Joseph A Piccirilli

University of Chicago, Chicago, Illinois, United States

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Publications (116)945.94 Total impact

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    ABSTRACT: Spinach is an in vitro-selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.
    Nature Chemical Biology 06/2014; · 12.95 Impact Factor
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    ABSTRACT: To catalyze pre-mRNA splicing, U6 small nuclear RNA positions two metals that interact directly with the scissile phosphates. U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. Domain V ligands are organized by base-triple interactions, which also juxtapose the 3' splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.
    Nature Structural & Molecular Biology 04/2014; · 11.90 Impact Factor
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    ABSTRACT: 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.
    The Journal of Organic Chemistry 03/2014; · 4.56 Impact Factor
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    ABSTRACT: Although there have been great strides in defining the mechanisms of RNA strand cleavage by 2'-O-transphosphorylation, long-standing questions remain. How do different catalytic modes such as acid/base and metal ion catalysis influence transition state charge distribution? Does the large rate enhancement characteristic of biological catalysis result in different transition states relative to solution reactions? Answering these questions is important for understanding biological catalysis in general, and revealing principles for designing small molecule inhibitors. Recent application of linear free energy relationships and kinetic isotope effects together with multi-scale computational simulations are providing tentative answers to these questions for this fundamentally important class of phosphoryl transfer reactions.
    Current Opinion in Chemical Biology. 01/2014; 21:96–102.
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    ABSTRACT: In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a dynamic machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, after the discovery of self-splicing group II intron RNAs, the snRNAs were proposed to catalyse splicing. However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported so far. By using metal rescue strategies in spliceosomes from budding yeast, here we show that the U6 snRNA catalyses both of the two splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction. Notably, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms and probably common evolutionary origins. Our results demonstrate that RNA mediates catalysis within the spliceosome.
    Nature 11/2013; · 38.60 Impact Factor
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    ABSTRACT: Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high-resolution structure of the catalytic domain of Sin, a particularly well-characterized family member, provided a detailed view of the catalytic site. To ask how the enzyme might protonate and stabilize the 3'O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg69 in Sin, was partially rescued by a 3' phosphorothiolate substrate strand. We conclude that Arg69 has an important role in stabilizing the 3' O leaving group and is the prime candidate for the general acid that protonates the 3' oxygen, in good agreement with the position it occupies in the high-resolution structure of the active site of Sin.
    Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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    ABSTRACT: 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 (18)O 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.
    Proceedings of the National Academy of Sciences 07/2013; · 9.81 Impact Factor
  • Nan-Sheng Li, Louise Scharf, Erin J Adams, Joseph A Piccirilli
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    ABSTRACT: beta-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.
    The Journal of Organic Chemistry 06/2013; · 4.56 Impact Factor
  • Armando R Hernández, Joseph A Piccirilli
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    ABSTRACT: Non-enzymatic copying of an RNA template is appealing as a transition from pre-life to an RNA world, but it has been difficult to demonstrate in the laboratory. Now, two separate studies focusing on RNA's backbone connectivity offer partial solutions to some of the problems raised with this hypothesis for the origin of life.
    Nature Chemistry 05/2013; 5(5):360-2. · 21.76 Impact Factor
  • Nan‐Sheng Li, Joseph A. Piccirilli
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    ABSTRACT: A novel synthetic approach to the new potentially important nucleoside analogues (V), (VI), (X), and (XI) is developed.
    ChemInform 12/2012; 43(50).
  • Nan-Sheng Li, John K Frederiksen, Joseph A Piccirilli
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    ABSTRACT: This work describes a general method for the synthesis of oligoribonucleotides containing a site-specific nonbridging phosphorodithioate linkage via automated solid-phase synthesis using 5'-O-DMTr-2'-O-TBS-ribonucleoside 3'-N,N-dimethyl-S-(2,4-dichlorobenzyl) phosphorothioamidites (2a-2d). The 3'-phosphorothioamidites (2a-2d) can be conveniently prepared in good yields (86-99%) via a one-pot reaction from the corresponding 5'-O-DMTr-2'-O-TBS-ribonucleosides (1a-1d).
    The Journal of Organic Chemistry 10/2012; · 4.56 Impact Factor
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    ABSTRACT: The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5'-oxygen atom at the scissile phosphate by sulfur (5'-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5'-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pK(a). On substitution of G8 by diaminopurine, the 5'-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pK(a) consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data.
