Table Of Content

Methods in Molecular Biology 1111 Atsushi Ogawa Editor Artifi cial Riboswitches Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Artificial Riboswitches Methods and Protocols Edited by Atsushi Ogawa Ehime University, Matsuyama, Ehime, Japan Editor Atsushi Ogawa Ehime University Matsuyama Ehime , Japan ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-754-9 ISBN 978-1-62703-755-6 (eBook) DOI 10.1007/978-1-62703-755-6 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2013958315 © Springer Science+Business Media New York 2 014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Prefa ce Ribonucleic acids or RNAs are versatile. mRNAs serve as mediators of hereditary information from DNAs to proteins, tRNAs carry amino acids into the ribosome on mRNA, and rRNAs in the ribosome promote peptide bond formation between the amino acids. In addition, many diverse noncoding RNAs directly regulate gene expression in cis and/or trans. There are also many other types of RNAs known in nature, including RNA enzymes (ribozymes). It is considered that in an ancient RNA world (where proteins and DNAs did not still exist), a greater variety of functional RNAs would have fl ourished, though almost all of them are now defunct. RNA functions are dependent on their sequences, which are composed of four kinds of nucleotides: A, G, U, and C. The sequences determine the higher order structures, which in turn make possible their unique functions. In other words, we could create new functional RNAs by appropriately lining up the nucleotides. Although RNAs are not as diverse as proteins, which have 20 amino acids as building block, RNAs have a large advantage in that they are easy to be amplifi ed with the following three steps: reverse transcription, poly- merase chain reaction (PCR), and transcription. We can therefore obtain functional RNAs from a randomized RNA pool by repeating selection–amplifi cation cycles (in vitro selection). In fact, many types of functional RNAs have been selected via in vitro selection. The representative examples are aptamers that tightly bind to their specifi c ligand molecules and ribozymes that catalyze specifi c biochemical reactions. In vitro selection also enables us to obtain a ligand-responsive ribozyme called aptazyme, which is generally a conjugate between two foregoing functional RNAs, an aptamer and a ribozyme. A riboswitch is also a functional RNA: a ligand-dependent and cis-acting gene regulator in mRNA. This regulatory RNA is composed of an aptamer domain, which binds to the specifi c ligand as described above, and an expression platform, which regulates transcription termination, translation initiation, self-cleavage, or splicing of its own mRNA. The interac- tion between the aptamer domain and the ligand induces conformational changes of the expression platform to up- or down-regulate gene expression. Natural riboswitches acting in response to endogenous metabolites have been identifi ed mainly in bacteria and rarely in eukaryotes over the last decade. In parallel with these discoveries, a variety of methods have also been reported for artifi cially constructing arbitrary molecule-dependent riboswitches with the corresponding in vitro-selected aptamers or self-cleaving aptazymes. This volume is mainly focused on the state-of-the-art methods developed in recent years for creating artifi cial riboswitches. Approximately half of the total chapters are devoted to screening or rational design methods for obtaining artifi cial riboswitches that function in either bacterial or eukaryotic translation systems. In these methods, an aptamer or an aptazyme is generally required for a gene regulator to acquire ligand responsiveness. Although an already identifi ed aptamer or aptazyme is available, several chapters cover in vitro selection methods for obtaining a new aptamer or aptazyme for a ligand molecule that the reader might be interested in (a small molecule, protein, or photo-r esponsive molecule). In this context, one chapter is devoted to a computational