个人简介

B.A. (Chemistry), 1982, University of Scranton; Fulbright Scholar, 1983, Technische Hochschule Aachen, Germany; Harvard Traveling Scholar, 1986-1989, ETH Zurich, Switzerland; Ph.D. (Chemical Biology), 1989, Harvard University

研究领域

Our group is broadly interested in the chemistry and biochemistry of nucleic acids with particular emphasis on RNA catalysis and structure. The laboratory integrates areas of organic chemistry, physical chemistry, enzymology and molecular biology to gain a fundamental understanding of nucleic acid structure and mechanisms of RNA catalysis. Using the principles and techniques of organic chemistry and molecular biology, we manipulate the structure of RNA molecules at precise locations in ways that are designed to answer very specific questions about biological function.

Our group is broadly interested in the chemistry and biochemistryof nucleic acids with particular emphasis onRNA and RNA catalysis. The laboratory integrates areasof organic chemistry, physical chemistry, enzymologyand molecular biology to gain a fundamental understandingof nucleic acid structure and mechanisms ofRNA catalysis. Using the principles and techniques oforganic chemistry and molecular biology, we manipulatethe structure of RNA molecules at precise locations inways that are designed to answer very specific questionsabout biological function. Mechanism of RNA Catalysis We employ these approaches toward gaining a fundamentalunderstanding of the role that divalent metal ionsplay in phosphoryl transfer reactions that occur duringRNA splicing, a fundamental step in genetic expression.One experimental system that we are using to addressthese issues is the self-splicing intervening sequenceRNA of the ciliated protozoan Tetrahymena. Shortenedforms of this RNA can act as enzymes, catalyzing thesequence specific cleavage of RNA and DNA substrateswith multiple turnover. We have used sulfur substitutionof the oxygen substituents on the phosphoryl groupundergoing transfer to reveal the transition state interactionsbetween the ribozyme and the scissile phosphate.Another area of interest is the development of new methodsand model systems for studying RNA molecules. Forexample, we have recently designed a series of nucleosideanalogues, in which the C2Õ-beta hydrogen atom ofthe ribose is replaced by CH3, CH2F, CHF2, or CF3.These analogues provide a systematic way to perturb theacidity of the 2'-OH group, thereby allowing us to probethe all important role of this functional group in RNAmediated biological processes. RNA-Protein Interactions Restrictocin is a small protein (149 amino acids) that isso toxic that a single molecule can kill an entire cell. Thisprotein from Aspergillus restrictus is a member of a groupof functionally homologous cytotoxins, which includesthe better-known sarcin, and the mechanism of toxicity isfascinating. The single protein is able to cross the cellmembrane and cleave the 23Ð28S ribosomal RNA at asingle phosphodiester bond. The cleavage site resides ina region of the ribosomal RNA known as the sarcin/ricinloop (SRL), which folds into a tetraloop motif and abulged-G motif. The SRL participates in the binding ofelongation factors during protein synthesis. Consideringthat the 28S ribosomal RNA contains thousands of phosphodiesterbonds, the apparent specificity of this ribonucleaseis remarkable. This single cleavage event inactivatesthe ribosome and consequently abolishes its abilityto carry out protein synthesis, which ultimately leadsto death of the cell. This scenario immediately prompts a number of questions:How does the protein cross the cell membrane?Does it really possess the attributed specificity? Is everyribosome in the cell inactivated or does a single inactivationevent lead to activation of an apoptotic pathway?Additionally, the potency of this protein immediatelysuggests a potential clinic use as an anticancer drug. Allof these are interesting questions that we hope to answer.In addition, this system has broader significance in biologyas a model system to study RNA-protein interactions,which are ubiquitous and mediate numerous importantevents during gene expression. The crystal structures ofrestrictocin and the SRL RNA have been solved in isolation,and Carl CorrellÕs lab (University of Chicago) hassolved a structure of an SRL analog in complex withrestrictocin. Upon complex formation the geometry ofthe tetraloop is dramatically rearranged by base restackingand base flipping. Remarkably, few functional studieshave been reported on this protein. Our initial focus willbe to determine the dynamic changes that occur in theSRL when it binds to restrictocin and to elucidate theenergetic contributions that enzyme-RNA substrate contactsplay in cleavage-site recognition and catalysis.

