Unraveling RNA Conformation Dynamics in Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episode Syndrome with Solid-State Nanopores.

This study investigates transfer ribonucleic acid (tRNA) conformational dynamics in the context of MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) using solid-state silicon nitride (SiN) nanopore technology. SiN nanopores in thin membranes with specific dimensions exhibit high signal resolution, enabling real-time and single-molecule electronic detection of tRNA conformational changes. We focus on human mitochondrial tRNALeu(UAA) (mt-Leu(UAA)) that decodes Leu codons UUA/UUG (UUR) during protein synthesis on the mt-ribosome. The single A14G substitution in mt-Leu(UAA) is the major cause of MELAS disease. Measurements of current blockades and dwell times reveal distinct conformational dynamics of the wild-type (WT) and the A14G variant of mt-Leu(UAA) in response to the conserved post-transcriptional m1G9 methylation. While the m1G9-modified WT transcript adopts a more stable structure relative to the unmodified transcript, the m1G9-modified MELAS transcript adopts a less stable structure relative to the unmodified transcript. Notably, these differential features were observed at 0.4 M KCl, but not at 3 M KCl, highlighting the importance of experimental settings that are closer to physiological conditions. This work demonstrates the feasibility of the nanopore platform to discern tRNA molecules that differ by a single-nucleotide substitution or by a single methylation event, providing an important step forward to explore changes in the conformational dynamics of other RNA molecules in human diseases.

Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology.

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA structure would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has an opposite and destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated by the observation that demethylation of m 1 G9, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving its structure and activity. These results have new therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.

Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity.

Monitoring the dynamic changes of cellular tRNA pools is challenging, due to the extensive post-transcriptional modifications of individual species. The most critical component in tRNAseq is a processive reverse transcriptase (RT) that can read through each modification with high efficiency. Here we show that the recently developed group-II intron RT Induro has the processivity and efficiency necessary to profile tRNA dynamics. Using our Induro-tRNAseq, simpler and more comprehensive than the best methods to date, we show that Induro progressively increases readthrough of tRNA over time and that the mechanism of increase is selective removal of RT stops, without altering the misincorporation frequency. We provide a parallel dataset of the misincorporation profile of Induro relative to the related TGIRT RT to facilitate the prediction of non-annotated modifications. We report an unexpected modification profile among human proline isoacceptors, absent from mouse and lower eukaryotes, that indicates new biology of decoding proline codons.

Reinvestigating the Synthesis and Characterization of Ethyl 3-[5-(2-Ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]-but-2-enoate.

We reinvestigated the reported method for the synthesis of ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]-but-2-enoate (MIBE), which was obtained by the reaction of 5-hydroxy-1-methyl-1H-indole with excess ethyl acetoacetate catalyzed by indium(III) chloride. Based on the NMR and MS data, we assigned the structure of the isolated product as (3E)-3-(2-ethoxy-2-oxoethylidene)-1,2,3,4-tetrahydro-7-hydroxy-1,4-dimethylcyclopent[b]indole-1-acetate (2a) rather than the reported MIBE.

A general and efficient approach to synthesize the phosphoramidites of 5'-18O labeled purine nucleosides.

5'-18O labeled RNA oligos are important probes to investigate the mechanism of 2'-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5'-18O labeled nucleosides starting from the corresponding commercially available 5'-O-DMT protected nucleosides. Using this method, we prepared 5'-18O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5'-18O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5'-18O-2'-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5'-18O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2'-O-transphosphorylation reactions.

Demethylation of dimethyl phosphonate esters with sodium ethanethiolate: Improved synthesis of 5'-methylene substituted 2',5'-deoxyribonucleotides.

We describe a new demethylation method for dimethyl phosphonate esters using sodium ethanethiolate. The new procedure allows demethylation of nucleoside dimethyl phosphonate esters without 1'-α-anomerization, providing an improved synthesis of 5'-methylene substituted 2',5'-deoxynucleotides.

Dissociative Transition State in Hepatitis Delta Virus Ribozyme Catalysis.

