Peptide Nucleic Acids: Protocols and Applications Book Reviews

This page contains excerpts from published reviews. Full details of our books are available, including how to buy.

Review by
Gerald Owenson University of Warwick, UK

Microbiology Today 26: 203. November 1999

The use of peptide nucleic acid is a novel and growing field. This book brings together a collection of protocols, ranging from the synthesis of PNA to its use in a number of molecular applications. The information is logically presented, with each chapter taking the form of a short publication. Although there is limited troubleshooting information, each method is presented in a clear and concise manner, together with appropriate results and discussion. However, due to the nature of the protocols, there is inevitably some overlap between chapters. This book would be an excellent source of information for individuals who are interested in PNA technology and its relevance to their own research. It would also be useful for the researcher who was perhaps not aware of PNA, but who would like to find out more. It is perhaps less relevant for workers already in the field as it is limited to standard protocols.

Review by
Vadim V. Demidov Center for Advanced Biotechnology, 36 Cummington Street, Boston, Massachusetts 02215, USA

Modern Drug Discovery (American Chemical Society). July/August 2000

More than a hundred years ago, Paul Ehrlich, the founder of modern chemotherapy, dreamed about "magic bullets" able to effectively cure patients without producing the side effects usually caused by insufficient specificity of most therapeutics. Not long ago, synthetic oligonucleotides and their numerous modifications were developed that were capable of selectively targeting loci in the human genome and/or specific enzymes responsible for disease. Among the most promising oligonucleotide analogs and mimics are the polyamide or peptide nucleic acids (PNAs), designed by Danish scientists P. E. Nielsen, M. Egholm, R. H. Berg, and 0. Buchardt. It is remarkable that, starting with the first paper in 1991 that introduced them, the number of publications in the field of PNA research now approaches 400! At present, PNAs have been the subject of almost 50 reviews in numerous scientific periodicals.

PNAs consist of a protein-like, polyamide backbone and standard or modified nucleobases. The structure of PNAs represents a radical departure from common oligonucleotide chemistry, giving the PNA molecules a set of exceptional chemical, physical, and biological properties. As compared to ordinary oligonucleotides and their analogs, the pseudopeptide backbone gives PNAs excellent chemical and biological stability, higher binding affinity, and better specificity. In addition, PNAs provide more favorable conditions for targeting DNA, RNA, and (deoxy)ribonucleoproteins along with the unique ability to invade duplex DNA. As a result, PNAs find more and more innovative applications both in vitro and in vivo that have practical potential for DNA diagnostics, gene therapy, and medicinal chemistry, and that could not be performed using oligonucleotides.

The recent publication of Peptide Nucleic Acids, the first book on PNAs, marks an important event: the transition of the emerging PNA technology from infancy to maturity. This is the comprehensive manual on practical PNA applications edited by two PNA co-inventors, who are also the leading scientists in PNA research. The book includes joint contributions from 45 recognized experts and covers virtually all aspects of the field, from introduction to PNAs and basic PNA chemistry to PNA-based biomolecular tools and antisense antigene technology. Key topics include hybridization-based techniques with the use of PNA probes and biosensors, PCR clamping, purifying, and selectively manipulating nucleic acids. Along with applied topics, this book of PNA protocols features some related basic themes such as thermodynamics of the PNA-nucleic acid interactions, characterization of the strand-displacement complexes formed by PNAs with duplex DNA, and interaction of PNAs with DNA-processing enzymes.

The book is focused on detailed, step-by-step protocols and exemplary real-world applications where the PNA molecules perform advantageously or make it possible to use methods that were unworkable before. Each chapter explains clearly and in practical terms the concepts behind the particular protocol detailed. All chapters have extensive reference sections and are well illustrated. The final chapter describes approaches for the discovery of new PNA-based drugs. At the end of the book there is a helpful appendix providing guidelines for rational design of PNA oligomers; their storage, handling, and quantification; and troubleshooting recommendations.

Notwithstanding the great value of this book, in which many up-to-date applications are adequately considered, a few important methods with promising biomedical potential have been missed. Among them are use of the strand-displacement complexes as artificial promoters and microscopy markers, PNA-assisted cleavage of duplex DNA with single-strand-specific nucleases, PNA-directed mutagenesis, and pregel hybridization. Some missing innovative methods, such as PNA-assisted DNA topological labeling and use of pseudocomplementary PNAs or PNA molecular beacons, were developed during or soon after the book was published. Still, this is essential reading if you wish to become familiar with novel, exciting technologies. Without doubt, Peptide Nucleic Acids: Protocols and Applications will serve as a reference guide for life scientists who may consider PNAs a useful tool in their studies. It should encourage and inspire researchers who are eager to develop innovative pharmaceuticals and to further extend the possibilities of PNAs. As one of the contributors to this book, David R. Corey, wrote: "It is becoming clear that imagination will be the primary limitation to the development of new applications for PNAs." So, let's make them real and practical!

Review by
Lars Branden Department of Biosciences, Novum Research Park, Karolinska Institutet, 141 57 Huddinge, Sweden.

Trends in Biotechnology 18(8). July 2000

This book is an upgraded guide to the properties and possibilities of the DNA mimic peptide nucleic acid (PNA), which is more complete than the 'Users guide' found on the PerSeptive Biosystems homepage, even though it has been recently revised (www.pebio.com/ds/pna/ users.html). As stated in the preface, the focus of this book is on the actual applications and protocols relating to PNA. The neutrality of the backbone and the flexibility of adding functional units to the PNA oligonucleotide make it a formidable tool, and its wide range of applications are well illustrated in the spectrum of protocols included in the book.