近期论文

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Lu J, Koo SC, Li NS and Piccirilli JA. Synthesis of 2'-O-photocaged ribonuleoside phosphoramidites. Nucleosides, Nucleotides and Nucleic Acids. 2015 34(2), 114-129. Shelke SA, Piccirilli JA. Origins of life: RNA made in its own mirror image. Nature. 2014 Oct 29. Roy S, Ly D, Li NS, Altman JD, Piccirilli JA, Moody DB, Adams EJ. Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells. Proc Natl Acad Sci U S A. 2014 Oct 8. Kellerman DL, York DM, Piccirilli JA, Harris ME. Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2'-O-transphosphorylation. Curr Opin Chem Biol. 2014 Jul 11;21C:96-102. Huang H, Suslov NB, Li NS, Shelke SA, Evans ME, Koldobskaya Y, Rice PA, Piccirilli JA. A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore. Nat Chem Biol. 2014 Jun 22. Fica SM, Mefford MA, Piccirilli JA, Staley JP. Evidence for a group II intron-like catalytic triplex in the spliceosome. Nat Struct Mol Biol. 2014 Apr 20. doi: 10.1038/nsmb.2815. Li NS, Tuttle N, Staley JP, Piccirilli JA. Synthesis and Incorporation of the Phosphoramidite Derivative of 2'-O-Photocaged 3'-S-Thioguanosine into Oligoribonucleotides: Substrate for Probing the Mechanism of RNA Catalysis. J Org Chem. 2014 Mar 17. [Epub ahead of print] Li NS, Piccirilli JA. Synthesis of stereopure acyclic 1,5-dimethylalkane chirons: building blocks of highly methyl-branched natural products. Tetrahedron. 2013 Nov 18;69(46);9633-9641 Fica SM, Tuttle N, Novak T, Li NS, Lu J, Koodathingal P, Dai Q, Staley JP, Piccirilli JA. RNA catalyses nuclear pre-mRNA splicing. Nature. 2013 Nov 14;503(7475):229-34. Keenholtz RA, Mouw KW, Boocock MR, Li NS, Piccirilli JA, Rice PA. Arginine as a General Acid Catalyst in Serine Recombinase-mediated DNA Cleavage. J Biol Chem. 2013 Oct 4;288(40):29206-29214. Gu H, Zhang S, Wong KY, Radak BK, Dissanayake T, Kellerman DL, Dai Q, Miyagi M, Anderson VE, York DM, Piccirilli JA, Harris ME. Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′-O-transphosphorylation. Proc Natl Acad Sci U S A. 2013 Aug 6;110(32):13002-7. Li NS, Scharf L, Adams EJ, Piccirilli JA. Highly stereocontrolled total synthesis of β-D-mannosyl phosphomycoketide: a natural product from Mycobacterium tuberculosis. J Org Chem. 2013 Jun 21;78(12) :5970-86. Hernandez AR, Piccirilli JA. Chemical origins of life: Prebiotic RNA unstuck. Nat Chem. 2013 May;5(5):360-2. Li NS, Frederiksen JK, Piccirilli JA. Automated Solid-Phase Synthesis of RNA Oligonucleotides Containing a Nonbridging Phosphorodithioate Linkage via Phosphorothioamidites. J Org Chem. 2012 Nov 2;77(21):9889-92 Kath-Schorr S, Wilson TJ, Li NS, Lu J, Piccirilli JA, Lilley DM. General acid-base catalysis mediated by nucleobases in the hairpin ribozyme. J Am Chem Soc. 2012 Sep 7. Sakaguchi, R., Giessing, A., Dai, Q., Lahoud, G., Liutkeviciute, Z., Klimasauskas, S., Piccirilli, J., Kirpekar, F. & Hou, Y. M. (2012). Recognition of guanosine by dissimilar tRNA methyltransferases. RNA 18, 1687-701. Li NS, Piccirilli JA. Efficient synthesis of 2′-C-α-aminomethyl-2′-deoxynucleosides. Chem Commun 2012 Sep 11;48(70):8754-6. Frederiksen JK, Li NS, Das R, Herschlag D, Piccirilli JA. Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding. RNA. 2012 Apr 26. Sengupta RN, Herschlag D, Piccirilli JA. Thermodynamic Evidence for Negative Charge Stabilization by a Catalytic Metal Ion within an RNA Active Site. ACS Chem Biol. 2012 Feb 17;7(2):294-9. Wong KY, Gu H, Zhang S, Piccirilli JA, Harris ME, York DM. Characterization of the reaction path and transition States for RNA transphosphorylation models from theory and experiment. Angew Chem Int Ed Engl. 2012 Jan 16;51(3):647-51. Dai Q, Sengupta R, Deb SK, Piccirilli JA. Synthesis of 2′-N-Methylamino-2′-deoxyguanosine and 2′-N,N-Dimethylamino-2′-deoxyguanosine and Their Incorporation into RNA by Phosphoramidite Chemistry.J Org Chem. 2011 Nov 4;76(21):8718-25. Piccirilli JA, Koldobskaya Y. Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment. Philos Trans R Soc Lond B Biol Sci. 2011 Oct 27;366(1580):2918-28. Li NS, Frederiksen JK, Piccirilli JA. Synthesis, Properties, and Applications of Oligonucleotides Containing an RNA Dinucleotide Phosphorothiolate Linkage. Acc Chem Res. 2011 Dec 20;44(12):1257-69. Leung EK, Suslov N, Tuttle N, Sengupta R, Piccirilli JA. The Mechanism of Peptidyl Transfer Catalysis by the Ribosome. Annu Rev Biochem. 2010 Jun 23. Annu Rev Biochem. 2011 Jun 7;80:527-55. Forconi M, Porecha RH, Piccirilli JA, Herschlag D. Tightening of Active Site Interactions En Route to the Transition State Revealed by Single-Atom Substitution in the Guanosine-Binding Site of the Tetrahymena Group I Ribozyme. J Am Chem Soc. 2011 May 25;133(20):7791-800. Gilbreth RN, Truong K, Madu I, Koide A, Wojcik JB, Li NS, Piccirilli JA, Chen Y, Koide S. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design. Proc Natl Acad Sci U S A. 2011, 108, 7751-7756. Plantinga MJ, Korennykh AV, Piccirilli JA, Correll CC. The ribotoxin restrictocin recognizes its RNA substrate by selective engagement of active site residues. Biochemistry. 2011 Apr 12;50(14):3004-13. Ramu, H., Vazquez-Laslop, N., Klepacki, D., Dai, Q., Piccirilli, J., Micura, R., Mankin, A.S. Nascent Peptide in the Ribosome Exit Tunnel Affects Functional Properties of the A-Site of the Peptidyl Transferase Center. Mol Cell. 2011, 41, 321-330. Koldobskaya Y, Duguid EM, Shechner DM, Suslov NB, Ye J, Sidhu SS, Bartel DP, Koide S, Kossiakoff AA, Piccirilli JA. A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination. Nat Struct Mol Biol. 2011 Jan;18(1):100-6. Li NS, Frederiksen JK, Koo SC, Lu J, Wilson TJ, Lilley DM, Piccirilli JA. A general and efficient approach for the construction of RNA oligonucleotides containing a 5′-phosphorothiolate linkage. Nucleic Acids Res. 2011 Mar;39(5):e31. Forconi M, Schwans JP, Porecha RH, Sengupta RN, Piccirilli JA, Herschlag D. 2′-Fluoro substituents can mimic native 2′-hydroxyls within structured RNA. Chem Biol. 2011 Aug 26;18(8):949-54.