Antisense Technology A Practical Approach

by Lichtenstein, Conrad; Nellen, Wolfgang

• ISBN13: 9780199635849
• ISBN10: 0199635846
• Format: Hardcover
• Copyright: 1998-03-05
• Publisher: Oxford University Press

Summary

This valuable text provides proven step-by-step protocols for antisense techniques in a range of different organisms and cell culture systems. In addition, it discusses the potential benefits and problems for various antisense methods which complement gene knock-out experiments. The book includes antisense techniques such as: analysis of nucleic acid structures; measurement and evaluation of antisense effects; selection, preparation, and the use of antisense oligonucleotides; in vitro RNA transcription; construction strategies, testing and optimization of catalytic antisense RNAs based on hammerhead ribozymes; synthesis and evaluation of 2-5 A- antisense chimeras for targeted degradation of RNA. The numerous hints and tips for success and advice on trouble shooting, make this authoritative text invaluable to all those researchers who work on gene expression, RNA transcription, or protein function.

Table of Contents

List of Contributors	xvii	(4)
Abbreviations	xxi
1. Nucleic acid structures	1	(24)
Detlev Riesner
1. Introduction	1	(1)
2. Structure of RNA:RNA and RNA:DNA double-strands and intramolecular structure of RNA	1	(4)
Intermolecular double-strand formation	1	(1)
Intramolecular RNA structure	2	(3)
3. Prediction of intramolecular structures and double-strand formation	5	(6)
Estimation of the equilibrium between intermolecular and intramolecular structures	6	(2)
Programs to predict intramolecular secondary structures	8	(3)
4. Experimental determination of secondary structure	11	(12)
RNA structure mapping by DMS modification	11	(5)
Analysis of structural transitions and complex formation of RNA by temperature-gradient gel electrophoresis (TGGE)	16	(7)
Acknowledgements	23	(1)
References	23	(2)
2. Evaluation of antisense effects	25	(14)
Wolfgang Nellen
Conrad Lichtenstein
1. Introduction	25	(1)
2. Protein expression studies	26	(1)
3. Analysis of steady state RNA levels	27	(1)
4. Analysis of RNA transcription rates	28	(3)
Nuclear run-ons	28	(3)
Targets for strand-specific run-on assays	31	(1)
5. Polymerase assays for antisense activity	31	(2)
6. RNA localization studies	33	(1)
7. Antisense-independent gene silencing	34	(3)
Design of primers	35	(2)
Acknowledgements	37	(1)
References	38	(1)
3. Selecting, preparing, and handling antisense oligodeoxyribonucleotides	39	(36)
Jean-Jacques Toulme
Christian Cazenave
Serge Moreau
1. Introduction	39	(2)
2. Chemical synthesis of oligodeoxynucleotides	41	(4)
Main features of solid phase phosphoramidite chemistry	41	(1)
The synthesis cycle	42	(1)
Deprotection and purification	43	(2)
3. Synthesis of modified oligonucleotides	45	(6)
Phosphorothioates	45	(2)
Methylphosphonates	47	(1)
2'-O-methyloligoribonucleotides	48	(1)
Terminal modification of oligonucleotides	49	(2)
4. Properties of oligonucleotides	51	(13)
Affinity	51	(8)
Specificity	59	(2)
Nuclease sensitivity	61	(2)
Triplex formation	63	(1)
5. In vitro use of antisense oligonucleotides	64	(8)
In cell-free extracts	64	(3)
In injected Xenopus oocytes	67	(1)
In cultured cells	68	(4)
Acknowledgements	72	(1)
References	72	(3)
4. In vitro RNAs	75	(18)
Ute Weber
Hans J. Gross
1. Introduction	75	(1)
2. Rapid purification of T7 RNA polymerase	75	(4)
3. Template DNA	79	(3)
Essential features	79	(1)
Synthetic templates	80	(2)
Cloned templates	81	(1)
Preparation of the plasmid DNA template for run-off transcription	81	(1)
4. In vitro transcription with T7 RNA polymerase	82	(5)
Optimizing the transcription assay	82	(1)
Small scale in vitro transcription	83	(1)
Large scale in vitro transcription	84	(1)
