The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate helix register into a so-called “shifted” conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage.

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.

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.

Endonucleolytic ribozymes constitute a class of non-coding RNAs that catalyze single-strand RNA scission. With crystal structures available for all of the known ribozymes, a major challenge involves relating functional data to the physically observed RNA architecture. In the case of the hepatitis delta virus (HDV) ribozyme, there are three high-resolution crystal structures, the product state of the reaction and two precursor variants, with distinct mechanistic implications. Here, we develop new strategies to probe the structure and catalytic mechanism of a ribozyme. First, we use double-mutant cycles to distinguish differences in functional group proximity implicated by the crystal structures. Second, we use a corrected form of the Brønsted equation to assess the functional significance of general acid catalysis in the system. Our results delineate the functional relevance of atomic interactions inferred from structure, and suggest that the HDV ribozyme transition state resembles the cleavage product in the degree of proton transfer to the leaving group.

Spinach RNA aptamer contains a G-quadruplex motif that serves as a platform for binding and fluorescence activation of a GFP-like fluorophore. Here we show that Pb2+ induces formation of Spinach's G-quadruplex and activates fluorescence with high selectivity and sensitivity. This device establishes the first example of an RNA-based sensor that provides a simple and inexpensive tool for Pb2+ detection.

•Isotopically labeled nucleotides enable kinetic isotope effect measurements on chemical reactions involving nucleic acids.•State-of-the-art synthetic methods allow access to a variety of site-specifically labeled nucleotides.•A survey of isotope effect studies employing labeled nucleotides underscores their value in interrogating enzymatic transition states.•Isotope effect analysis provides a powerful strategy to investigate RNA catalysisExperimental analysis of kinetic isotope effects represents an extremely powerful approach for gaining information about the transition state structure of complex reactions not available through other methodologies. The implementation of this approach to the study of nucleic acid chemistry requires the synthesis of nucleobases and nucleotides enriched for heavy isotopes at specific positions. In this review, we highlight current approaches to the synthesis of nucleic acids enriched site specifically for heavy oxygen and nitrogen and their application in heavy atom isotope effect studies. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment.

Oligoribonucleotides containing 3′-S-phosphorothiolate linkages possess properties that can reveal deep mechanistic insights into ribozyme-catalyzed reactions. “Photocaged” 3′-S- RNAs could provide a strategy to stall reactions at the chemical stage and release them after assembly steps have occurred. Toward this end, we describe here an approach for the synthesis of 2′-O-(o-nitrobenzyl)-3′-thioguanosine phosphoramidite starting from N2-isobutyrylguanosine in nine steps with 10.2% overall yield. Oligonucleotides containing the 2′-O-(o-nitrobenzyl)-3′-S-guanosine nucleotide were then constructed, characterized, and used in a nuclear pre-mRNA splicing reaction.

β-d-Mannosyl phosphomycoketide (C32-MPM), a naturally occurring glycolipid found in the cell walls of Mycobacterium tuberculosis, acts as a potent antigen to activate T-cells upon presentation by CD1c protein. The lipid portion of C32-MPM contains a C32-mycoketide, consisting of a saturated oligoisoprenoid chain with five chiral methyl branches. Here we develop several stereocontrolled approaches to assemble the oligoisoprenoid chain with high stereopurity (>96%) using Julia–Kocienski olefinations followed by diimide reduction. By careful choice of olefination sites, we could derive all chirality from a single commercial compound, methyl (2S)-3-hydroxy-2-methylpropionate (>99% ee). Our approach is the first highly stereocontrolled method to prepare C32-MPM molecule with >96% stereopurity from a single >99% ee starting material. We anticipate that our methods will facilitate the highly stereocontrolled synthesis of a variety of other natural products containing chiral oligoisoprenoid-like chains, including vitamins, phytol, insect pheromones, and archaeal lipids.

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2′-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the 18O KIEs on the 2′O nucleophile, 5′O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium- or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5′O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/Lys41), together with proton transfer from His119, render departure of the 5′O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2′-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure.

Starting from methyl 3,5-di-O-benzyl-2-keto-α-D-ribofuranoside, a convergent, six-step synthesis is developed to give efficiently all four 2′-C-α-aminomethyl-2′-deoxynucleosides (U, C, A, G) in 38%, 42%, 12%, 12% yield, respectively. Convergence is achieved by the glycosylation of persilylated nucleobases with methyl 2-α-phthalimidomethyl ribofuranoside.

