native plasma in seconds. As a result, DR patients are differentiated from non DR controls with a sensitivity of 94% and specificity of 90% using a classification model built on the plasma MFs. Furthermore, DR progression is monitored by a panel of plasma metabolic signatures with gradual changes. This work provides an advanced molecular tool for the metabolomic characterization of DR and may guide the clinical decision making in DR for personalized medicine in the future.

Nanolayered metals are shown to have significantly improved radiation resistance, owing to the copious interplay between their densely populated internal boundaries with irradiation-induced defects. Therefore, they have become promising candidates for structural applications in advanced nuclear reactors. Here we fabricated bulk nanolaminated graphene (in the form of reduced graphene oxide)-aluminum composite, and explored their structural and property evolution under high energy helium irradiation. We carefully designed and implemented the micro-/nano-mechanical test, so that the mechanical behavior of small scale specimens resembled that of the bulk. Compared to the unreinforced matrix, the graphene–aluminum composite showed substantially lower irradiation-induced strengthening magnitude, a considerable total elongation, smaller lattice swelling, and a completely different deformation mechanism after irradiation. A combination of detailed experimental observations and large-scale atomistic simulations indicated that both remarkable irradiation resistance and distinct mechanism of graphene–aluminum composite are related to the excellent sink strength of the graphene layers for irradiation-generated defects. This study offers a novel and effective strategy to produce scalable radiation damage-tolerant bulk metal matrix composite with meticulously tailored microstructures.

Due to the paucity of known RNA structures, experimental phasing is crucial for obtaining three-dimensional structures of RNAs by X-ray crystallography. Covalent attachment of heavy atoms to RNAs is one of the most useful strategies to facilitate phase determination. However, this approach is limited by the inefficiency or inability to synthesize large RNAs (>60 nucleotides) site-specifically labeled with heavy atoms using traditional methods. Here, we applied our recently reported method, PLOR (position-selective labeling of RNA) to incorporate 5-iodouridine at specific positions in the adenine riboswitch RNA aptamer domain, which was then used for crystallization and subsequent de novo SAD phasing. PLOR is a powerful tool to improve the efficiency of obtaining RNA structures de novo by X-ray crystallography.

Position-selective incorporation of modified nucleotides is useful for RNA study. This protocol describes how to prepare site-specifically labeled RNA using PLOR, an interrupted in vitro transcription-based approach. Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid–liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13C15N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.