Ribonucleases and small nucleolytic ribozymes are both able to catalyze RNA strand cleavage through 2'-O-transphosphorylation, provoking the question of whether protein and RNA enzymes facilitate mechanisms that pass through the same or distinct transition states. Here, we report the primary and secondary 18O kinetic isotope effects for hepatitis delta virus ribozyme catalysis that reveal a dissociative, metaphosphate-like transition state in stark contrast to the late, associative transition states observed for reactions catalyzed by specific base, Zn2+ ions, or ribonuclease A. This new information provides evidence for a discrete ribozyme active site design that modulates the RNA cleavage pathway to pass through an altered transition state.

Structural Basis for Fluorescence Activation by Pepper RNA.

Pepper is a fluorogenic RNA aptamer tag that binds to a variety of benzylidene-cyanophenyl (HBC) derivatives with tight affinity and activates their fluorescence. To investigate how Pepper RNA folds to create a binding site for HBC, we used antibody-assisted crystallography to determine the structures of Pepper bound to HBC530 and HBC599 to 2.3 and 2.7 Å resolutions, respectively. The structural data show that Pepper folds into an elongated structure and organizes nucleotides within an internal bulge to create the ligand binding site, assisted by an out-of-plane platform created by tertiary interactions with an adjacent bulge. As predicted from a lack of K+ dependence, Pepper does not use a G-quadruplex to form a binding pocket for HBC. Instead, Pepper uses a unique base-quadruple·base-triple stack to sandwich the ligand with a U·G wobble pair. Site-bound Mg2+ ions support ligand binding structurally and energetically. This research provides insight into the structural features that allow the Pepper aptamer to bind HBC and show how Pepper's function may expand to allow the in vivo detection of other small molecules and metals.

Stereospecific lasofoxifene derivatives reveal the interplay between estrogen receptor alpha stability and antagonistic activity in ESR1 mutant breast cancer cells.

Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend estrogen receptor alpha (ERα) cellular lifetime/accumulation. They are antagonists in the breast but agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders/downregulators (SERDs) reduce ERα cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ERα cellular lifetime and accumulation influences antagonistic activity, we studied a series of methylpyrollidine lasofoxifene (Laso) derivatives that maintained the drug's antagonistic activities while uniquely tuning ERα cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ERα cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ERα ligand binding domain in complex with the methylated Laso derivatives or representative SERMs and SERDs show that molecules that favor a highly buried helix 12 antagonist conformation achieve the greatest transcriptional suppression activities in breast cancer cells harboring WT/Y537S ESR1. Together these results show that chemical reduction of ERα cellular lifetime is not necessarily the most crucial parameter for transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our studies show how small chemical differences within a scaffold series can provide compounds with similar antagonistic activities, but with greatly different effects of the cellular lifetime of the ERα, which is crucial for achieving desired SERM or SERD profiles.

Structural basis for substrate binding and catalysis by a self-alkylating ribozyme.

Ribozymes that react with small-molecule probes have important applications in transcriptomics and chemical biology, such as RNA labeling and imaging. Understanding the structural basis for these RNA-modifying reactions will enable the development of better tools for studying RNA. Nevertheless, high-resolution structures and underlying catalytic mechanisms for members of this ribozyme class remain elusive. Here, we focus on a self-alkylating ribozyme that catalyzes nitrogen-carbon bond formation between a specific guanine and a 2,3-disubstituted epoxide substrate and report the crystal structures of a self-alkylating ribozyme, including both alkylated and apo forms, at 1.71-Å and 2.49-Å resolution, respectively. The ribozyme assumes an elongated hairpin-like architecture preorganized to accommodate the epoxide substrate in a hook-shaped conformation. Observed reactivity of substrate analogs together with an inverse, log-linear pH dependence of the reaction rate suggests a requirement for epoxide protonation, possibly assisted by the ether oxygens within the substrate.

Synthesis of Oligoribonucleotides Containing a 2'-Amino-5'-S-phosphorothiolate Linkage.

