BioMEMS and Biomedical Nanotechnology

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List of Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii
I. Application of Microarray Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1. Electronic Microarray Technology and Applications in Genomics
and Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Ying Huang, Dalibor Hodko, Daniel Smolko, and Graham Lidgard
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Overview of Electronic Microarray Technology . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.1 NanoChip r Array and NanoChip r Workstation . . . . . . . . . . . . . . . . . . 5
1.2.2 Capabilities of the NanoChip r Electronic Microarrays. . . . . . . . . . . . . 7
1.3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3.1 Single Nucleotide Polymorphisms (SNPs)—Based Diagnostics . . . . . 10
1.3.2 Forensic Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3.3 Gene Expression Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.3.4 Cell Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.3.5 Electronic Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.6 Miniaturization of Electronic Microarray Technology
and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.3.7 Applications in Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.4 Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
References................................................................................. 19
2. Gene Expression Profiling Utilizing Microarray Technology
and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Dominick Sinicropi, Maureen Cronin, and Mei-Lan Liu
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.2 Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2.1 Detection Systems.............................................................. 25
2.2.2 Real-Time RT-PCR Data Analysis .......................................... 31
2.2.3 Qualification of Gene Panels Using Real-Time RT-PCR................ 32
2.2.4 Real-Time RT-PCR Summary................................................ 34
viii CONTENTS
2.3 Microarrays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.3.1 Technology Platforms ......................................................... 35
2.3.2 Target Amplification and Labeling.......................................... 37
2.3.3 Applications ..................................................................... 40
2.4 Comparison of Gene Expression Profiling Methods . . . . . . . . . . . . . . . . . . . . . 41
2.4.1 Comparison of cDNA Arrays with Other Gene Expression
Profiling Methods .............................................................. 41
2.4.2 Comparison of Oligonucleotide Arrays with Other Gene
Expression Profiling Methods................................................ 42
2.4.3 Comparison of cDNA and Oligonucleotide Microarray
Expression Profiles............................................................. 44
2.5 Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Acknowledgements .................................................................... 45
References ............................................................................... 45
3. Microarray and Fluidic Chip for Extracellular Sensing . . . . . . . . . . . . . . . . . . . 47
Mihrimah Ozkan, Cengiz S. Ozkan, Shalini Prasad, Mo Yang, and Xuan Zhang
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2 Antibody Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.3 Nucleic Acid Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.4 Ion Channel Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.5 Enzyme Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.6 Cell Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.7 Cellular Microorganism Based Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.8 Fluorescence Based Cell Biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.9 Cellular Metabolism Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.10 Impedance Based Cellular Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.11 Intracellular Potential Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.12 Extracllular Potential Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.13 Cell Patterning Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.14 Dielectrophoresis for Cell Patterning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.15 Basis of Dielectrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.16 Microelectrodes and Dielectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.17 Dielectric Properties of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.18 Effect of Electric Fields on Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.19 Cell Types and the Parameters for Dielectrophoretic Patterning . . . . . . . . . . 65
3.20 Biosensing System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.21 Chip Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.22 Environmental Chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.23 Experimental Measurement System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.24 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.24.1 Neuron Culture ................................................................ 67
3.24.2 Primary Osteoblast Culture ................................................. 68
3.25 Signal Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.26 Selection of Chemical Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
CONTENTS ix
3.26.1 Ethanol.......................................................................... 69
3.26.2 Hydrogen Peroxide ........................................................... 69
3.26.3 Pyrethroid ...................................................................... 70
3.26.4 Ethylene Diamene Tetra Acetic Acid (EDTA)........................... 70
3.27 Chemical Agent Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.27.1 Signature Pattern for Control Experiments............................... 70
3.28 Electrical Sensing Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.29 Ethanol Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.29.1 Single Neuron Sensing....................................................... 71
3.29.2 Single Osteoblast Sensing................................................... 71
3.30 Hydrogen Peroxide Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.30.1 Single Neuron Sensing....................................................... 72
3.30.2 Single Osteoblast Sensing................................................... 73
3.31 Pyrethroid Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.31.1 Single Neuron Sensing....................................................... 74
3.31.2 Single Osteoblast Sensing................................................... 75
