Peptide Nucleic Acids Methods and Protocols

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 Synthesisof 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. . . . . . . . . . . . . . . . . .

PNA Technology.- Chemistry.- Solid Phase Synthesis of PNA Oligomers.- Synthesis of PNA-Peptide Conjugates.- Parallel Synthesis of PNA-Peptide Conjugate Libraries.- Thermodynamics of PNA Interactions with DNA and RNA.- PNA Technology.- Measurement of PNA Binding to Double-Stranded DNA.- PNA-Mediated Immobilization of Supercoiled DNA.- PNA Openers and Their Applications.- Colorimetric Detection of PNA-DNA Hybridization Using Cyanine Dyes.- Diagnostics.- PNA-Mediated PCR Clamping.- PNA Clamping Techniques for the Determination of Oncogene Mutations.- PNA Fluorescent In Situ Hybridization for Rapid Microbiology and Cytogenetic Analysis.- Detection of Point Mutations Using PNA-Containing Electrophoresis Matrices.- Antisense and Antigene.- Lipid-Mediated Introduction of Peptide Nucleic Acids Into Cells.- Synthesis of Cell-Penetrating Peptide-PNA Constructs.- Antisense Inhibition of Bacterial Gene Expression and Cell Growth.- In Vitro Transcription from Peptide Nucleic Acid/DNA Strand Displacement Loops.- Animal Studies.- In Vitro and In Vivo Studies on the Pharmacokinetics and Metabolism of PNA Constructs in Rodents.