Preface A decade has passed since Methods in Enzymology addressed methods and techniques used in RNA processing. As has been evident since its inception, research in RNA processing progresses at a rapid pace. Its expansion into new areas of investigation has been phenomenal with novel discoveries being made in a variety of subspecialty areas. The subfield of RNA-ligand interactions concerns research problems in RNA structure, in the molecular recognition of structured RNA by diverse ligands, and in the mechanistic details of RNA's functional role following ligand binding. At the beginning of this new millennium, we celebrate the explosive development of exciting new tools and procedures whereby investigators explore RNA structure and function from the perspective of understanding RNA-ligand interactions. New insights into RNA processing are accompanied with improve- ments in older techniques as well as the development of entirely new methods. Previous Methods in Enzymology volumes in RNA processing have focused on basic methods generally employed in all RNA processing systems (Volume 180) or on techniques whose applications might be considerably more specific to a particular system (Volume 181). RNA- Ligand Interactions, Volumes 317 and 318, showcase many new methods that ihave led to significant advances in this subfield. The types of ligands described in these volumes certainly include proteins; however, ligands composed of RNA, antibiotics, other small molecules, and even chemical elements are also found in nature and have been the focus of much research work. Given the great diversity of RNA-ligand interactions described in these volumes, we have assembled the contributions according to whether they pertain to structural biology methods (Volume 317) or to biochemistry and molecular biology techniques (Volume 318). Aside from the particular systems for which these techniques have been devel- oped, we consider it likely that the methods described will enjoy uses that extend beyond RNA-ligand interactions to include other areas of RNA processing. This endeavor has been fraught with many difficult decisions regarding the selection of topics for these volumes. We were delighted with the number of chapters received. The authors have taken great care and dedication to present their contributions in clear language. Their willing- ness to share with others the techniques used in their laboratories is xiii xiv PREFACE apparent from the quality of their comprehensive contributions. We thank them for their effort and appreciate their patience as the volumes were assembled. DANIEL W. CeLANDER JOHN N. ABELSON Contributors to Volume 317 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. RAJENDRA K. AGRAWAL (18, 19), Howard Hughes Medical Institute, Health Research, Inc., at the Wadsworth Center, and Depart- ment of Biomedical Sciences, State Univer- sity of New York, Albany, New York 12201-0509 Russ B. ALTMAN (28), Departments of Medi- cine and Computer Science, Stanford Uni- versity, Stanford, California 94305-5479 MICHAEL A. BADA (28), Stanford University, Stanford, California 94305-5479 PETER BAYER (14), MRC Laboratory of Mo- lecular Biology, Cambridge CB2 2QH, En- gland LEONIr) BEIGELMAN (3), Ribozyme Pharma- ceuticals, Inc., Boulder, Colorado 80301 GREGOR BLAHA (19), AG Ribosomen, Max- Planck-Institut far Molekulare Genetik, D- 14195 Berlin, Germany MARC BOUDVILLAIN (10), Howard Hughes Medical Institute, Columbia University, New York, New York 10032 MICHAEL BRENOW1TZ (22), Department of Biochemistry, Center for Synchrotron BiD- sciences, Albert Einstein College of Medi- cine, Bronx, New York 10461 JOHN M. BURKE (25), University of Vermont, Burlington, Vermont 05405 NILS BURKHARDT (17), Gebi~ude 405, BAYER AG-Wuppertal, Abteilung MST, D-42096 Wuppertal, Germany JAMIE