BioMEMS and biomedical nanotechnology 2. Micro/nano technology for genomics and proteomics

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 . . . . . . . . . .