Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.

An Optimized Enzyme-Nucleobase Pair Enables In Vivo RNA Metabolic Labeling with Improved Cell-Specificity.

Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the in vivo expression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests. The selective incorporation of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report using this approach in a metastatic human breast cancer mouse model for profiling cell-specific nascent RNA.

Ordered immobilization of serine proteases enabled by a linchpin directed modification platform.

We report a chemoselective and site-selective precision engineering of lysine in proteases. The mild and physiological reaction conditions keep their auto-degradation under control. Furthermore, it enables single-site ordered immobilization, enhancing protein digestion and peptide mapping efficiency.

Using 5' 32 P-labeled Primer and Reverse Transcription to Probe RNA Structure.

RNA molecules perform numerous cellular functions necessary for cell viability, some of which can depend on the RNA's structure. Therefore, it is important to study RNA structure and RNA folding to better understand the molecular basis of these functions. RNA chemical mapping strategies to elucidate RNA structural changes involve using chemical reagents that form adducts or cleave RNA. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) measures RNA flexibility by modification of the 2' hydroxyl groups of flexible nucleotides. These RNA adducts can be detected using 32 P-labeled primers and reverse transcription (RT) followed by PAGE analysis. This strategy can reveal the base-paired regions of the RNA and provide insight into tertiary structure and solvent accessibility. This protocol provides a method to interrogate RNA structure using furoyl acylimidazole (FAI). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Reverse transcription (RT) primer labeling with 32 P radionuclide Basic Protocol 2: Characterization of RNA structure with radiolabeled primer and reverse transcription (RT).

Overview of Chemical Methods to Probe RNA Structure with Radionucleotides.

Structural features of RNA play an important role in its capability to perform various functions in biological systems. To probe structural features, chemical probes are used to conjugate or cleave RNA at solvent-accessible sites, differentiating between flexible and constrained regions. These conjugates or cleaved products are then detected using reverse transcription (RT), where enzymatic RNA-dependent DNA primer extension is abruptly halted at the conjugation site or cleavage site. Here, we provide an overview of methods to probe RNA structure in vitro using radioactively labeled DNA primers, which provide a highly sensitive method to visualize RT stop sites with gel electrophoresis. © 2023 Wiley Periodicals LLC.

Cycloaddition enabled mutational profiling of 5-vinyluridine in RNA.

We report the detection of 5-vinyluridine (5-VUrd) in RNA at single nucleotide resolution via mutational profiling. Maleimide cycloadducts with 5-VUrd in RNA cause a stop in primer extension during reverse transcription, and the full-length cDNA product from reverse transcription contains misincorporation across the cycloadduct site.

Mutually Orthogonal Bioconjugation of Vinyl Nucleosides for RNA Metabolic Labeling.

We report a strategy for the orthogonal conjugation of the vinyl nucleosides, 5-vinyluridine (5-VU) and 2-vinyladenosine (2-VA), via selective reactivity with maleimide and tris(2-carboxyethyl)phosphine (TCEP), respectively. The orthogonality was investigated using density functional theory (DFT) and confirmed by reactions with vinyl nucleosides. Further, these chemistries were used to modify RNA for fluorescent cell imaging. These reactions allow for the expanded use of RNA metabolic labeling to study nascent RNA expression within different RNA populations.

Tibialis posterior (TP) tendon transfer for foot drop: A single center experience.

BACKGROUND: Foot drop defined as a significant weakness of ankle and toe dorsiflexion. It leads to high stepping gait, functional impairment and deformity of the foot. Objective of this study was to assess the functional outcome of tibialis posterior (TP) transfer for patient with foot drop in a single center. METHODS: This is a retrospective study included 20 patients operated for foot drop of >1 year duration in the last 5 years. Preoperative assessment of muscles of all the three compartment of leg along with radiological assessment of ankle to rule out tarsal disintegration and ankle instability was done. Postoperatively gait, active dorsi/plantar flexion and the range of movement of the ankle and toes were assessed. RESULTS: Tibialis posterior transfer was performed on 20 patients (16 males and 4 females, mean age 31.4 years). Commonest cause of foot drop was Hansen's disease followed by post traumatic peroneal nerve damage and post injection sciatic neuropathy. At mean follow-up of 2 years, all patients, except one, could walk with heel-toe gait without any orthotic support. There was no pain, ruptures or infections of the transferred tendons. 19 of the 20 operated ankles had mean active dorsiflexion of 7.5°, the active plantar flexion of 36.25°, and the total range of movement 43.75°. The active dorsiflexion of the toes ranged from 5-20°. CONCLUSION: Dynamic tibialis posterior transfer gives good results in terms of normal gait, high patients' satisfaction with minimal donor site morbidity and low complication rate.

Decline in Pulmonary Function Tests after Menopause.

OBJECTIVES: Menopause is the permanent cessation of menstruation resulting from the loss of ovarian follicular activity. There is limited and conflicting evidence for an association between lung function and menopause. The purpose of this study is to evaluate Pulmonary Function Test (PFT) in postmenopausal women. METHODS: Digital Spirometer was used to measure PFTs in premenopausal (n = 49) and postmenopausal (n = 46) women. RESULTS: Significant decline in many PFT parameters was observed. CONCLUSIONS: Menopausal status is associated with low lung function.