H. CATE (12), Whitehead Institute, Cambridge, Massachusetts 02142-1479 ROBERT CEDERGREN (27), D~partement de Biochimie, Universit~ de Montreal, Mon- treal, Quebec H3C 3J7, Canada MARK R. CHANCE (22), Department of Physi- oloKy and Biophysics, Center for Synchro- tron Biosciences, Albert Einstein College of Medicine, Bronx, New York 10461 V1 T. CHU (10), Department of Biochemistry and Molecular Biophysics, Columbia Uni- versity, New York, New York 10032 MARIA COSTA (29), Centre de G~ndtique Mo- l~culaire du CNRS, F-91190 Gif-sur- Yvette, France DONALD M. CROTHERS (9), Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107 MICHAEL L. DERAS (22), Department of Bio- physics, Johns Hopkins University, Balti- more, Maryland 21218-2864 JENNIFER A. DOUDNA (12), Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114 FmTZ ECKSTEIN (5), Max-Planck-Institut far Experimentelle Medizin, D-37075 Gi~t- tingen, Germany XINOWANG FANO (24), Department of Bio- chemistry and Molecular Biology, Univer- sity of Chicago, Chicago, Illinois 60637 JOACHIM FRANK (18, 19), Howard Hughes Medical Institute, Health Research, Inc., at the Wadsworth Center, and Department of Biomedical Sciences, State University of New York, Albany, New York 12201-0509 BARBARA L. GOLDEN (8), Department of BiD- chemistry, Purdue University, West Lafa- yette, Indiana 47906-1153 ROBERT A. GRASSUCCI (18), Howard Hughes Medical Institute, Health Research, Inc., at the Wadsworth Center, Albany, New York 12201-0509 PETER HAEBERLI (3), Ribozyme Pharmaceu- ticals, Inc., Boulder, Colorado 80301 PAUL J. HAGERMAN (26), Department of BiD- chemistry and Molecular Genetics, Univer- sity of Colorado Health Sciences Center, Denver, Colorado 80262 ix X CONTRIBUTORS TO VOLUME 317 MARK R. HANSEN (15), Department of Chem- istry and Biochemistry, University of Colo- rado, Boulder, Colorado 80309-0215 PAUL HANSON (15), Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215 AMY B. HEAGLE (18), Howard Hughes Medi- cal Institute, Health Research, Inc., at the Wadsworth Center, Albany, New York 12201-0509 ECKHARD JANKOWSKY (10), Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032 ALEXANDER KARPEISKY (3), Ribozyme Phar- maceuticals, Inc., Boulder, Colorado 80301 ANNE I. KOSEK (6), Departments of Molecu- lar Biophysics and Biochemistry, and Chemistry, Yale University, New Haven, Connecticut 06520-8114 JON LAPHAM (9), Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107 FABRICE LECLERC (27), Department of Chem- istry and Chemical Biology, Harvard Uni- versity, Cambridge, Massachusetts 02138 DAVID M. J. LILLEY (23), Department of BiD- chemistry, University of Dundee, Dundee DD1 4HN, United Kingdom BELSIS LLORENTE (27), Centro de Quimica Farmac~utica, Atabey, Habana, Cuba STEPHEN R. LYNCH (16), Department of Struc- tural Biology, Stanford University School of Medicine, Stanford, California 94305-5126 CHRISTIAN MASSIRE (29), Institut de Biologie Mol~culaire et Cellulaire du CNRS, F-67084 Strasbourg, France JASENKA MATULIC-ADAMIC (3), Ribozyme Pharmaceuticals, Inc., Boulder, Colorado 8O3O1 DAVID B. McKAY (11), Department of Struc- tural Biology, Stanford University School of Medicine, Stanford, California 94305 FRANCOIS MICHEL (29), Centre de Gdn~tique Moldculaire du CNRS, F-91190 Gif-sur- Yvette, France MELISSA J. MOORE (7), Department of Bio- chemistry, W. M. Keck Institute for Cellular Visualization, Brandeis University, Wal- tham, Massachusetts 02454 JAMES B. MURRAY (13), Department of Chemistry and Biochemistry, and Center for the Molecular Biology of RNA, University of California, Santa Cruz, California 95064 KNUD H. NIERHAUS (17, 19), AG Ribosomen, Max-Planck-Institut far Molekulare Gen- etik, D-14195 Berlin, Germany LORI ORTOLEVA-DONNELLY (6), Depart- ments of Molecular Biophysics and Bio- chemistry, and Chemistry, Yale University, New