Better than the real thing: peptide nucleic acids: Peptide Nucleic Acids: Protocols and Applications edited by Peter E. Nielsen and Michael Egholm

Abstract

Horizon Scientific Press, 1999, $59.99 hbk (266 pages)ISBN 1 898486 16 6Reagents capable of sequence-specific recognition of nucleic acids are becoming more and more important for research, diagnosis and therapy. Rapid progress is being made in the elucidation of the human genome sequence, as well as that of other organisms, including human pathogens. As this information emerges, there is an increasing need for new tools to probe gene structure and function and for novel reagents to allow development of gene-based diagnostics and therapeutics.Over the past few years, peptide nucleic acids (PNAs) have emerged as one of the most promising new types of molecules for recognition of nucleic acids. PNAs are actually neither peptide nor nucleic acid, but have a hybrid structure consisting of repeating N-(2-aminoethyl)-glycine units linked by amide bonds. The purine (A, G) and pyrimidine (T, C) nucleobases are attached to this backbone via methylene carbonyl linkages. There are no sugar or phosphate groups. Hence, the unmodified PNAs are not charged at neutral pH.Originally, PNAs were designed to recognize and bind to duplex DNA in the major groove via Hoogsteen bonding to form triple helices. Although certain PNA sequences can do this, it was soon discovered that PNA oligomers can bind to single-stranded nucleic acids to form duplexes, either PNA–DNA or PNA–RNA, with affinity and specificity substantially exceeding that of comparable DNA or RNA oligonucleotides. In addition, triplex formation involv- ing PNAs can occur either with PNA–DNA–DNA triplexes or the more favored PNA–DNA–PNA triplexes. The stability of the PNA interaction with DNA is such that strand invasion of DNA by PNAs is thermodynamically favored, and can take place via either duplex, triplex or double duplex formation.The foregoing constitutes just a short list of the capabilities of PNAs. In fact, the known properties of PNAs and their potential applications in biomedicine are rapidly expanding as researchers continue to examine these fascinating molecules. It is therefore quite timely that Peter Nielsen and Michael Egholm, two of the inventors of PNAs, have put together a book reviewing the state of the art in PNA research.The emphasis here is on timely. The editors have clearly made an effort to include up-to-the-minute material and to publish it quickly. As a result, the topics include most, if not all, of the recent PNA advances, all by key researchers in the field. The book begins with a useful overview by the editors putting the development and use of PNAs in perspective, and serving as an introduction for those new to this area.This is followed by a series of chapters focusing on the fundamental chemistry of PNA synthesis. Several of these cover newer chemical strategies such as the synthesis of PNA–DNA and PNA–peptide chimeras and PNA conjugation to labeling compounds such as biotin, fluorescein and rhodamine. The emphasis here is on the protocols. True to the book’s subtitle, these chapters read like a good manual, with substantial detail as well as useful discussion providing experimental and technical insight. Although these sections will be most useful to synthetic chemists, they will also serve biologists by highlighting the versatility of PNAs and identifying the possible syntheses and novel molecules that can be made.Another section focuses on hybridization-based techniques, and these chapters provide a sophisticated discussion of the nucleic acid-binding properties of the PNAs, both in a section devoted to the thermodynamics of PNA–nucleic acid interactions as well in a series of application chapters. In this portion of the book, a wide variety of emerging properties and applications of PNAs are examined. The use of PNAs for in situ hybridization analysis of human tissue for research and diagnostic purposes is reviewed, as well as the application of PNAs to chromosomal analysis by FISH, which will be of particular interest to geneticists. Other chapters in this section focus on the unique properties of PNAs in array analyses, in Southern and northern hybridization protocols, in detecting single nucleotide polymorphisms, and in biosensor devices.The book also highlights novel nucleic acid manipulations with PNAs, including PNA-directed rare genome cleavage, duplex DNA capture, nucleic acid purification, and PNA clamping for PCR manipulation. One very new application of PNAs presented is in vivo labeling and tracking of plasmid DNA using labeled PNA clamps.The book concludes with a section on the use of PNAs as antisense and antigene reagents. Besides their unique binding properties, PNAs also show impressive in vivo stability, due to their resistance to nucleases and proteases. This feature renders them intriguing reagents for in vivo applications. The chapters in this section review the strand-displacement properties of PNAs for targeting DNA, the use of PNAs as potential antibiotics via antisense activity in bacteria, and the ability of PNAs to inhibit cellular DNA-metabolizing enzymes such as telomerase, with potential utility in cancer therapy. One area of omission in this otherwise comprehensive text is coverage of the emerging work on PNAs as antisense agents in vivo in experimental animals, although some of this work is mentioned in the introductory chapter. Overall, this book should serve as a useful primer for readers interested in exploring the potential utility of PNAs for their particular research interest. Even scientists experienced in the area will benefit from the compendium of protocols and the extensive references.