siRNA Design: Methods and Protocols

siRNA Design......Page 3
Preface......Page 5
Contents......Page 9
Contributors......Page 11
1.1. siRNA- and shRNA-Mediated Gene Silencing......Page 14
1.2. History of Development of Algorithms for Predicting siRNA Silencing Ef fi ciency......Page 16
1.3. List of siRNA Sequence Features Associated with Speci fi c Silencing Ef fi ciency......Page 19
1.4. Optimal Asymmetry in Terminal Duplex Stability......Page 20
1.7. Speci fi city Problems......Page 22
3. Methods......Page 24
4. Notes......Page 25
References......Page 27
1. Introduction......Page 30
2.1. The Reported Guidelines for Designing siRNA Sequences......Page 32
2.2. Problems with the Previous Guidelines......Page 35
3.1.1. Model for Decision Tree Learning......Page 36
3.2. Prediction Analysis of siRNA Target Sequences Based on Bayes’ Theorem......Page 37
3.2.1. Prediction of Gene Degradation Ratio for a Given siRNA Candidate......Page 38
3.2.2. Evaluation Criteria Used in the Proposed Method......Page 39
3.2.3. Veri fi cation Models of the Proposed Method......Page 40
3.3. Procedure for Selecting Effective siRNAs Based on the Average Silencing Probability......Page 45
3.4. siRNA Sequence Selection Based on a Hidden Markov Model......Page 46
3.4.1. The Viterbi Algorithm for Selection of the Optimal siRNA Nucleotide......Page 47
3.4.3. Evaluation Criteria Used in the Proposed Method......Page 48
3.5.3. Testing Data......Page 49
4.2. Evaluation of the Bayes’ Theorem Method......Page 50
4.2.1. Case 1: Combination of Both Independent Nucleotide Occurrences......Page 52
4.2.2. Case 2: Combination of Independent and Deductive Nucleotide Occurrences......Page 55
4.2.4. Case 4: Combination of Markov Model and Deductive Nucleotide Occurrences......Page 57
4.2.5. Comparative Analysis by the Normalization......Page 59
Case 1: Independent Nucleotide Occurrences at Individual Positions......Page 60
Case 2: Dependent Nucleotide Occurrences Based on the Simple Markov Model......Page 61
4.4. Evaluation of the HMM Method......Page 62
4.4.1. Evaluation for MG1 to MG5 Based on a Large Number of Ineffective siRNAs......Page 63
4.4.2. Characteristics for the Combinations of Two Successive Nucleotides......Page 65
References......Page 67
1. Introduction......Page 69
2.1. Selecting Functional siRNAs......Page 70
2.2. Reducing Seed-Dependent Off-Target Effects......Page 73
3.1. Designing siRNAs for Human/Mouse/Rat Genes......Page 75
3.2. Designing shRNA Expression Constructs with pol III Promoters......Page 77
4. Notes......Page 78
References......Page 79
1. Introduction......Page 81
2.3. In Vitro Transcription and End-Labeling of Target RNA......Page 83
2.6. Tissue Culture and Transfection of siRNA Duplexes......Page 84
3.1. Designing siRNAs and Control Oligonucleotides......Page 85
3.2. In Vitro Transcription and End-Labeling of Target RNA......Page 86
3.3. Magnetic Microbead Preparation, Conjugation of RNA, and Oligo Screening......Page 88
3.5. Transfection of Adherent Cells with siRNA Duplexes......Page 89
3.6. Con fi rmation of Knockdown of HCV RNA by Quantitative RT-PCR and Northern Blot Analyses......Page 91
4. Notes......Page 94
References......Page 97
1.1. Nucleic Acids in Gene Silencing Technology......Page 99
1.2. siRNAs Need Chemical Engineering......Page 100
2.1. Choosing an siRNA Template......Page 101
2.3.1. Backbone Modi fi cation......Page 102
2.3.2. 2  ¢ -OH Modi fi cation......Page 103
2.3.4. Base Modi fi cations......Page 104
3.1 Maximizing siRNA Activity......Page 105
3.2. Enhancing siRNA Stability......Page 106
3.4. Abrogating siRNA Immunogenicity......Page 108
3.5. Reducing siRNA Off-Targets Effects......Page 110
4. Recap Guide: Building Better siRNAs......Page 111
4.3. Modifying the siRNA Duplex Ends......Page 113
References......Page 114