    Journal of the American Chemical Society 09/2012; 134(40):16717-24. · 10.68 Impact Factor
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    ABSTRACT: Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.
    RNA 07/2012; 18(9):1687-701. · 5.09 Impact Factor
  • Nan-Sheng Li, Joseph A Piccirilli
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    ABSTRACT: 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.
    Chemical Communications 07/2012; 48(70):8754-6. · 6.38 Impact Factor
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    ABSTRACT: Within the three-dimensional architectures of RNA molecules, divalent metal ions populate specific locations, shedding their water molecules to form chelates. These interactions help the RNA adopt and maintain specific conformations and frequently make essential contributions to function. Defining the locations of these site-bound metal ions remains challenging despite the growing database of RNA structures. Metal-ion rescue experiments have provided a powerful approach to identify and distinguish catalytic metal ions within RNA active sites, but the ability of such experiments to identify metal ions that contribute to tertiary structure acquisition and structural stability is less developed and has been challenged. Herein, we use the well-defined P4-P6 RNA domain of the Tetrahymena group I intron to reevaluate prior evidence against the discriminatory power of metal-ion rescue experiments and to advance thermodynamic descriptions necessary for interpreting these experiments. The approach successfully identifies ligands within the RNA that occupy the inner coordination sphere of divalent metal ions and distinguishes them from ligands that occupy the outer coordination sphere. Our results underscore the importance of obtaining complete folding isotherms and establishing and evaluating thermodynamic models in order to draw conclusions from metal-ion rescue experiments. These results establish metal-ion rescue as a rigorous tool for identifying and dissecting energetically important metal-ion interactions in RNAs that are noncatalytic but critical for RNA tertiary structure.
    RNA 04/2012; 18(6):1123-41. · 5.09 Impact Factor
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    Raghuvir N Sengupta, Daniel Herschlag, Joseph A Piccirilli
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    ABSTRACT: 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 pK(a) 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.
    ACS Chemical Biology 02/2012; 7(2):294-9. · 5.44 Impact Factor
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    ABSTRACT: The elucidation of RNA reaction mechanisms has a wide range of implications, from the origin of life to biotechnology. In their Communication (DOI:10.1002/anie.201104147), K.-Y. Wong, J. A. Piccirilli, M. E. Harris, D. M. York, et al. report a joint theoretical and experimental study on kinetic isotope effects in models of RNA cleavage transesterification. The characteristics of the mechanism and the transition states are distinctly different in the reaction with the native compound relative to two thio-substituted analogues.
    Angewandte Chemie International Edition 12/2011; · 11.34 Impact Factor
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    Angewandte Chemie International Edition 11/2011; 51(3):647-51. · 11.34 Impact Factor
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    Joseph A Piccirilli, Yelena Koldobskaya
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    ABSTRACT: All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3',5'-phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA-protein world.
    Philosophical Transactions of The Royal Society B Biological Sciences 10/2011; 366(1580):2918-28. · 6.23 Impact Factor
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    ABSTRACT: 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 -OCH(3), -NH(2), -NHMe, and -NMe(2), 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 (G(NHMe)) and 2'-N,N-dimethylamino-2'-deoxyguanosine (G(NMe(2))) and their corresponding phosphoramidites. The key step in obtaining the nucleosides involved S(N)2 displacement of 2'-β-triflate from an appropriate guanosine derivative by methylamine or dimethylamine. We readily obtained the G(NMe(2)) phosphoramidite and incorporated it into RNA. However, the G(NHMe) 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.
    The Journal of Organic Chemistry 09/2011; 76(21):8718-25. · 4.56 Impact Factor

Publication Stats

2k Citations
945.94 Total Impact Points

Institutions

  • 1999–2014
    • University of Chicago
      • • Department of Biochemistry & Molecular Biology
      • • Department of Chemistry
      Chicago, Illinois, United States
  • 2007–2011
    • Stanford University
      • Department of Biochemistry
      Stanford, CA, United States
  • 2009
    • University of Michigan
      • Department of Chemistry
      Ann Arbor, MI, United States
  • 1991–2009
    • Howard Hughes Medical Institute
      Maryland, United States
  • 2005
    • University of Illinois at Chicago
      • Department of Chemistry
      Chicago, IL, United States
  • 2002
    • Lewis & Clark College
      • Department of Chemistry
      Portland, OR, United States
  • 1987–1991
    • École Polytechnique Fédérale de Lausanne
      • Laboratoire de chimie inorganique et bioinorganique
      Lausanne, VD, Switzerland
  • 1990
    • ETH Zurich
      • Laboratory of Organic Chemistry
      Zürich, ZH, Switzerland