Exemplifying and illustrating recent exciting advances in PNA chemistry, the second edition of Peptide Nucleic Acids: Methods and Protocols serves as a vital complement to the first edition of the book. Since the discovery of peptide nucleic acids, many interesting new derivatives and analogues in terms of nucleic acid recognition specificity and affinity have emerged. Also, as this detailed volume presents, great ingenuity in exploiting the unique properties of PNAs for a wide variety of applications within drug discovery, medical diagnostics, chemical biology and nanotechnology has unfolded. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Peptide Nucleic Acids: Methods and Protocols, Second Edition serves as a source of useful specific methods and protocols as well as a source of inspiration for future developments.

Peptide nucleic acids (PNAs) have now existed for slightly more than ten years, with the interest in and applications of this pseudopeptide DNA mimic steadily increasing during the entire period. PNAs have rapidly attracted the attention of scientists from a diversity of fields ranging from (bio)organic and biophysical chemistry to prebiotic evolution, and from molecular biology to genetic diagnostics and drug development. Many of the applications take advantage of the unique properties of PNA-an uncharged pseudopeptide-that distinguish this DNA mimic from more traditional DNA analogs. Rather than trying to create a comprehensive collection of all published methods and protocols involving PNA-many of which have not yet been validated- I have decided to concentrate on select protocols that are either very well established by several groups around the world, such as PCR-clamping and in situ hybridization, or on new methods that may have broader future impact. Basic methods for PNA oligomer synthesis and analyses have also been included. I am very grateful to those friends and colleagues who have enthusiastically contributed their work, discussions, and writing, and thereby made this book possible. Peter E. Nielsen v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix IINTRODUCTION 1 PNA Technology Peter E. Nielsen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 II CHEMISTRY 2 Solid Phase Synthesis of PNA Oligomers Frederik Beck. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3 Synthesis of PNA-Peptide Conjugates Satish Kumar Awasthi and Peter E. Nielsen. . . . . . . . . . . . . . . . . . 43 4 Parallel Synthesis of PNA-Peptide Conjugate Libraries Satish Kumar Awasthi and Peter E. Nielsen. . . . . . . . . . . . . . . . . .

Exemplifying and illustrating recent exciting advances in PNA chemistry, the second edition of Peptide Nucleic Acids: Methods and Protocols serves as a vital complement to the first edition of the book. Since the discovery of peptide nucleic acids, many interesting new derivatives and analogues in terms of nucleic acid recognition specificity and affinity have emerged. Also, as this detailed volume presents, great ingenuity in exploiting the unique properties of PNAs for a wide variety of applications within drug discovery, medical diagnostics, chemical biology and nanotechnology has unfolded. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Peptide Nucleic Acids: Methods and Protocols, Second Edition serves as a source of useful specific methods and protocols as well as a source of inspiration for future developments.

The concept of using photochemical probes in the study of biological systems was developed by Westheimer who published the first photoaffinity labeling experiments more than twenty years ago (J.Bio1. Chem. 237, (1962) 3006). Since then the concept has been used successfully in various areas of biochemistry and recently several new interesting and exciting aspects of the concept have been developed. It is the general opinion by scientists in the "field" that the full potential of photochemical probes in biochemical studies has far from been exploited yet. This is mostly due to the interdisciplinary character of the concept involving photochemistry, synthetic chemistry as well as biochemistry/molecular biology. The perspective of the NATO advanced workshop on "Photochemical Probes in Biochemistry," held in Holte (Copenhagen) Denmark 14-19, August, 1988, was several fold. The workshop was to give an account of the "state of the art" of using photochemical probes in biochemistry as well as to bring together specialists in photochemistry, syn thetic chemistry and molecular biology in order to analyze advantages as well as the inherent problems and pitfalls of the concept and provide suggestions and guidelines for fu ture research. Furthermore, it is the hope of the editor that the present publication which gives an account of the lectures presented at the workshop, will provide an introduction to scientists who are not familiar with photochemical probes, but to whom these could help answer central and pertinent questions."

The concept of using photochemical probes in the study of biological systems was developed by Westheimer who published the first photoaffinity labeling experiments more than twenty years ago (J.Bio1. Chem. 237, (1962) 3006). Since then the concept has been used successfully in various areas of biochemistry and recently several new interesting and exciting aspects of the concept have been developed. It is the general opinion by scientists in the "field" that the full potential of photochemical probes in biochemical studies has far from been exploited yet. This is mostly due to the interdisciplinary character of the concept involving photochemistry, synthetic chemistry as well as biochemistry/molecular biology. The perspective of the NATO advanced workshop on "Photochemical Probes in Biochemistry", held in Holte (Copenhagen) Denmark 14-19, August, 1988, was several fold. The workshop was to give an account of the "state of the art" of using photochemical probes in biochemistry as well as to bring together specialists in photochemistry, syn­ thetic chemistry and molecular biology in order to analyze advantages as well as the inherent problems and pitfalls of the concept and provide suggestions and guidelines for fu­ ture research. Furthermore, it is the hope of the editor that the present publication which gives an account of the lectures presented at the workshop, will provide an introduction to scientists who are not familiar with photochemical probes, but to whom these could help answer central and pertinent questions.