1. Introduction......Page 122
2.1. General......Page 124
2.2. Synthesis of Reporter Plasmids Containing siRNA and miRNA Targets......Page 125
2.4. Quantitative Real-Time PCR Assay......Page 126
2.6. Global Gene Expression Pro fi ling......Page 127
3.1. Bioinformatic Design of Highly Functional siRNAs......Page 128
3.2. In Vitro Activity Screen of siRNAs......Page 130
3.3. The Addition of a 3 ¢ -Overhang Modi fi cation by UNA to Increase the siRNA Speci fi city......Page 131
3.4. 5  ¢ -End Chemical Modi fi cation to Increase the Strand Selection Bias During RISC Assembly......Page 132
3.6. Design of Luciferase Reporter Constructs......Page 133
3.6.1. Dual Luciferase Assay......Page 134
3.7.1. Isolation and Transfection of Human Peripheral Blood Mononuclear Cells......Page 135
3.7.2. Detection of Human Interferon- a and Human Interferon- b Using ELISA......Page 136
3.8. Microarray Analysis to Determine Global Off-Target Events......Page 138
3.8.1. Microarray Protocol and Data Analysis......Page 139
4. Notes......Page 140
References......Page 142
1. Introduction......Page 146
2.2. Veri fi cation of siRNA Annealing......Page 148
2.6. Western Blotting for Ago2......Page 149
2.7. 5  ¢ RACE Assay......Page 150
2.10. DNA Microarray......Page 151
3. Methods......Page 152
3.1.2. asiRNA Transfection......Page 154
3.1.4. Reverse Transcription......Page 155
3.2. Assessment of the Requirement of Ago2 for asiRNA-Mediated Gene Silencing......Page 156
3.3. Mapping of the Target mRNA Cleavage Site by 5 ¢ RACE Assay......Page 157
3.4. Assessment of the Antisense-Mediated Silencing and the Sense-Strand-Mediated Off-Target Silencing Activity of asiRNA Using a Reporter Assay System......Page 159
3.5.1. Comparison of the Potency of asiRNA and siRNA to Compete Against Exogenously Introduced siCREB3......Page 160
3.6.1. Analysis of Genome-Wide Transcription Pro fi les......Page 161
3.6.2. Analysis of Sense-Strand Seed Sequence Mediated Off-Target Effects by DNA Microarray......Page 162
References......Page 163
1. Introduction......Page 164
2. Materials......Page 165
2.3. Polyacrylamide Gels ( See Note 8)......Page 166
2.7. General Reagents and Solutions......Page 167
3.2. siRNA Annealing ( See Notes 9 and 10)......Page 168
3.3. Analysis of siRNA Stability in the Presence of 10% FBS ( See Note 11)......Page 169
3.6. Mapping of Nuclease Sensitive Sites ( See Note 14)......Page 170
3.7. Preparation of an Imidazole Ladder......Page 171
3.9. Gene Silencing Assay Using a GFP-Tagged Target ( See Notes 15 – 21)......Page 172
4. Notes......Page 173
References......Page 178
1. Introduction......Page 180
3. Methods......Page 181
3.1. Finding Partially Complementary Dual-Targeting siRNA Candidates......Page 182
3.3. Predicting Both Duplex Strands’ siRNA Ef fi cacy......Page 183
3.5. Scoring Duplex Candidates......Page 184
4. Notes......Page 185
References......Page 187
1. Introduction......Page 189
1.1.2. Rational Design of siRNAs Recruiting TLR7/8......Page 190
1.2. siRNAs Recruiting TLR9......Page 192
2.1. Cell Culture......Page 193
2.2. TNF- a and IFN- a Enzyme-Linked ImmunoSorbent A ssay......Page 194
3.3.2. For the THP-1 Assay......Page 195
3.4. TLR Stimulation of the Cells......Page 196
3.5. Cytokine Production Analysis by ELISA......Page 197
4. Notes......Page 198
References......Page 199
1. Introduction......Page 202
3.1. Choosing a Region of Sequence Against Which an esiRNA Is To Be Designed......Page 206
3.2. In Silico Procedure for Predicting the Ef fi ciency of Constituent siRNAs......Page 207
3.3.1. Steps for Finding Unintended mRNAs Containing a Near-Perfect Match to an siRNA Sequence......Page 208
3.4. Combining Ef fi ciency and Off-Target Predictions for Evaluating and Selecting an Optimal Region for Endoribonuclease-Based Digestion......Page 209