Haven, Connecticut 06520-8114 TAD PAN (20), Department of Biochemistry and Molecular Biology, University of Chi- cago, Chicago, Illinois 60637 ARTHUR PARDI (15), Department of Chemis- try and Biochemistry, University of Colo- rado, Boulder, Colorado 80309-0215 PAWEL PENCZEK (18), Howard Hughes Medi- cal Institute, Health Research, Inc., at the Wadsworth Center, and Department of BiD- medical Sciences, State University of New York, Albany, New York 12201-0509 JOSEPH D. PUGLISI (16), Department of Struc- tural Biology, Stanford University School of Medicine, Stanford, California 94305-5126 ANNA MARIE PYLE (10), Department of BiD- chemistry and Molecular Biophysics, and Howard Hughes Medical Institute, Colum- bia University, New York, New York 10032 CHARLES C. QUERY (7), Department of Cell Biology, Albert Einstein College of Medi- cine, Bronx, New York 10461 CORIE Y. RALSTON (22), Department of Physi- ology and Biophysics, Center for Synchro- tron Biosciences, Albert Einstein College of Medicine, Bronx, New York 10461 MICHAEL I. RECI-rr (16), Department of Struc- tural Biology, Stanford University School of Medicine, Stanford, California 94305-5126 BEATRIX ROHRDANZ (17), AG Ribosomen, Max-Planck-lnstitut far Molekulare Gen- etik, D-14195 Berlin, Germany CONTRIBUTORS TO VOLUME 317 xi SEAN P. RYDER (6), Department of Molecular Biophysics and Biochemistry, Yale Univer- sity, New Haven, Connecticut 06520-8114 STEPHEN A. SCARINGE (1), Dharmacon Re- search, Inc., Boulder, Colorado 80301 BIANCA SCLAVI (22), Department of Physiol- ogy and Biophysics, Center for Synchrotron Biosciences, Albert Einstein College of Medicine, Bronx, New York 10461 LINCOLN G. SCOTT (2), The Scripps Research Institute, La Jolla, California 92037 WILLIAM G. SCOTT (13), Department of Chemistry and Biochemistry, and Center for the Molecular Biology of RNA, University of California, Santa Cruz, California 95064 VALERIE M. SHELTON (24), Department of Chemistry, University of Chicago, Chicago, Illinois 60637 TOBIN R. SOSNICK (24), Department of Bio- chemistry and Molecular Biology, Univer- sity of Chicago, Chicago, Illinois 60637 RuI SOUSA (4), Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760 CHRISTIAN M. T. SPAHN (17, 19), Wadsworth Center, New York State Department of Health, New York, New York 12201-0509 ULRICH STELZL (19), AG Ribosomen, Max- Planck-lnstitut fiir Molekulare Genetik, D- 14195 Berlin, Germany ScoTt A. S'rROBEL (6), Departments of Mo- lecular Biophysics and Biochemistry, and Chemistry, Yale University, New Haven, Connecticut 06520-8114 MICHAEL SULLIVAN (22), Department of Physiology and Biophysics, Center for Syn- chrotron Biosciences, Albert Einstein Col- lege of Medicine, Bronx, New York 10461 DAVID SWEEDLER (3), Ribozyme Pharmaceu- ticals, Inc., Boulder, Colorado 80301 THOMAS J. TOLBERT (2), The Scripps Re- search Institute, La Jolla, California 92037 DANIEL K. TREIBER (21), The Scripps Re- search Institute, La Jolla, California 92037 FRANCISCO J. TRIANA-ALONSO (17), Centro de Investigaciones BiomOdicas, Universidad de Carabobo, LaMorita, Maracay, Vene- zuela GABRIELE VARANI (14), MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England LUCA VARANI (14), MRC Laboratory of Mo- lecular Biology, Cambridge CB2 2QH, En- gland L. CLAUS S. VORTLER (5), Max-Planck-lnsti- tut fiir Experimentelle Medizin, D-37075 GOttingen, Germany NILS G. WALTER (25), Department of Chemis- try, University of Michigan, Ann Arbor, Michigan 48109-1055 JOSEPH E. WEDEKIND (11), Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 ERIC WESTHOF (29), Institut de Biologie Mo- lOculaire et Cellulaire du CNRS, F-67084 Strasbourg, France JAMES R. WILLIAMSON (2, 21), The Scripps Research Institute, La Jolla, California 92037 SARAH A. WOODSON (22), Department of Bio- physics, Johns Hopkins University, Balti- more, Maryland 21218-2864 [ 11 5'-SILYL-2'-ACE RNA OLIGONUCLEOTIDE SYNTHESIS 3 [11 Advanced 5'-Sflyl-2'-Orthoester Approach to RNA O1igonucleotide Synthesis By STEPHEN A. SCARINGE Introduction The need for routine syntheses of RNA oligonucleotides has grown rapidly during the 1990s as research reveals the increasing breadth of RNA's biological functions. 