method for designing a starting library of RNA sequences for in vitro selection. Protocols for evaluating the activities of the v vi Preface resultant riboswitches are also presented. Some other chapters include protocols for construction of ligand-dependent, trans-acting gene regulators. Artifi cial riboswitches and other ligand-responsive gene regulators that are obtainable through the cutting-edge methods described here make it possible to switch protein syn- thesis ON or OFF with arbitrary ligand molecules, which can be freely chosen when select- ing the corresponding aptamers to be implemented in the gene regulators. Therefore, this book can be regarded as a collection of recipes for the gene circuit elements in synthetic biology and metabolic engineering. However, I would recommend this cookbook not only to bioengineers who aim to reprogram cell behaviors and molecular biologists who leverage these regulators for genetic studies but also to all researchers who just want to regulate the expression of a specifi c gene by an arbitrary molecule in various organisms, to detect a specifi c molecule with reporter protein expression in vitro or in vivo, or to design ligand- dependent RNA switches by using aptamers or aptazymes. All chapters are written by experts from all around the world who are active in the front lines of the relevant research areas. I would like to express my gratitude to the contributors, all of whom were willing to write their lab protocols in an easily comprehensible manner, despite their busy research lives. I believe that the readers will be able to easily understand and follow the experimental procedures, thanks to the intelligible explanations and notes, and to construct their own artifi cial riboswitches. Last but not least, I am also grateful to Prof. John Walker for giving me the opportunity to edit this book. Matsuyama, Ehime, J apan Atsushi Ogawa Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Computational Design of RNA Libraries for In Vitro Selection of Aptamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Yaroslav G. Chushak, Jennifer A. Martin, Jorge L. Chávez, Nancy Kelley-L oughnane, and Morley O. Stone 2 In Vitro Selection of RNA Aptamers for a Small-Molecule Dye. . . . . . . . . . . . 17 Asako Murata and Shin-ichi Sato 3 Development of Photoswitchable RNA Aptamer–Ligand Complexes. . . . . . . . 29 Gosuke Hayashi and Kazuhiko Nakatani 4 Identification of RNA Aptamers Against Recombinant Proteins with a Hexa-Histidine Tag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Shoji Ohuchi 5 Artificial Riboswitch Selection: A FACS-Based Approach. . . . . . . . . . . . . . . . . 57 Zohaib Ghazi, Casey C. Fowler, and Yingfu Li 6 FRET-Based Optical Assay for Selection of Artificial Riboswitches. . . . . . . . . . 77 Svetlana V. Harbaugh, Molly E. Chapleau, Yaroslav G. Chushak, Morley O. Stone, and Nancy Kelley-Loughnane 7 Portable Two-Way Riboswitches: Design and Engineering . . . . . . . . . . . . . . . 93 Ye Jin and Jian-Dong Huang 8 Generation of Orthogonally Selective Bacterial Riboswitches by Targeted Mutagenesis and In Vivo Screening . . . . . . . . . . . . . . . . . . . . . . . 107 Helen A. Vincent, Christopher J. Robinson, Ming-Cheng Wu, Neil Dixon, and Jason Micklefield 9 Dual Genetic Selection of Synthetic Riboswitches in Escherichia coli . . . . . . . . 131 Yoko Nomura and Yohei Yokobayashi 10 Nucleotide Kinase-Based Selection System for Genetic Switches . . . . . . . . . . . 141 Kohei Ike and Daisuke Umeno 11 Measuring Riboswitch Activity In Vitro and in Artificial Cells with Purified Transcription–Translation Machinery . . . . . . . . . . . . . . . . . . . . . 153 Laura Martini and Sheref S. Mansy 12 Rational Design of Artificial ON-Riboswitches . . . . . . . . . . . . . . . . . . . . . . . . 165 Atsushi Ogawa 13 Engineering Protein-Responsive mRNA Switch in Mammalian Cells. . . . . . . . 183 Kei Endo and Hirohide Saito vii viii Contents 14 Guanine-Tethered Antisense Oligonucleotides as Synthetic Riboregulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Masaki Hagihara 15 In Vitro Selection of Allosteric Ribozymes that Sense the Bacterial Second Messenger c-di-GMP . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Kazuhiro Furukawa, Hongzhou Gu, and Ronald R. Breaker 16 Dual-Selection for Evolution of In Vivo Functional Aptazymes as Riboswitch Parts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Jonathan A. Goler, James M. Carothers, and Jay D. Keasling 17 In Vivo Screening for Aptazyme-Based Bacterial Riboswitches. . . . . . . . . . . . . 237 Charlotte Rehm and Jörg S. Hartig 18 Engineered Riboswitch as a Gene-Regulatory Platform for Reducing Antibiotic Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Libing Liu and Shu Wang 19 Construction of Ligand-Responsive MicroRNAs that Operate Through Inhibition of Drosha Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Chase L. Beisel, Ryan J. Bloom, and Christina D. Smolke 20 A Three-Dimensional Design Strategy for a Protein- Responsive shRNA Switch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Shunnichi Kashida and Hirohide Saito Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Contributors CHASE L. BEISEL • Department of Chemical and Biomolecular Engineering, North Carolina State University , R aleigh , N C , U SA RYAN J. BLOOM • Department of Bioengineering, S tanford University , P alo Alto , C A , U SA RONALD R. BREAKER • Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, Yale University , N ew Haven, C T, U SA ; D epartment of Molecular Biophysics and Biochemistry , Y ale University , N ew Haven, C T, U SA JAMES M. CAROTHERS • University of California , B erkeley , C A , U SA ; U .S. Department of Energy Joint BioEnergy Institute , E meryville, C A , U SA MOLLY E. CHAPLEAU • UES, Inc. , D ayton, O H , U SA JORGE L. CHÁVEZ • UES, Inc. , D ayton, O H , U SA YAROSLAV G. CHUSHAK • The Henry M. Jackson Foundation , B ethesda, M D , U SA NEIL DIXON • Manchester Institute of Biotechnology and School of Chemistry , T he University of Manchester , M anchester, U K KEI ENDO • Department of Reprogramming Science, Center for iPS Cell Research and Application, K yoto University , K yoto, J apan CASEY C. FOWLER • Department of Biochemistry and Biomedical Sciences, M cMaster University , H amilton, O N , C anada ; D epartment of Chemistry and Chemical Biology, McMaster University , H amilton, O N , C anada ; M ichael G. DeGroote Institute for Infectious Disease Research , M cMaster University , H amilton, O N , C anada KAZUHIRO FURUKAWA • Department of Molecular, Cellular and Developmental Biology, Yale University , N ew Haven, C T, U SA ZOHAIB GHAZI • Department of Biochemistry and Biomedical Sciences, M cMaster University , H amilton, O N , C anada ; D epartment of Chemistry and Chemical Biology, McMaster University , H amilton, O N , C anada ; M ichael G. DeGroote Institute for Infectious Disease Research , M cMaster University , H amilton, O N , C anada JONATHAN A. GOLER • University of California , B erkeley , C A , U SA ; U .S. Department of Energy Joint BioEnergy Institute , E meryville, C A , U SA HONGZHOU GU • Department of Molecular, Cellular and Developmental Biology, H oward Hughes Medical Institute, Yale University , N ew Haven, C T, U SA MASAKI HAGIHARA • Graduate School of Science and Technology , H irosaki University , Hirosaki , J apan SVETLANA V. HARBAUGH • The Henry M. Jackson Foundation , B ethesda, M D , U SA JÖRG S. HARTIG • Department of Chemistry , U niversity of Konstanz , K onstanz, G ermany GOSUKE HAYASHI • Department of Chemistry and Biotechnology, Research Center for Advanced Science and Technology , T he University of Tokyo , T okyo, J apan JIAN-DONG HUANG • Department of Biochemistry and Shenzhen Institute of Research and Innovation, T he University of Hong Kong , P okfulam, H ong Kong ; T he Center for Synthetic Biology Engineering Research , S henzhen Institutes of Advanced Technology , Shenzhen, P eople’s Republic of China KOHEI IKE • Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, C hiba University , C hiba, J apan ix