4. Notes......Page 210
References......Page 211
1. Introduction......Page 214
2. Cellular Processing of shRNAs......Page 216
3. shRNA Structures and Factors Effecting Activity......Page 218
4. Promoters for shRNA Transcription......Page 221
4.1. RNA Polymerase III Promoters......Page 222
4.2. RNA Polymerase II Promoters......Page 224
4.3. Inducible shRNA Expression......Page 225
5. Vectors Used for shRNA Delivery......Page 227
6. Potential Toxicity of shRNAs In Vivo......Page 229
7. Combinatorial-RNAi......Page 230
8. Summary......Page 233
References......Page 234
1. Introduction......Page 242
2.1. shRNA Vector Construction......Page 246
2.2. Arti fi cial miRNA Vector Construction......Page 248
2.5. Lentiviral Vector Production......Page 249
3. Methods......Page 250
3.1. shRNA Vector and Multi-shRNA Vector Construction......Page 251
3.2. Arti fi cial miRNA Vector or amiRNA Polycistron Construction......Page 254
3.5. Lentiviral Vector Production ( See Fig.  4)......Page 256
3.6. Titration of Lentiviral Vectors......Page 258
4. Notes......Page 259
References......Page 262
1. Introduction......Page 267
2.3. Assess Comparative Knockdown by Co-transfection......Page 275
2.4. Stem–Loop RT-PCR for Mature shRNA Detection......Page 276
2.6. Preparation of DNA-DOTAP:Chol Lipoplexes......Page 277
3.2. Construction of Bi-shRNA Expression Vector......Page 278
3.3. Compare Knockdown Ef fi ciency by Co-transfection......Page 279
3.4. Demonstrate Bi-shRNA Expression by Stem–Loop RT-PCR ( See Fig.  3)......Page 280
3.5. Demonstrate Target Gene Cleavage by 5 ¢ RLM-RACE ( See Fig.  4)......Page 281
3.6. Preparation of DNA-DOTAP:Chol Lipoplexes......Page 283
References......Page 285
1. Introduction......Page 287
3.1. Design of sshRNAs......Page 289
3.2. Chemical Modi fi cation of sshRNAs......Page 291
3.3. Avoiding Multimeric Forms of shRNAs......Page 293
4. Notes......Page 294
References......Page 297
1. Introduction......Page 299
2. Long dsRNA Application in Mammals......Page 302
3. Preparation of Long dsRNA for RNAi Experiments......Page 303
3.1. dsRNA Produced by In Vitro Transcription......Page 304
3.1.1. dsRNA Produced from a Vector or a Transgene......Page 305
4. Effects of Long dsRNAs in Somatic Cells and Their Analysis......Page 307
4.1. dsRNA Transfection......Page 309
4.2. dsRNA Expression......Page 313
5. Effects of Long dsRNA in Mammalian Oocytes and Early Embryos......Page 316
5.1. Transgenic RNA......Page 317
References......Page 319
1. Introduction......Page 323
3.1.1. Selection of Suitable RNAi Target Sequences......Page 325
3.1.2. Assessing the Predicted RNAi Speci fi city......Page 326
3.1.3. Assessing the Predicted RNAi Ef fi ciency......Page 329
3.1.5. Ranking of RNAi Reagents......Page 332
3.2.1. Design of Long dsRNAs......Page 333
3.2.2. Design of siRNAs......Page 340
3.2.3. Design of shRNAs......Page 342
3.2.5. Design of Genome-Wide RNAi Libraries......Page 343
4. Notes......Page 347
References......Page 349
1. Introduction......Page 355
2.2. Construction of shRNA Expression Plasmids......Page 356
2.3. Con fi rmation of Insert Size by Colony PCR......Page 358
3.2. Construction of shRNA Expression Plasmids......Page 359
4. Notes......Page 360
References......Page 362
1. Introduction......Page 364
2. Materials......Page 370
3.1. Choosing Conserved Regions for Selecting amiRNA Targeting Sites......Page 371
3.3. Cloning In Silico......Page 374
3.5. Analysis of Transgenic Insertion......Page 377
3.7. Analysis of Small RNA......Page 378
3.8. Characterizing Resistance......Page 379
4. Notes......Page 380
References......Page 382
1. Introduction......Page 385
2. Materials......Page 387
3.1. Single Gene Silencing by MIGS......Page 388
3.2. Multi-Gene Silencing with MIGS......Page 390
4. Notes......Page 391
References......Page 393
INDEX......Page 394