1 RNA synthesis can be accomplished for most applications via either biochemical methods, e.g., T7 transcription, or chemi- cal methods. Transcription and chemical methodologies complement each other well and enable a wide range of RNAs to be synthesized. Current chemical synthetic methods 2 enable the synthesis of RNA in acceptable yields and quality, but none are as routine and dependable as DNA. The need for an improved oligoribonucleotide synthesis technology has contin- ued to persist. This article describes a recently developed technological advance in the chemical synthesis of RNA oligonucleotides utilizing a novel 5'-O-silyl ether protecting group in conjunction with an acid-labile 2'-0- orthoester. 3 Using this technology, numerous RNA oligonucleotides have been routinely synthesized in high yields and of unprecedented quality. The advantageous properties of 5'-silyl-2'-orthoester RNA chemistry make it possible to synthesize RNA oligonucleotides with a quality only previously observed in DNA. The ribonucleoside phosphoramidites couple in >99% stepwise yields in less than 90 sec. Consequently, yields are rou- tinely 1.5-3 times that observed with older RNA synthesis chemistries. At the same time, the overall purity is significantly increased. For some applications, the RNA is of sufficient purity to use without further pro- cessing. After synthesis of an RNA oligonucleotide, the 2'-orthoester pro- t S. Altman, Proc. Natl. Acad. Sci. U.S.A. 90, 10898 (1993); B. A. Sullenger and T. R. Cech, Science 262, 1566 (1993); T. Cech, Curr. Opin. Struct. Biol. 2, 605 (1992); N. Usman and R. Cedergren, Trends Biochem. Sci. 17, 334 (1992). 2 F. Wincott, A. DiRenzo, C. Shaffer, S. Grimm, D. Tracz, C. Workman, D. Sweedler, C. Gonzalez, S. Scaringe, and N. Usman, Nucleic Acids Res. 23, 2677 (1995); N. Usman, K. I(. Ogilvie, M Y. Jiang, and R. J. Cedergren, J. Am. Chem. Soc. 109, 7845 (1987); T. Wu, K. K. Ogilvie, and R. T. Pon, Nucleic Acids Res. 17, 3501 (1989); T. Tanaka, S. Tamatsukuri, and M. Ikehara, Nucleic Acids Res. 14, 6265 (1986); J. A. Hayes, M. J. Brunden, P. T. Gilham, and G. R. Gough, Tetrahedron Lett. 26, 2407 (1985); M. V. Rao, C. B. Reese, V. Schehlman, and P. S. Yu, J. Chem. Soc. Perkin Trans. I, 43 (1993). 3 S. A. Scaringe, F. E. Wincott, and M. H. Caruthers, J. Am. Chem. Soc. 129, 11820 (1998). Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. METHODS IN ENZYMOLOGY, VOL. 317 0076-6879/00 $30.00 4 SEMISYNTHETIC METHODOLOGIES [ 1] /~'MeO~.N~,~O,v,~o ~ Uridine Cytidine Adenosine '1 H Guanosine FIG. 1. Protected ribonucleoside phosphoramidites for 5'-silyl-2'-orthoester RNA synthe- sis chemistry. tected RNA is water soluble and significantly more stable to degradation than the final fully deprotected RNA product. These features of the 2'- orthoester group enable the RNA to be easily handled in aqueous solutions. Furthermore, the 2'-orthoester groups interrupt secondary structure. This property has made it possible to analyze and purify RNA oligonucleotides of every sequence to date regardless of secondary structure. This includes 10- to 15-base-long homopolymers of guanosine. Finally, when the RNA is ready for use, the 2'-orthoester groups are completely removed in less than 30 min under extremely mild conditions in common aqueous buffers. These unique properties of the 5'-silyl ether and 2'-orthoester protecting groups have made it possible to routinely synthesize high-quality RNA oligonucleotides. [1] 5'-SlLYL-2'-ACE