3.32 EDTA Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
3.32.1 Single Neuron Sensing....................................................... 76
3.32.2 Single Osteoblast Sensing................................................... 76
3.33 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.34 Visualization of Physiological Changes Due to the Effect
of the Chemical Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.34.1 Effect of Ethanol on Neurons............................................... 80
3.34.2 Effect of Ethanol on Osteoblasts ........................................... 80
3.34.3 Effect of Hydrogen Peroxide on Neurons ................................ 83
3.34.4 Effect of Hydrogen Peroxide on Osteoblasts ............................ 84
3.34.5 Effect of Pyrethroid on Neurons ........................................... 86
3.34.6 Effect of Pyrethroid on Osteoblasts........................................ 88
3.34.7 Effect of EDTA on Neurons................................................. 89
3.34.8 Effect of EDTA on Osteoblasts............................................. 91
3.35 Discussion and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
References ............................................................................... 98
4. Cell Physiometry Tools based on Dielectrophoresis . . . . . . . . . . . . . . . . . . . . . . . 103
Ronald Pethig
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.2 Dielectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.3 Dielectric Polarizability of Bioparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.4 Dynamics of Interfacial Polarization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.5 Surface Charge Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.6 Other Physiometric Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.7 Traveling Wave Dielectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
4.8 Controlling Possible DEP-Induced Damage to Cells . . . . . . . . . . . . . . . . . . . . . 120
Concluding Comments.................................................................. 123
References................................................................................. 124
x CONTENTS
5. Hitting the Spot: The Promise of Protein Microarrays . . . . . . . . . . . . . . . . . . . . 127
Joanna S. Albala
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
5.2 Generation of Protein Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
5.2.1 Content............................................................................. 128
5.2.2 Surface Chemistry ............................................................... 129
5.2.3 Microarray Production .......................................................... 129
5.2.4 Detection........................................................................... 130
5.3 Protein Arrays for Analysis of Proteins Involved in Recombination
& DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.3.1 Protein Expression Microarrays............................................... 130
5.3.2 Protein Interaction Arrays ...................................................... 132
5.4 Summary: Protein arrays-Hope or hype? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Acknowledgements...................................................................... 133
References................................................................................. 133
6. Use of Electric Field Array Devices for Assisted Assembly of DNA
Nanocomponents and Other Nanofabrication Applications . . . . . . . . . . . . . . . 137
Michael J. Heller, Cengiz S. Ozkan, and Mihrimah Ozkan
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
6.2 Active Microelectronic Array Hybridization Technology . . . . . . . . . . . . . . . . . 141
6.3 Electric Field Assisted Nanofabrication Process . . . . . . . . . . . . . . . . . . . . . . . . . 146
6.4 Integration of Optical Tweezers for Manupilation of Live Cells . . . . . . . . . . . 153
Conclusions ............................................................................... 156
Abbreviations ............................................................................. 156
Acknowledgements...................................................................... 157
References................................................................................. 157
7. Peptide Arrays in Proteomics and Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . 161
Ulrich Reineke, Jens Schneider-Mergener, and Mike Schutkowski
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
7.2 Generation of Peptide Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
7.2.1 Coherent Surfaces and Surface Modification ............................... 163
7.2.2 Generation of Micro-Structured Surfaces ................................... 173
7.2.3 Peptide Array Preparation ...................................................... 182
7.2.4 Techniques for Array Production with Pre-Synthesized Peptides....... 200
7.3 Library Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
7.3.1 Protein Sequence-Derived Libraries.......................................... 204
7.3.2 De Novo Approaches............................................................ 210
7.4 Assays for Peptide Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
7.4.1 Screening .......................................................................... 215
7.4.2 Read-Out........................................................................... 219
7.5 Applications of Peptide Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
7.5.1 Antibodies ......................................................................... 222
7.5.2 Protein-Protein Interactions .................................................... 224
7.5.3 Enzyme-Substrate and Enzyme-Inhibitor Interactions .................... 226
CONTENTS xi
7.5.4 Application of Peptide Arrays: Miscellaneous ............................. 228
7.5.5 Peptidomimetics.................................................................. 231
7.6 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
References................................................................................. 265
8. From One-Bead One-Compound Combinatorial Libraries
to Chemical Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Kit S. Lam, Ruiwu Liu, Jan Marik, and Pappanaicken R. Kumaresan
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
8.2 OBOC Peptide Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
8.3 Encoded OBOC Small Molecule Combinatorial Libraries . . . . . . . . . . . . . . . . 287
8.4 Peptide and Chemical Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
8.4.1 Immobilization Methods for Pre-Synthesized Libraries .................. 289
8.4.2 In Situ Synthesis of Microarrays .............................................. 292