Protein and RNA enzymes that catalyze phosphoryl transfer reactions frequently contain active site metal ions that interact with the nucleophile and leaving group. Mechanistic models generally hinge upon the assumption that the metal ions stabilize negative charge buildup along the reaction coordinate. However, experimental data that test this assumption directly remain difficult to acquire. We have used an RNA substrate bearing a 3′-thiol group to investigate the energetics of a metal ion interaction directly relevant to transition state stabilization in the Tetrahymena group I ribozyme reaction. Our results show that this interaction lowers the pKa of the 3′-thiol by 2.6 units, stabilizing the bound 3′-thiolate by 3.6 kcal/mol. These data, combined with prior studies, provide strong evidence that this metal ion interaction facilitates the forward reaction by stabilization of negative charge buildup on the leaving group 3′-oxygen and facilitates the reverse reaction by deprotonation and activation of the nucleophilic 3′-hydroxyl group.

RNA represents a prominent class of biomolecules. Present in all living systems, RNA plays many essential roles in gene expression, regulation, and development. Accordingly, many biological processes depend on the accurate enzymatic processing, modification, and cleavage of RNA. Understanding the catalytic mechanisms of these enzymes therefore represents an important goal in defining living systems at the molecular level.In this context, RNA molecules bearing 3′- or 5′-S-phosphorothiolate linkages comprise what are arguably among the most incisive mechanistic probes available. They have been instrumental in showing that RNA splicing systems are metalloenzymes and in mapping the ligands that reside within RNA active sites. The resulting models have in turn verified the functional relevance of crystal structures. In other cases, phosphorothiolates have offered an experimental strategy to circumvent the classic problem of kinetic ambiguity; mechanistic enzymologists have used this tool to assign precise roles to catalytic groups as general acids or bases. These insights into macromolecular function are enabled by the synthesis of nucleic acids bearing phosphorothiolate linkages and the unique chemical properties they impart. In this Account, we review the synthesis, properties, and applications of oligonucleotides and oligodeoxynucleotides containing an RNA dinucleotide phosphorothiolate linkage.Phosphorothioate linkages are structurally very similar to phosphorothiolate linkages, as reflected in the single letter of difference in nomenclature. Phosphorothioate substitutions, in which sulfur replaces one or both nonbridging oxygens within a phosphodiester linkage, are now widely available and are used routinely in numerous biochemical and medicinal applications. Indeed, synthetic phosphorothioate linkages can be introduced readily via a sulfurization step programmed into automated solid-phase oligonucleotide synthesizers.In contrast, phosphorothiolate oligonucleotides, in which sulfur replaces a specific 3′- or 5′-bridging oxygen, have presented a more difficult synthetic challenge, requiring chemical alterations to the attached sugar moiety. Here we begin by outlining the synthetic strategies used to access these phosphorothiolate RNA analogues. The Arbuzov reaction and phosphoramidite chemistry are often brought to bear in creating either 3′- or 5′-S-phosphorothiolate dinucleotides. We then summarize the responses of the phosphorothiolate derivatives to chemical and enzymatic cleavage agents, as well as mechanistic insights their use has engendered. They demonstrate particular utility as probes of metal-ion-dependent phosphotransesterification, general acid-base-catalyzed phosphotransesterification, and rate-limiting chemistry. The 3′- and 5′-S-phosphorothiolates have proven invaluable in elucidating the mechanisms of enzymatic and nonenzymatic phosphoryl transfer reactions. Considering that RNA cleavage represents a fundamental step in the maturation, degradation, and regulation of this important macromolecule, the significant synthetic challenges that remain offer rich research opportunities.

