Joseph A Piccirilli

University of Chicago, Chicago, Illinois, United States

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Publications (142)1186.41 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH2) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed MC, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.
    RNA 11/2015; DOI:10.1261/rna.053710.115 · 4.94 Impact Factor
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    ABSTRACT: The Varkud satellite (VS) ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.
    Nature Chemical Biology 09/2015; 11(11). DOI:10.1038/nchembio.1929 · 13.00 Impact Factor

  • Biochimica et Biophysica Acta 08/2015; DOI:10.1016/j.bbapap.2015.08.006 · 4.66 Impact Factor
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    ABSTRACT: As one of its goals, synthetic biology seeks to increase the number of building blocks in nucleic acids. While efforts towards this goal are well advanced for DNA, they have hardly begun for RNA. Herein, we present a crystal structure for an RNA riboswitch where a stem C:G pair has been replaced by a pair between two components of an artificially expanded genetic-information system (AEGIS), Z and P, (6-amino-5-nitro-2(1H)-pyridone and 2-amino-imidazo[1,2-a]-1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNA molecule nor the details of its interaction with a hypoxanthine ligand. This was confirmed in solution by in-line probing, which also measured a 3.7 nM affinity of the riboswitch for guanine. These data show that the Z:P pair mimics the natural Watson-Crick geometry in RNA in the first example of a crystal structure of an RNA molecule that contains an orthogonal added nucleobase pair. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Angewandte Chemie International Edition 07/2015; 54(34):9853-9856. DOI:10.1002/ange.201504731 · 11.26 Impact Factor
  • Selene C Koo · Jun Lu · Nan-Sheng Li · Edward Leung · Subha R Das · Michael E Harris · Joseph A Piccirilli ·
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    ABSTRACT: 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 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.
    Journal of the American Chemical Society 06/2015; 137(28). DOI:10.1021/jacs.5b01189 · 12.11 Impact Factor
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    ABSTRACT: Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2'-O-transphosphorylation by the HDV ribozyme is sensitive to the identity of the N(-1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2'O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(-1) nucleobase contributes to HDV catalysis, and to test the utility of this approach for RNA structure-function studies. Internal competition reactions containing all four substrate sequence variants at the N(-1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed basepairing interaction. Mutants A78U, A78G and A79G retain significant catalytic activity, but do not alter the specificity for the N(-1) nucleobase. Effects of nucleobase analog substitutions at N(-1) indicate that U is preferred due to the ability to donate an H-bond in the Watson-Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site, and illustrate multiple-substrate kinetic analyses as a practical approach for characterizing structure-function relationships in RNA reactions. Published by Elsevier Inc.
    Analytical Biochemistry 05/2015; 483(1). DOI:10.1016/j.ab.2015.04.024 · 2.22 Impact Factor
  • Michael E Harris · Joseph A Piccirilli · Darrin M York ·
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    ABSTRACT: The well-studied mechanism of ribonuclease A is believed to involve concerted general acid-base catalysis by two histidine residues, His12 and His119. The basic features of this mechanism are often cited to explain rate enhancement by both protein and RNA enzymes that catalyze RNA 2'-O-transphosphorylation. Recent kinetic isotope effect analyses and computational studies are providing a more chemically detailed description of the mechanism of RNase A and the rate limiting transition state. Overall, the results support an asynchronous mechanism for both solution and ribonuclease catalyzed reactions in which breakdown of a transient dianoinic phosphorane intermediate by 5'O-P bond cleavage is rate limiting. Relative to non-enzymatic reactions catalyzed by specific base, a smaller KIE on the 5'O leaving group and a less negative βLG are observed for RNase A catalysis. Quantum mechanical calculations consistent with these data support a model in which electrostatic and H-bonding interactions with the non-bridging oxygens and proton transfer from His119 render departure of the 5'O less advanced and stabilize charge buildup in the transition state. Both experiment and computation indicate advanced 2'O-P bond formation in the rate limiting transition state. However, this feature makes it difficult to resolve the chemical steps involved in 2'O activation. Thus, modeling the transition state for RNase A catalysis underscores those elements of its chemical mechanism that are well resolved, as well as highlighting those where ambiguity remains. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 04/2015; DOI:10.1016/j.bbapap.2015.04.022 · 4.66 Impact Factor
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    Saurja DasGupta · Sandip A Shelke · Nan-Sheng Li · Joseph A Piccirilli ·
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    ABSTRACT: 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.
    Chemical Communications 04/2015; 51(43). DOI:10.1039/C5CC01526J · 6.83 Impact Factor
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    Benjamin P. Weissman · Nan-Sheng Li · Darrin York · Michael Harris · Joseph A. Piccirilli ·
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    ABSTRACT: Experimental 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. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 03/2015; 80(11). DOI:10.1016/j.bbapap.2015.03.007 · 2.75 Impact Factor
  • Haoyuan Chen · Joseph A. Piccirilli · Michael E. Harris · Darrin M. York ·