Purification of the transcripts	85	(2)
5. Modified transcripts	87	(3)
The 5' terminus of the RNA	88	(1)
5' cap structure	89	(1)
Radiolabelled RNA	89	(1)
Non-radioactive labels for RNA	89	(1)
Modified RNA for cross-linking experiments	90	(1)
Modifications stabilizing the RNA	90	(1)
Acknowledgements	90	(1)
References	91	(2)
5. Catalytic antisense RNA based on hammerhead ribozymes	93	(34)
Martin Tabler
Georg Sczakiel
1. Introduction	93	(3)
Origin of hammerhead ribozymes	93	(2)
Exploitation of the hammerhead structure for generation of ribozymes that act as specific endoribonucleases	95	(1)
2. The ribozyme reaction	96	(6)
The catalytic cycle	96	(4)
Stoichiometric cleavage	100	(2)
Conclusion of theoretical considerations	102	(1)
3. Design of the ribozyme and target site selection	102	(4)
Cleavable motifs	102	(1)
Prediction of accessible target regions by computer analysis	102	(1)
The length of the antisense arms	103	(3)
4. Construction strategies for catalytic antisense RNAs	106	(7)
Incorporation of DNA cassettes	106	(4)
Asymmetric hammerhead ribozymes	110	(1)
PCR-based generation strategies of asymmetric hammerhead ribozymes utilizing pre-made helix I box vectors	110	(3)
Direct PCR amplification	113	(1)
5. Testing and optimizing the catalytic antisense RNA in vitro	113	(11)
Quick cleavage assay for testing recombinant DNA encoding catalytic antisense RNA	113	(3)
Determination of association rates	116	(2)
Optimization of the length of helix III	118	(4)
Determination of cleavage rates	122	(2)
6. Concluding remarks	124	(1)
References	125	(2)
6. 2-5A-antisense chimeras for targeted degradation of RNA	127	(30)
R.H. Silverman
A. Maran
R.K. Maitra
C.F. Waller
K. Lesiak
S. Khamnei
G. Li
W. Xiao
P.F. Torrence
1. Introduction	127	(1)
2. Background	127	(2)
The 2-5A system: an RNA degradation pathway that functions in interferon action	127	(1)
The 2-5A-antisense strategy	128	(1)
3. 2-5A-antisense chimeras: construction, composition, and synthetic approach	129	(14)
Reagents and chemicals for composite oligonucleotide synthesis	141	(1)
Assembly procedure: synthesis of oligonucleotides	142	(1)
HPLC methods	143	(1)
4. Biological and biochemical evaluation of 2-5A-antisense chimeras	143	(11)
Targeted cleavage of mRNA species in cell-free systems	144	(6)
Degradation of target RNA molecules in intact cells induced by 2-5A-antisense	150	(4)
References	154	(3)
7. In vitro applications of antisense RNA and DNA	157	(16)
Stephen H. Munroe
1. Introduction	157	(1)
2. Synthesis of RNA in vitro	157	(1)
3. Working with RNA under RNase-free conditions	158	(1)
4. Protein catalysed RNA:RNA annealing	159	(6)
5. Antisense inhibition of pre-mRNA splicing	165	(5)
Introduction	165	(1)
mRNA splicing in vitro	165	(3)
Role of snRNPs in pre-mRNA splicing	168	(2)
6. Antisense inhibition of mRNA translation	170	(1)
Acknowledgements	171	(1)
References	171	(2)
8. Antisense applications in Dictyostelium: a lower eukaryotic model system	173	(18)
Martin Hildebrandt
1. General considerations	173	(2)
History of antisense-mediated gene inactivation in Dictyostelium	173	(1)
Advantages of antisense-mediated gene inactivation	174	(1)
Mechanism of antisense-mediated gene inactivation	175	(1)
2. Construction of vectors expressing antisense RNAs	175	(9)
Examples of antisense-mediated gene inactivation	175	(1)
Choice of the appropriate vector	176	(1)
Choice of the appropriate promoter	177	(5)
Choice of sequence for antisense constructs	182	(2)
3. Methods for antisense transformation	184	(4)
Acknowledgements	188	(1)
References	189	(2)
9. Applications of antisense technology in plants	191	(30)
Anja G.J. Kuipers
Evert Jacobsen
Richard G.F. Visser
1. Introduction	191	(1)
2. Characteristic of antisense RNA in plants	191	(2)
3. Alternative for creating mutants	193	(1)