RNA OLIGONUCLEOTIDE SYNTHESIS 5 ~H sse 20 1 - Base H% ^ Base , ,, "y'( 5_ FIG. 2. General synthesis scheme from TIPS-protected nucleoside to fully protected nucleo- side phosphoramidite, where R is cyclododecyl and Base is either N-isobutyryladenine, N- acetylcytosine, N-isobutyrylguanine, or uracil [reaction (i); tris(2-acetoxyethoxy)orthoformate, pyridinium toluene sulfonate; reaction (ii); TEMED-HF, acetonitrile; reaction (iii); DOD-C1, imidazole, THF; reaction (iv); bis(N,N-diisopropylamine)methoxyphosphine, tetrazole, DCM]. Synthesis of Nucleoside Phosphoramidites The Y-hydroxyl, 2'-hydroxyl, and amine protecting groups used in 5'- silyl-2'-orthoester chemistry continue to be refined and optimized. At this time the ribonucleoside phosphoramidites use the bis(trimethylsiloxy)- cyclododecyloxysilyl ether (DOD) protecting group on the Y-hydroxyl and the bis(2-acetoxyethoxy)methyl (ACE) orthoester protecting group on the 2'-hydroxyl (Fig. i). The exocyclic amines are protected with the following acyl groups: acetyl for cytidine and isobutyryl for adenosine and guanosine. Synthesis of these compounds proceeds according to the general outline in Fig. 2 [reactions (i)-(iv)]. The N-acyl-5'-O-3'-O-tetraisopropyldisiloxanyl-protected ribonucleo- side starting materials (1) (N-acyl-TIPS nucleosides) can be synthesized according to the literature 4 or obtained commercially (Aldrich, Milwaukee, WI, or Monomer Sciences, Huntsville, AL). The remaining reactions can be effected utilizing the following generalized protocols. 4 G. S. Ti, B. L. Gaffney, and R. A. Jones, J. Am. Chem. Soc. 104, 1316 (1982); W. T. Mar- kiewicz and M. Wiewiorowski, Nucl. Acids Res. Spec. Pub. 4,185, (1978); W. T. Markiewicz, E. Biala, R. W. Adamiak, K. Grzeskowiak, R. Kierzek, A. Kraszewski, J. Stawinski, and M. Wieworowski, NucL Acids Res. Symp. 7, 115 (1980). 6 SEMISYNTHETIC METHODOLOGIES [ 1] Synthesis of 2'-O-ACE Protected Nucleoside (2): Reaction (i) The ACE orthoester is introduced onto the 2'-hydroxyl by reacting the N-acyI-TIPS nucleoside with the trisorthoformate reagent under acid catalysis. The 2'-hydroxyl displaces one of the alcohols on the orthoformate reagent (Fig. 3) to produce the desired product (2). As described later, the reaction proceeds under high vacuum to remove the 2-acetoxyethanol by- product and drive the reaction forward. An improved method for introduc- ing the 2'-O-ACE orthoester is currently being developed and will be reported shortly. Procedure. N-acyl-TIPS-nucleoside (1) (1 equivalent, 10 mmol) is re- acted neat with tris(2-acetoxyethoxy) orthoformate (322.31 g/mol, 5.6 equivalent, 18.04 g) and pyridinium p-toluene sulfonate (251.31 g/mol, 0.2 equivalent, 0.50 g) at 55 ° for 3 hr under high vacuum (<0.015 mm Hg). The reaction is cooled to room temperature, neutralized with N,N,N',N'- tetramethylethylenediamine (TEMED) (150 ml/mol, 0.5 equivalent, 0.75 ml), diluted with 50 ml dichloromethane (DCM) and 150 ml hexanes, and purified on 300 g silica gel (Merck-VWR Scientific) with a hexane/ethyl acetate gradient. Column chromatography removes the neutralized catalyst but does not yield pure product because the excess orthoformate reagent generally eluted with the nucleoside product. However, the excess reagent does not interfere with the following reaction and it is easily removed during purification of the next nucleoside intermediate (3). Therefore, the semipurified product is carried through to the next reaction. Removal of 5'-Y-TIPS Protecting Group: Reaction (ii) The 5'-3'-TIPS group is removed with fluoride ions, e.g., tetrabutyl- ammonium fluoride or amine hydrofluoride salts. These salts can chro- matograph with the product and complicate purification. (Tetrabutylam- monium fluoride and triethylammonium hydrofluoride are known to cause this problem.) Therefore, a