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    ABSTRACT: Divalent metal ions, due to their ability to stabilize high concentrations of negative charge, are important for RNA folding and catalysis. Detailed models derived from the structures and kinetics of enzymes and from computational simulations have been developed. However, in most cases the specific catalytic modes involving metal ions and their mechanistic roles and effects on transition state structures remain controversial. Valuable information about the nature of the transition state is provided by measurement of kinetic isotope effects (KIEs). However, KIEs reflect changes in all bond vibrational modes that differ between the ground state and transition state. QM calculations are therefore essential for developing structural models of the transition state and evaluating mechanistic alternatives. Herein, we present computational models for Zn(2+) binding to RNA 2'O-transphosphorylation reaction models that aid in the interpretation of KIE experiments. Different Zn(2+) binding modes produce distinct KIE signatures, and one binding mode involving two zinc ions is in close agreement with KIEs measured for non-enzymatic catalysis by Zn(2+) aquo ions alone. Interestingly, the KIE signatures in this specific model are also very close to those in RNase A catalysis. These results allow a quantitative connection to be made between experimental KIE measurements and transition state structure and bonding, and provide insight into RNA 2'O-ransphosphorylation reactions catalyzed by metal ions and enzymes. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 03/2015; 1854(11). DOI:10.1016/j.bbapap.2015.02.022 · 2.75 Impact Factor
  • Jun Lu · Selene C Koo · Nan-Sheng Li · Joseph A Piccirilli ·
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    ABSTRACT: The chemical synthesis and incorporation of the phosphoramidite derivatives of 2 '-O-photocaged ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-nitrobenzyl group into oligoribonucleotides are described. The efficiency of UV irradiated uncaging of these 2'-O-photocaged oligoribonucleotides was found in the order of α-methyl-o-nitrobenzyl < 4,5-dimethoxy-2-nitrobenzyl < 2'-O-o-nitrobenzyl.
    Nucleosides Nucleotides &amp Nucleic Acids 02/2015; 34(2):114-29. DOI:10.1080/15257770.2014.965256 · 1.02 Impact Factor
  • Sandip A Shelke · Joseph A Piccirilli ·
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    ABSTRACT: An RNA enzyme has been generated that can assemble a mirror-image version of itself. The finding helps to answer a long-standing conundrum about how RNA molecules could have proliferated on prebiotic Earth.
    Nature 10/2014; 515(7527). DOI:10.1038/nature13935 · 41.46 Impact Factor
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    Sobhan Roy · Dalam Ly · Nan-Sheng Li · John D Altman · Joseph A Piccirilli · D Branch Moody · Erin J Adams ·
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    ABSTRACT: CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens.
    Proceedings of the National Academy of Sciences 10/2014; 111(43). DOI:10.1073/pnas.1408549111 · 9.67 Impact Factor
  • Daniel L Kellerman · Darrin M York · Joseph A Piccirilli · Michael E Harris ·
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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 08/2014; 21:96–102. DOI:10.1016/j.cbpa.2014.06.010 · 6.81 Impact Factor
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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; 10(8). DOI:10.1038/nchembio.1561 · 13.00 Impact Factor
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    Sebastian M Fica · Melissa A Mefford · Joseph A Piccirilli · Jonathan P Staley ·
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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; 21(5). DOI:10.1038/nsmb.2815 · 13.31 Impact Factor
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    Nan-Sheng Li · Nicole Tuttle · Jonathan P Staley · Joseph A Piccirilli ·
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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; 79(8). DOI:10.1021/jo4028374 · 4.72 Impact Factor
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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; 503(7475). DOI:10.1038/nature12734 · 41.46 Impact Factor
  • Nan-Sheng Li · Joseph A. Piccirilli ·
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    ABSTRACT: An efficient synthetic method towards stereopure acyclic 1,5-dimethylalkane building blocks from methyl (2R)-3-hydroxy-2-methylpropionate (R)-1 (>99% ee) and methyl (2S)-3-hydroxy-2-methylpropionate (S)-1 (>99% ee) through a series of chemical transformations, including Julia-Kocienski olefination and diimide reduction, is described. Through this strategy, two fragments of beta-D-mannosyl phosphomycoketide (C-32-MPM) and four stereopure 1,5-dimethylalkane C-10 chirons are prepared. These C-32-MPM fragments and C-10 chirons have shown great potential application as building blocks for the synthesis of highly methyl-branched natural products containing chiral oligoisoprenoid-like chains. (c) 2013 Elsevier Ltd. All rights reserved.
    Tetrahedron 11/2013; 69(46):9633-9641. DOI:10.1016/j.tet.2013.09.020 · 2.64 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; 288(40). DOI:10.1074/jbc.M113.508028 · 4.57 Impact Factor

Publication Stats

4k Citations
1,186.41 Total Impact Points


  • 1997-2015
    • University of Chicago
      • • Department of Biochemistry & Molecular Biology
      • • Department of Chemistry
      Chicago, Illinois, United States
  • 2010
    • University of Dundee
      • Division of Cancer Research
      Dundee, Scotland, United Kingdom
  • 1991-2009
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
    • University of Cambridge
      • Department of Pharmacology
      Cambridge, England, United Kingdom
  • 2008
    • Rosalind Franklin University of Medicine and Science
      • Biochemistry and Molecular Biology
      North Chicago, Illinois, United States
  • 1992-2001
    • Stanford University
      • Department of Biochemistry
      Palo Alto, California, United States
  • 1987-1991
    • Eawag: Das Wasserforschungs-Institut des ETH-Bereichs
      Duebendorf, Zurich, Switzerland