The ability of fluorine in a C-F bond to act as a hydrogen bond acceptor is controversial. To test such ability in complex RNA macromolecules, we have replaced native 2′-OH groups with 2′-F and 2′-H groups in two related systems, the Tetrahymena group I ribozyme and the ΔC209 P4-P6 RNA domain. In three cases the introduced 2′-F mimics the native 2′-OH group, suggesting that the fluorine atom can accept a hydrogen bond. In each of these cases the native hydroxyl group interacts with a purine exocyclic amine. Our results give insight about the properties of organofluorine and suggest a possible general biochemical signature for tertiary interactions between 2′-hydroxyl groups and exocyclic amino groups within RNA.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (70 K)Download as PowerPoint slideHighlights► We tested the ability of organofluorine to accept hydrogen bonds in two RNA systems ► In three cases a 2′-F, but not a 2′-H, can effectively substitute the native 2′-OH ► In these cases the native 2′-OH accepts a hydrogen bond from a purine amino group ► We suggest that a 2′-F can accept a hydrogen bond from an RNA purine amino group

Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein−RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes.

The 2′-hydroxyl groups within RNA contribute in essential ways to RNA structure and function. Previously, we designed an atomic mutation cycle (AMC) that uses ribonucleoside analogues bearing different C-2′-substituents, including −OCH3, −NH2, −NHMe, and −NMe2, to identify hydroxyl groups within RNA that donate functionally significant hydrogen bonds. To enable AMC analysis of the nucleophilic guanosine cofactor in the Tetrahymena ribozyme reaction and at other guanosines whose 2′-hydroxyl groups impart critical functional contributions, we describe here the syntheses of 2′-methylamino-2′-deoxyguanosine (GNHMe) and 2′-N,N-dimethylamino-2′-deoxyguanosine (GNMe2) and their corresponding phosphoramidites. The key step in obtaining the nucleosides involved SN2 displacement of 2′-β-triflate from an appropriate guanosine derivative by methylamine or dimethylamine. We readily obtained the GNMe2 phosphoramidite and incorporated it into RNA. However, the GNHMe phosphoramidite posed a significantly greater challenge due to lack of a suitable −2′-NHMe protecting group. After testing several strategies, we established that allyloxycarbonyl (Alloc) provided suitable protection for 2′-N-methylamino group during the phosphoramidite synthesis and the subsequent RNA synthesis. This work enables AMC analysis of guanosine’s 2′-hydroxyl group within RNA.

To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary 18O kinetic isotope effects (KIEs) and solvent D2O isotope effects were measured to probe the mechanism of base-catalyzed 2′-O-transphosphorylation of the RNA dinucleotide 5′-UpG-3′. The observed 18O KIEs for the nucleophilic 2′-O and in the 5′-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus−oxygen bond fission to the leaving group (18kLG = 1.034 ± 0.004) and phosphorus−nucleophile bond formation (18kNUC = 0.984 ± 0.004). A breakpoint in the pH dependence of the 2′-O-transphosphorylation rate to a pH independent phase above pH 13 has been attributed to the pKa of the 2′−OH nucleophile. A smaller nucleophile KIE is observed at pH 12 (18kNUC = 0.995 ± 0.004) that is interpreted as the combined effect of the equilibrium isotope effect (ca. 1.02) on deprotonation of the 2′-hydroxyl nucleophile and the intrinsic KIE on the nucleophilic addition step (ca. 0.981). An alternative mechanism in which the hydroxide ion acts as a general base is considered unlikely given the lack of a solvent deuterium isotope effect above the breakpoint in the pH versus rate profile. These results represent the first direct analysis of the transition state for RNA strand cleavage. The primary 18O KIE results and the lack of a kinetic solvent deuterium isotope effect together provide strong evidence for a late transition state and 2′-O nucleophile activation by specific base catalysis.

In the structures of the HDV ribozyme a cytosine nucleobase resides at the active site poised to participate directly in catalysis. Defining the functional role of the nucleobase requires nucleoside analogues that perturb the functional groups in a strategic manner. Herein, we have developed efficient methods for the synthesis of five C-nucleoside phosphoramidite derivatives that, when used in combination, provide strategies for probing the potential functional role of cytosine’s keto group and imino nitrogen. Phosphoramidites 15a and 15b were synthesized in 11 steps starting from 2-amino-5-bromopyrimidine (1a) and 2-amino-5-bromopyridine (1b), respectively, with overall yields of 10.8% and 6.6%, respectively. Phosphoramidite 21 was prepared from intermediate 11b in seven steps with an overall yield of 33.7%. Phosphoramidites 23 and 25 were prepared from 2,4-diamino-5-(β-d-ribofuranosyl)-1,3-pyrimidine (22) and pseudoisocytidine (24), respectively, with an overall yield of 15.9% (six steps) and 37.9% (four steps), respectively. These phosphoramidites were incorporated into oligonucleotides by solid-phase synthesis.