4. Inhibition of gene expression in plants	194	(1)
5. Factors affecting the effectiveness of antisense inhibition	195	(11)
Composition of the antisense gene	195	(7)
Heterologous sequences	202	(1)
Promoter choice	202	(2)
T-DNA copy number	204	(2)
External factors affecting the antisense effect	206	(1)
6. Specificity of antisense inhibition	206	(1)
7. Stability of antisense inhibition	207	(1)
8. Optimization of antisense inhibition	207	(2)
9. Application of antisense technology in plants	209	(6)
Applications in crop improvement	209	(2)
Virus-resistance by antisense RNA	211	(2)
Characterization of gene expression and dissection of metabolic pathways	213	(1)
Assignment of gene function	213	(2)
10. Antisense inhibition and co-suppression	215	(1)
References	216	(5)
10. Antisense molecules and ribozymes: medical applications	221	(20)
Michael Strauss
1. Introduction	221	(1)
2. Studies in cell culture	221	(12)
Applications of antisense oligodeoxyribonucleotides	223	(3)
Using synthetic ribozymes	226	(1)
Using expression constructs	226	(4)
Selection of target sites for ribozyme cleavage and isolation of ribozymes	230	(3)
3. In vivo studies in animals	233	(2)
4. Generation of transgenic mice with antisense/ribozyme constructs	235	(1)
5. Clinical application	236	(1)
6. Conclusions	236	(1)
References	237	(4)
11. Non-antisense effects of oligodeoxynucleotides	241	(24)
C.A. Stein
Arthur Krieg
1. Introduction	241	(2)
2. Non-antisense effects of oligodeoxynucleotides	243	(14)
Anti-HIV effects of PS oligos	243	(4)
Protein kinase C	247	(1)
Growth factors	247	(1)
Extracellular matrix elements	248	(1)
Effects of PS oligodeoxynucleotides on transcription factor activities	249	(1)
PS and chimeric oligodeoxynucleotides can specifically or non-specifically inhibit protein tyrosine kinases and polynucleotide kinase	249	(1)
PO and PS oligodeoxynucleotides containing G-rich regions block interferon-gamma binding and/or secretion	250	(1)
Identification of a mitogenic CpG motif in PO and PS oligodeoxynucleotides	250	(7)
3. Immune stimulation by nucleic acids	257	(1)
Polynucleotides	257	(1)
Nucleotides	257	(1)
4. Problems in the interpretation of data derived from in vitro and in vivo use of oligodeoxynucleotides	258	(3)
Acknowledgements	261	(1)
References	261	(4)
12. Antisense rescue	265	(8)
Jeffrey T. Holt
1. Introduction	265	(1)
2. The antisense rescue approach	266	(6)
Acknowledgements	272	(1)
References	272	(1)
13. Shotgun antisense mutagenesis	273	(8)
Timothy P. Spann
Debra A. Brock
Richard H. Gomer
1. Introduction	273	(2)
Overview of shotgun antisense	273	(1)
The need for large quantities of cDNA	273	(2)
2. Isolation of mRNA	275	(1)
3. Synthesis of cDNA	275	(1)
4. Vector preparation for directional cloning	276	(1)
5. Ligation of cDNA into pBluescript	276	(1)
6. PCR amplification of the cDNA	277	(1)
7. Gel purification of PCR product	278	(1)
8. Restriction enzyme digestion and second gel purification of PCR product	278	(1)
9. Ligation of amplified cDNA with antisense vector	278	(1)
Choice of antisense vector	278	(1)
Ligation	278	(1)
10. Use of shotgun antisense library	278	(1)
Acknowledgements	279	(1)
References	279	(2)
14. Detection of sense:antisense duplexes by structure-specific anti-RNA antibodies	281	(16)
Noemi Lukacs
1. Introduction	281	(1)
2. dsRNA-specific antibodies and their suggested applications in antisense research	281	(2)
3. Analysis of duplex-forming capacity of antisense sequences in vitro	283	(3)
Sandwich ELISA	283	(1)
dsRNA immunoblotting	284	(2)
4. Detection of dsRNA by immunofluorescence in situ	286	(4)
5. Enrichment of dsRNA by immunoaffinity chromatography	290	(3)
6. Potential applications of dsRNA-specific antibodies for in vivo studies	293	(1)
Acknowledgements	294	(1)
References	294	(3)
A1. List of Suppliers	297	(4)
Index	301