8.4.3 CD, Microfluidics, Fiber Optic Microarray, Multiplex Beads ........... 295
8.5 Detection Methods in Chemical Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
8.5.1 Identification and Characterization of Bound Proteins.................... 296
8.5.2 Detection Methods to Identify Post-Translational Modification
of Proteins or to Quantitate Enzyme Activity in Analytes................ 297
8.6 Application of Chemical Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
8.6.1 Protein Binding Studies......................................................... 298
8.6.2 Post-Translational Modification, Enzyme-Substrate
and Inhibitor Studies ............................................................ 299
8.6.3 Cell-Binding Studies ............................................................ 300
8.6.4 Drug Discovery and Cell Signaling .......................................... 300
8.6.5 Diagnostic Studies ............................................................... 301
8.6.6 Non-Biological Applications .................................................. 301
8.7 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Acknowledgements...................................................................... 303
Abbreviations ............................................................................. 303
References................................................................................. 304
II. Advanced Microfluidic Devices and Human Genome Project . . . . . . . . . . . . . . . . 309
9. Plastic Microfluidic Devices for DNA and Protein Analyses . . . . . . . . . . . . . . . 311
Z. Hugh Fan and Antonio J. Ricco
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
9.1.1 Detection........................................................................... 311
9.1.2 Materials........................................................................... 312
9.2 Electrokinetic Pumping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
9.3 Plastic Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
9.3.1 Pumping and Detection ......................................................... 315
9.3.2 Device Fabrication ............................................................... 316
9.4 DNA Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
9.4.1 Integrating PCR and DNA Fragment Separations ......................... 318
xii CONTENTS
9.4.2 DNA Sequencing ............................................................... 320
9.4.3 DNA Sample Purification ..................................................... 321
9.5 Protein Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
9.5.1 Isoelectric Focusing for Studying Protein Interactions .................. 323
9.5.2 Enzymatic Digestion for Protein Mapping................................. 324
Concluding Remarks................................................................... 326
Acknowledgements .................................................................... 326
References ............................................................................... 326
10. Centrifuge Based Fluidic Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Jim V. Zoval and M.J. Madou
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
10.2 Why Centrifuge as Fluid Propulsion Force? . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
10.3 Compact Disc or Micro-Centrifuge Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
10.3.1 How it Works .................................................................. 333
10.4 Some Simple Fluidic Function Demonstrated on a CD . . . . . . . . . . . . . . . . . . 334
10.4.1 Mixing of Fluid ............................................................... 334
10.4.2 Valving ......................................................................... 335
10.4.3 Volume Definition (Metering) and Common
Distribution Channels ........................................................ 338
10.4.4 Packed Columns .............................................................. 339
10.5 CD Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
10.5.1 Two-Point Calibration of an Optode-Based Detection System ...... 339
10.5.2 CD Platform for Enzyme-Linked Immunosorbant
Assays (ELISA) ............................................................... 340
10.5.3 Multiple Parallel Assays ..................................................... 341
10.5.4 Cellular Based Assays on CD Platform ................................... 342
10.5.5 Automated Cell Lysis on a CD ............................................. 344
10.5.6 Integrated Nucleic Acid Sample Preparation and
PCR Amplification ........................................................... 356
10.5.7 Sample Preparation for MALDI MS Analysis .......................... 358
10.5.8 Modified Commercial CD/DVD Drives in
Analytical Measurements ................................................... 359
Conclusion............................................................................... 361
Acknowledgements .................................................................... 362
References ............................................................................... 362
11. Sequencing the Human Genome: A Historical Perspective
On Challenges For Systems Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Lee Rowen
11.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
11.2 Approaches Used to Sequence the Human Genome . . . . . . . . . . . . . . . . . . . . . 366
11.2.1 Overview........................................................................ 366
11.2.2 Strategy Used for Sequencing Source Clones............................ 368
11.2.3 Construction of the Chromosome Tiling Paths .......................... 379
11.2.4 Data Sharing ................................................................... 379
CONTENTS xiii
11.3 Challenges for Systems Integration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
11.3.1 Methodological Challenges for Sequencing Source
Clones: 1990–1997 ........................................................... 381
11.3.2 Challenges for Sequencing the Entire Human
Genome: 1998–2003.......................................................... 386
11.4 Are there Lessons to be Learned from the Human Genome Project? . . . . . . 395
Acknowledgements .................................................................... 397
References ............................................................................... 398
III. Nanoprobes for Imaging, Sensing and Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
12. Hairpin Nanoprobes for Gene Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Philip Santangelo, Nitin Nitin, Leslie LaConte, and Gang Bao
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
12.2 Nanoprobe Design Issues for Homogeneous Assays . . . . . . . . . . . . . . . . . . . . 405