Oligoribonucleotides containing a photocaged 2'-amino-5'-S-phophorothiolate linkage have potential applications as therapeutic agents and biological probes to investigate the RNA structure and function. We envisioned that oligoribonucleotides containing a 2'-amino-5'-S-phosphorothiolate linkage could provide an approach to identify the general base within catalytic RNAs by chemogenetic suppression. To enable preliminary tests of this idea, we developed synthetic approaches to a dinucleotide, trinucleotide, and oligoribonucleotide containing a photocaged 2'-amino-5'-S-phosphorothiolate linkage. We incorporated the photocaged 2'-amino-5'-S-phosphorothiolate linkage into an oligoribonucleotide substrate for the hepatitis delta virus (HDV) ribozyme and investigated the pH dependence of its cleavage following UV irradiation both in the presence and absence of the ribozyme. The substrate exhibited a pH-rate profile characteristic of the modified linkage but reacted slower when bound to the ribozyme. Cleavage inhibition by the HDV ribozyme could reflect a non-productive ground-state interaction with the modified substrate's nucleophilic 2'-NH2 or a poor fit of the modified transition state at the ribozyme's active site.

The SARS-CoV-2 Programmed -1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography.

The programmed -1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses' genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1-10 and NSPs 1-16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the -1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the -1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.

The Positively Charged Active Site of the Bacterial Toxin RelE Causes a Large Shift in the General Base pKa.

The bacterial toxin RelE cleaves mRNA in the ribosomal A site. Although it shares a global fold with other microbial RNases, the active site contains several positively charged residues instead of histidines and glutamates that are typical of ribonucleases. The pH dependences of wild-type and mutant RelE indicate it uses general acid-base catalysis, but either the general acid (proposed to be R81) or the general base must have a substantially downshifted pKa. However, which group is shifted cannot be determined using available structural and biochemical data. Here, we use a phosphorothiolate at the scissile phosphate to remove the need for a general acid. We show this modification rescues nearly all of the defect of the R81A mutation, supporting R81 as the general acid. We also find that the observed pKa of the general base is dependent on the charge of the side chain at position 81. This indicates that positive charge in the active site contributes to a general base pKa downshifted by more than 5 units. Although this modestly reduces the effectiveness of general acid-base catalysis, it is strongly supplemented by the role of the positive charge in stabilizing the transition state for cleavage. Furthermore, we show that the ribosome is required for cleavage but not binding of mRNA by RelE. Ribosome functional groups do not directly contact the scissile phosphate, indicating that positioning and charge interactions dominate RelE catalysis. The unusual RelE active site catalyzes phosphoryl transfer at a rate comparable to those of similar enzymes, but in a ribosome-dependent fashion.

Confluence of theory and experiment reveals the catalytic mechanism of the Varkud satellite ribozyme.

The Varkud satellite ribozyme catalyses site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here, we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free-energy relationship measurements with molecular dynamics, molecular solvation theory and ab initio quantum mechanical/molecular mechanical free-energy simulations to gain insight into the catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfer in the transition state. Further, we provide evidence for a critical Mg2+ in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg2+ adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the Varkud satellite, hairpin and hammerhead ribozyme classes.

Reinvestigating the synthesis and efficacy of small benzimidazole derivatives as presequence protease enhancers.

Presequence protease (PreP) is a proteostatic enzyme that plays a key role in the maintenance of mitochondrial health. Defects in PreP stability are associated with neurological disorders in humans, and altered activity of this enzyme modulates the progress of Alzheimer's disease-like pathology in mice. As agonists that boost PreP proteolytic activity represent a promising therapeutic avenue, we sought to determine the structural basis for the action of benzimidazole derivatives (3c and 4c), first reported by Vangavaragu et al. (Eur. J. Med. Chem. 76 (2014) 506-516) that enhance the activity of PreP. However, we found the published procedure for the synthesis of 3c yielded aldimine A instead. We then developed an alternative synthesis and obtained 3c, termed compound C, and an alternative benzimidazole derivative, termed compound B. We tested compounds A, B and C for their ability to enhance the activities of human PreP. In contrast to the previous report, we observed that none of the compounds A, B, or C (3c) modulated the catalytic activity of human PreP. Here we report our findings on the mis-identification of the reported benzimidazoles and the lack of biological activity of such compounds on human PreP. Thus, PreP modulators for PreP-based therapies remain to be discovered.

Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes.

Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2' nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2'-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2' of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct.