very polar amine salt of hydrofluoric acid is used to ensure that during chromatography these salts do not elute with the product. H ase '4" 1 FIG. 3. Reaction of protected nucleoside with tris(2-acetoxyethoxy) orthoformate. Base is either N-isobutyryladenine, N-acetylcytosine, N-isobutyrylguanine, or uracil. [1] 5'-SILYL-2'-ACE RNA OLIGONUCLEOTmE SYNTHESIS 7 Procedure. To TEMED (150 ml/mol, 5 equivalent, 7.50 ml) in acetoni- trile (CH3CN) (100 ml) is slowly added 48% hydrofluoric acid (36 ml/mol, 3.5 equivalent, 1.26 ml) at 0 °. This solution is then added to compound 2. The reaction proceeds at room temperature with mixing. After 6 hr, the CH3CN is removed under vacuum, but not to dryness. The residue is resuspended in 100 ml of DCM and purified on 300 g of silica gel with an ethyl acetate/methanol gradient. The overall yield from 1 [reactions (i) and (ii)] was 40-70%. The 2'-ACE uridine nucleoside is a clear oil and the remaining three nucleosides are white foams. 5'-O-Silylation: Reaction (iii) The steric hindrance of the bis(trimethylsiloxy)cyclododecyloxysilyl chloride (DOD-CI) silylating reagent permits the silyl chloride to react preferentially with the primary 5'-hydroxyl group over the secondary 3'- hydroxyl. The silyl chloride will react with the 3'-hydroxyl but factors such as a slow rate of addition and low temperature enhance the selectivity and increase yields. Procedure. To a solution of 2'-O-ACE-nucleoside (3) (1 equivalent, 10 retool) and imidazole (68.08 g/mol, 4 equivalent, 2.72 g) in tetrahydro- furan (50 ml) at 0 ° is added bis(trimethylsiloxy)cyclododecyloxysilyl chlo- ride (DOD-CI) (424 g/mol, 1.5 equivalent, 6.36 g in 20 ml tetrahydrofuran) over 30 min with stirring. The reaction is worked up by adding 70 ml of ethyl acetate, washing with saturated sodium chloride and drying the organic phase over sodium sulfate. The solvent is removed from the organic phase and the residue resuspended in 50 ml DCM and 150 ml hexanes. The 5'- silyl-2'-ACE nucleoside product (4) is purified on 300 g silica gel with a hexane/ethyl acetate gradient in the presence of 20% acetone. The products are isolated as oils or oily foams in 75-85% yields. Y-O-Phosphitylation: Reaction (iv) The final nucleoside phosphoramidite products are synthesized using the bis(N,N,-diisopropylamine)methoxyphosphine method. 5 Procedure. To a solution of a 5'-O-silyl-2'-O-ACE-nucleoside (1 equivalent, 10 mmol) in 25 ml of dry dichloromethane is added bis(N,N- diisopropylamine)methoxyphosphine (262 g/mol, 1.5 equivalent, 3.93 g) and then tetrazole (70 g/mol, 0.8 equivalent, 0.56 g) with stirring. After 4 hr the reaction is washed with saturated sodium chloride and the organic phase dried over sodium sulfate. The nucleoside phosphoramidite product (5) is purified on 300 g silica gel with a hexane/dichloromethane gradient 5 A. D. Barone, J Y. Tang, and M. H. Caruthers, Nucleic Acids Res. 12, 4051 (1984). [...]... both analyses.) 7) and is ready for analysis Alternatively, the RNA can be dried in vacuo, e.g., Speed-Vac, and 2'-deprotected prior to analysis HPLC Analysis At this point the 2'-ACE protected RNA is water soluble and can be analyzed via anion-exchange HPLC or PAGE HPLC analyses of a 35-mer both 2'-protected and fully deprotected are illustrated in Fig 8 The purity of this synthesis is representative... phosphorylation of glucose catalyzed by hexokinase (HXK) Glucose 6-phosphate (G6P) is then taken in a tandem oxidation catalyzed by glucose-6-phosphate dehy11 S Grzesiek, J Anglister, H Ren, and A Bax, J Am Chem Soc 115, 4369 (1993) 12 M A Markus, K T Dayie, P Matsudaira, and G Wagner, J Magn Reson B 105,192 (1994) 13 X Shan, K H Gardner, D R Muhandiram, N S Rao, C H Arrowsmith, and L E Kay, J Am Chem... phosphoribosyltransferase (APT) assay utilizing myokinase (ADK), pyruvate