In the first step of self-splicing, group I introns utilize an exogenous guanosine nucleophile to attack the 5′-splice site. Removal of the 2′-hydroxyl of this guanosine results in a 106-fold loss in activity, indicating that this functional group plays a critical role in catalysis. Biochemical and structural data have shown that this hydroxyl group provides a ligand for one of the catalytic metal ions at the active site. However, whether this hydroxyl group also engages in hydrogen-bonding interactions remains unclear, as attempts to elaborate its function further usually disrupt the interactions with the catalytic metal ion. To address the possibility that this 2′-hydroxyl contributes to catalysis by donating a hydrogen bond, we have used an atomic mutation cycle to probe the functional importance of the guanosine 2′-hydroxyl hydrogen atom. This analysis indicates that, beyond its role as a ligand for a catalytic metal ion, the guanosine 2′-hydroxyl group donates a hydrogen bond in both the ground state and the transition state, thereby contributing to cofactor recognition and catalysis by the intron. Our findings continue an emerging theme in group I intron catalysis: the oxygen atoms at the reaction center form multidentate interactions that function as a cooperative network. The ability to delineate such networks represents a key step in dissecting the complex relationship between RNA structure and catalysis.

Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigen-binding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab–RNA complex at 1.95 Å. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementarity-determining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab–RNA interface also differs significantly from Fab–protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology.

Group II introns are mobile genetic elements that have been implicated as agents of genetic diversity, and serve as important model systems for investigating RNA catalysis and pre-mRNA splicing. In the absence of an atomic-resolution structure of the intron, detailed understanding of its catalytic mechanism has remained elusive. Previous identification of a divalent metal ion stabilizing the leaving group in both splicing steps suggested that the group II intron may employ a “two-metal ion” mechanism, a catalytic strategy used by a number of protein phosphoester transfer enzymes. Using metal rescue experiments, we now reveal the presence of a second metal ion required for nucleophile activation in the exon-ligation step of group II intron splicing. Coupled with biochemical and structural evidence of at least two metal ions at the group I intron reaction center, these results suggest a mechanistic paradigm for describing catalysis by large ribozymes.

2′-Amino-2′-deoxynucleosides and oligonucleotides containing them have proven highly effective for an array of biochemical applications. The guanosine analogue and its phosphoramidite derivatives have been accessed previously from 2′-amino-2′-deoxyuridine by transglycosylation, but with limited overall efficiency and convenience. Using simple modifications of known reaction types, we have developed useful protocols to obtain 2′-amino-2′-deoxyguanosine and two of its phosphoramidite derivatives with greater convenience, fewer steps, and higher yields than reported previously. These phosphoramidites provide effective synthons for the incorporation of 2′-amino-2′-deoxyguanosine into oligonucleotides.

•Fabs that bind RNA have great potential as biomedical research tools.•Fab BRG binds to a 12-nt ssRNA with high affinity and specificity.•The Fab BRG stabilizes a hairpin conformation of the RNA.•Complementarity-determining region-H3 and tyrosine play dominant roles in RNA recognition.•Fab provides a versatile scaffold for RNA binding.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 20 nM 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 2.38 Å. 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.Download high-res image (137KB)Download full-size image

Spinach RNA aptamer contains a G-quadruplex motif that serves as a platform for binding and fluorescence activation of a GFP-like fluorophore. Here we show that Pb2+ induces formation of Spinach's G-quadruplex and activates fluorescence with high selectivity and sensitivity. This device establishes the first example of an RNA-based sensor that provides a simple and inexpensive tool for Pb2+ detection.

Starting from methyl 3,5-di-O-benzyl-2-keto-α-D-ribofuranoside, a convergent, six-step synthesis is developed to give efficiently all four 2′-C-α-aminomethyl-2′-deoxynucleosides (U, C, A, G) in 38%, 42%, 12%, 12% yield, respectively. Convergence is achieved by the glycosylation of persilylated nucleobases with methyl 2-α-phthalimidomethyl ribofuranoside.