12.3 In Vitro Gene Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
12.3.1 Pathogen Detection ........................................................... 409
12.3.2 Mutation Detection and Allele Discrimination .......................... 409
12.4 Intracellular RNA Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
12.4.1 Cytoplasmic and Nuclear RNA............................................. 411
12.4.2 RNA Secondary Structure ................................................... 418
12.5 Living Cell RNA Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
12.5.1 Cellular Delivery of Probes.................................................. 419
12.5.2 Intracellular Probe Stability ................................................. 424
12.5.3 Intracellular mRNA Detection .............................................. 428
12.6 Opportunities and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Acknowledgements .................................................................... 433
References ............................................................................... 433
13. Fluorescent Lanthanide Labels with Time-Resolved Fluorometry
In DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Takuya Nishioka, Jingli Yuan, and Kazuko Matsumoto
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
13.2 Lanthanide Fluorescent Complexes and Labels . . . . . . . . . . . . . . . . . . . . . . . . . 438
13.3 Time-Resolved Fluorometry of Lanthanide Complexes . . . . . . . . . . . . . . . . . 441
13.4 DNA Hybridization Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
Conclusion............................................................................... 445
References ............................................................................... 445
14. Role of SNPs and Haplotypes in Human Disease and Drug Development . . 447
Barkur S. Shastry
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
14.2 SNP Discovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
14.3 Detection of Genetic Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
14.4 Disease Gene Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
xiv CONTENTS
14.5 Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
14.6 Haplotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
14.7 Drug Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Concluding Remarks................................................................... 454
References ............................................................................... 454
15. Control of Biomolecular Activity by Nanoparticle Antennas . . . . . . . . . . . . . . 459
Kimberly Hamad-Schifferli
15.1 Background and Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
15.1.1 ATP Synthase as a Molecular Motor ...................................... 459
15.1.2 Biological Self Assembly of Complex Hybrid Structures ............ 461
15.1.3 DNA as a Medium for Computation ...................................... 463
15.1.4 Light Powered Nanomechanical Devices................................. 463
15.2 Nanoparticles as Antennas for Controlling Biomolecules . . . . . . . . . . . . . . . . 465
15.2.1 Technical Approach........................................................... 468
15.2.2 Dehybridization of a DNA Oligonucleotide Reversibly by
RFMF Heating of Nanoparticles ........................................... 469
15.2.3 Determination of Effective Temperature by RFMF
Heating of Nanoparticles .................................................... 469
15.2.4 Selective Dehybridization of DNA Oligos by RFMF
Heating of Nanoparticles .................................................... 471
Conclusions and Future Work ....................................................... 473
References ............................................................................... 474
16. Sequence Matters: The Influence of Basepair Sequence
on DNA-protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
Yan Mei Wang, Shirley S. Chan, and Robert H. Austin
16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
16.2 Generalized Deformations of Objects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
16.3 Sequence Dependent Aspects to the Double Helix Elastic Constants . . . . . . 484
16.4 Sequence Dependent Bending of the Double Helix and the
Structure Atlas of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
16.5 Some Experimental Consequences of Sequence Dependent Elasticity . . . . . 486
16.5.1 Phage 434 Binding Specificity and DNase I Cutting Rates ........... 486
16.5.2 Nucleosome Formation: Sequence and Temperature Dependence... 491
Conclusions.............................................................................. 494
References ............................................................................... 494
17. Engineered Ribozymes: Efficient Tools for Molecular
Gene Therapy and Gene Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
Maki Shiota, Makoto Miyagishi, and Kazunari Taira
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
17.2 Methods for the Introduction of Ribozymes into Cells . . . . . . . . . . . . . . . . . . . 498
17.3 Ribozyme Expression Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
17.3.1 The Pol III System ............................................................ 499
CONTENTS xv
17.3.2 Relationship Between the Higher-Order Structure of
Ribozymes and their Activity .............................................. 500
17.3.3 Subcellular Localization and Efficacy of Ribozymes................... 501
17.3.4 Mechanism of the Export of tRNA-Ribozymes from the
Nucleus to the Cytoplasm ................................................... 504
17.4 RNA-Protein Hybrid Ribozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
17.4.1 Accessibility to Ribozymes of their Target mRNAs.................... 505
17.4.2 Hybrid Ribozymes that Efficiently Cleave their Target mRNAs,
Regardless of Secondary Structure ........................................ 505
17.5 Maxizymes: Allosterically Controllable Ribozymes. . . . . . . . . . . . . . . . . . . . . 508
17.5.1 Shortened Hammerhead Ribozymes that Function as Dimers........ 508
17.5.2 Design of an Allosterically Controllable Maxizyme.................... 509
17.5.3 Inactivation of an Oncogene in a Mouse Model......................... 512
17.5.4 Generality of the Maxizyme Technology ................................ 512
17.6 Identification of Genes Using Hybrid Ribozymes . . . . . . . . . . . . . . . . . . . . . . . 513
17.7 Summary and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
References ............................................................................... 516
About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521

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