kinase (PYKF), and L-lactate dehydrogenase (LDH) to couple APT activity with N A D H oxidation N A D + production is measured by the change in absorbance at 340 nm (Ae34o = 6220 cm -1 M-l) (B) The uracil phosphoribosyltransferase (UPP) assay based on the change in extinction coefficient at 271 nm between uracil and U M P (A8 271 = 2763... (1997) 5 G Wagner, J Biomol NMR 3, 375 (1993) 6 T Dieckermann and J Feigon, Curr Opin Struct Biol 4, 745 (1994) 7 G Varani, F Aboul-ela, and F H Allain, Prog NMR Spectrosc 29, 51 (1996) 8 y Oda, H Nakamura, T Yamazaki, K Nagayama, M Yoshida, S Kanaya, and M Ikehara, J Biomol NMR 2, 137 (1992) 9 V L Hsu and I M Armitage, Biochemistry 31, 12778 (1992) 10 T J Tolbert and J R Williamson, J Am Chem Soc... 3-Phosphoglycerate mutase Enolase (Phosphopyruvate hydratase) Pyruvate kinase Glutamate dehydrogenase CTP synthase L-Lactate dehydrogenase PRPP Abbreviation ATP UTP GTP EC - CTP Source Vendor Baker's yeast Baker's yeast Sigma Sigma HXK PGH 2.7.1.1 5.3.1.9 ZWF GDN RPI1 1.1.1.49 L rnesenteroides Sigma Sigma 1.1.1.44 Torula yeast Sigma 5.3.1.6 Spinach PRSA 2.7.6.1 APT UPP GPT 2.4.2.7 JM109/pTTA6 2.4.2.9 JM109/pT'FU2... Biomol NMR 3, 375 (1993) 6 T Dieckermann and J Feigon, Curr Opin Struct Biol 4, 745 (1994) 7 G Varani, F Aboul-ela, and F H Allain, Prog NMR Spectrosc 29, 51 (1996) 8 y Oda, H Nakamura, T Yamazaki, K Nagayama, M Yoshida, S Kanaya, and M Ikehara, J Biomol NMR 2, 137 (1992) 9 V L Hsu and I M Armitage, Biochemistry 31, 12778 (1992) 10 T J Tolbert and J R Williamson, J Am Chem Soc 118, 7929 (1994) METHODS... 6-phosphogluconate (6PG) was regenerated by reductive amination of excess ~-ketoglutarate and ammonia with NADPH catalyzed by glutamic dehydrogenase (GLUD) 19 A number of advantages are associated with the synthesis of NTPs using the glycolysis and pentose phosphate pathways First, glucose can be obtained in a variety of isotopic labeled forms available commercially Second, all but four of the enzymes required to convert... spectrophotometer (Hitachi, San Jose, CA) Procedure 1 Adenine phosphoribosyltransferase (APT) activity is measured by coupling the APT reaction to N A D H oxidation with the enzymes myoki- 26 SEMISYNTHETIC METHODOLOGIES [9.] Adenine ppi A PRPP ~ J ~ AMP Uracil ADK ATP Lactate i f LDH ~,~ Pyruvate = ~ ' ~ ) • ADP l ADP PYKF PRPP ~ ppi J : UMP Glutamine PEP C UTP NAD+ NADH ~ffPY~G: ATP CTP ADP FIG 3 (A) The adenine... (Glucose6-phosphate isomerase) Glucose-6-phosphate dehydrogenase Phosphogluconate dehydrogenase Phosphoriboisomerase (Ribose-5phosphate isomerase) Phosphoribosylpyrophosphate synthetase (Ribose-phosphatic pyrophosphokinase) Adenine phosphoribosyltransferase Uracil phosphoribosyltransferase Xanthine-guanine phosphoribosyltransferase Nucleoside-monophosphate kinase Myokinase (Adenylate kinase) Guanylate kinase 3-Phosphoglycerate... synthase (PYRG) assay based on the change in extinction coefficient at 291 nm between UTP and CTP (Ae291 = 1338 cm -1 M-l) Substrates are denoted in boldface type and enzymes are denoted in italics nase (ADK), pyruvate kinase (PYKF), and L-lactate dehydrogenase (LDH) as shown in Fig 3A 2s The conversion of N A D H to N A D + is monitored at 340 nm 2 The assay solution (1 ml) contains 0.2 m M N A D H . Universidad de Carabobo, LaMorita, Maracay, Vene- zuela GABRIELE VARANI (14), MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England LUCA VARANI (14), MRC Laboratory of Mo- lecular. prior to analysis. HPLC Analysis At this point the 2'-ACE protected RNA is water soluble and can be analyzed via anion-exchange HPLC or PAGE. HPLC analyses of a 35-mer both 2'-protected. progresses at a rapid pace. Its expansion into new areas of investigation has been phenomenal with novel discoveries being made in a variety of subspecialty areas. The subfield of RNA-ligand interactions