Evidence That Nucleophile Deprotonation Exceeds Bond Formation in the HDV Ribozyme Transition State.

Steric constraints imposed by the active sites of protein and RNA enzymes pose major challenges to the investigation of structure-function relationships within these systems. As a strategy to circumvent such constraints in the HDV ribozyme, we have synthesized phosphoramidites from propanediol derivatives and incorporated them at the 5'-termini of RNA and DNA oligonucleotides to generate a series of novel substrates with nucleophiles perturbed electronically through geminal fluorination. In nonenzymatic, hydroxide-catalyzed intramolecular transphosphorylation of the DNA substrates, pH-rate profiles revealed that fluorine substitution reduces the maximal rate and the kinetic p Ka, consistent with the expected electron-withdrawing effect. In HDV ribozyme reactions, we observed that the RNA substrates undergo transphosphorylation relatively efficiently, suggesting that the conformational constraints imposed by a ribofuranose ring are not strictly required for ribozyme catalysis. In contrast to the nonenzymatic reactions, however, substrate fluorination modestly increases the ribozyme reaction rate, consistent with a mechanism in which (1) the 2'-hydroxyl nucleophile exists predominantly in its neutral, protonated form in the ground state and (2) the 2'-hydroxyl bears some negative charge in the rate-determining step, consistent with a transition state in which the extent of 2'-OH deprotonation exceeds the extent of P-O bond formation.

Synthesis of 5'-Thio-3'-O-ribonucleoside Phosphoramidites.

The chemical synthesis of phosphoramidite derivatives of all four 5'-deoxy-5'-thioribonucleosides is described. These phosphoramidites contained trityl (A, G, C, and U), dimethoxytrityl (A and G), or tert-butyldisulfanyl (G) as the 5'-S-protecting group. The application of several of these phosphoramidites for solid-phase synthesis of oligoribonucleotides containing a 2'-O-photocaged 5'-S-phosphorothiolate linkage or 5'-thiol-labeled RNAs is also further investigated.

Efficient Synthetic Approach to Linear Dasatinib-DNA Conjugates by Click Chemistry.

A pair of synthetic approaches to linear dasatinib-DNA conjugates via click chemistry are described. The first approach involves the reaction of excess azido dasatinib derivative with 5'-(5-hexynyl)-tagged DNAs, and the second involves the reaction of excess alkynyl-linked dasatinib with 5'-azido-tagged DNA. The second approach using alkynyl-derived dasatinib and 5'-azido-tagged DNA yielded the corresponding dasatinib-DNA conjugates in higher yield (47% versus 10-33% for the first approach). Studies have shown these linear dasatinib-DNA conjugates-derived gold nanoparticles exhibit efficacy against leukemia cancer cells with reduced toxicity toward normal cells compared to that of free dasatinib.

Specific Recognition of a Single-Stranded RNA Sequence by a Synthetic Antibody Fragment.

Antibodies that bind RNA represent an unrealized source of reagents for synthetic biology and for characterizing cellular transcriptomes. However, facile access to RNA-binding antibodies requires the engineering of effective Fab libraries guided by the knowledge of the principles that govern RNA recognition. Here, we describe a Fab identified from a minimalist synthetic library during phage display against a branched RNA target. The Fab (BRG) binds with 20nM dissociation constant to a single-stranded RNA (ssRNA) sequence adjacent to the branch site and can block the action of debranchase enzyme. We report the crystal structure in complex with RNA target at 2.38Å. The Fab traps the RNA in a hairpin conformation that contains a 2-bp duplex capped by a tetraloop. The paratope surface consists of residues located in four complementarity-determining regions including a major contribution from H3, which adopts a helical structure that projects into a deep, wide groove formed by the RNA. The amino acid composition of the paratope reflects the library diversity, consisting mostly of tyrosine and serine residues and a small but significant contribution from a single arginine residue. This structure, involving the recognition of ssRNA via a stem-loop conformation, together with our two previous structures involving the recognition of an RNA hairpin loop and an RNA tertiary structure, reveals the capacity of minimalist libraries biased with tyrosine, serine, glycine, and arginine to form binding surfaces for specific RNA conformations and distinct levels of RNA structural hierarchy.