The introduction gives the reader a good overview of the properties of the PNA molecule, as well as some of its drawbacks (e.g. the solubility problem of purine-rich sequences). The subsequent chapters cover the synthesis of PNA and different PNA hybrids. They illustrate the development in the efforts being made to synthesize PNA in order for it to be as readily achievable as nucleic acid synthesis. The reader is given good, solid advice when it comes to the type of linker that should be used for the different types of PNA-DNA hybrids.

Some aspects of the radiolabeling of PNA molecules by using protein-specific enzymes are discussed, as well as the possibilities of using the free amino-terminus for fluorescence labeling. Some of the applications reflect the thermodynamic properties of PNA, and this is exemplified in Chapter 3.1 by Holmen and Norden.

The use of PNA as a probe for hybridization in different detection systems is clearly illustrated in Chapter 3.2. In addition, PNA-assisted rare cleavage is a good illustration of what PNA can be used for with regard to blocking enzyme function. The PNA-DNA-PNA hybrid shields the DNA site from methylases and therefore a non-methylated site can be created, making the targeted sequence sensitive to restriction-enzyme cleavage after PNA removal. The labeling of plasmids with different types of fluorescent molecules, such as rhodamine, has long been a problem if one has needed to combine this with expression from a reporter gene in the plasmid. Philip Felgner and his colleagues (Chapter 4.5) have solved this by using a specifically engineered site in the reporter plasmids, enabling a PNA clamp to bind to the plasmid. The clamp is labeled either with a fluorescent label or with biotin. This technique has made it possible to track the fate of plasmids in the cells at the same time as maintaining gene expression.

In conclusion, this book is a necessary requirement for anyone wanting to delve into the field of PNA. It will also be an aid to developing the new genetic tool that PNA represents, thus enabling researchers to create new solutions to problems in nucleic acid detection and gene medicine.

Review by
Michael Loeffelholz, Ph.D. State Hygienic Laboratory, University of Iowa

Current Microbiology 40: 288. April 2000

Peptide nucleic acid (PNA), first described in 1991, is a synthetic molecule consisting of a polyamide (peptide) backbone to which purine and pyrimidine bases are attached. In many respects PNA mimics and even outperforms the behavior of oligonucleotide probes. PNA forms strong hybrids with complementary DNA and RNA. The neutral charge of the polyamide backbone facilitates hybridization and thus allows for rapid and robust assays. This book is an invaluable resource for molecular biologists, biochemists, and molecular microbiologists involved in the development of hybridization-based techniques and DNA diagnostics. The editors have compiled protocols from leadiug international experts in the field of PNA. The style is consistently clear, concise, and informative.

This book of 262 pages consists of 5 sections: introduction; chemistry; hybridization-based techniques; biomolecular tools; and antisense/antigen technology. An appendix containing valuable information on PNA design, handling, and analysis follows these. The section on chemistry contains four chapters on the synthesis of PNA or PNA-DNA chimeras, and a chapter on labeling of PNA. Four hybridization techniques are described in the next section: in situ hybridization; array technology; detection of single nucleotide polymorphisms; and biosensors. The chapter on in situ hybridization contains beautiful color photographs demonstrating the specific staining of Epstein-Barr virus-infected cells and sputum smears containing Mycobacterium tuberculosis. The section on biomolecular tools contains a chapter on PNA-assisted rare cleavage (PARC) of genomic DNA. PARC essentially converts standard restriction enzymes into rare genome cutters, thus expanding the capability of current microorganism strain typing procedures that rely on a limited repertoire of rare-cutting restriction enzymes. Additional applications of PNA covered in this section include PCR clamping and plasmid labeling. The final section covers antisense/antigene technology and includes protocols for characterization of PNA-DNA strand displacement complexes and PNA-mediated inhibition of telomerase.

The editors and authors deserve praise for this extensive, yet easily read compilation of protocols and applications. It is a valuable reference for any microbiology laboratory engaged in molecular biology research or the development of molecular methods.

Review by
UIf Diederichsen Institut für Organische Chemie der Universität Würzburg (Germany)

ChemBioChem 2(4): 293-294 April 2001

Peptide nucleic acids (PNAs) were invented in 1991 by the editors together with Buchardt and Berg in order to generate molecules that would effectively bind to DNA duplexes, resulting in the formation of triple-stranded complexes. PNAs gained so much importance in chemistry and biology that it is well worth writing a book about synthesis and applications of this class of artificial oligomers only eight years after their introduction. PNA turned out not only to be capable of forming triple-stranded complexes upon binding to DNA as planned, but, even better, an oligomer that is capable of forming stable double strands with DNA or RNA. PNA differs from oligonucleotides in many respects since the ribose-phosphodiester backbone present in DNA is completely substituted by an isosteric aminoethylglycine-polyamide backbone in PNA. Similar pairing selectivities of PNA and DNA are combined with different physical properties, resulting in beneficial applications in medicinal chemistry and diagnostics. Therefore, this book gives a wide overview starting with the introduction to PNAs and their hybridization potential, summarizes the synthetic strategies, and finally gives examples of applications in diagnostics, molecular biology, and medicinal chemistry. This is done in overall twenty contributions, mostly written by those researchers who are responsible for the respective inventions covered.

The sub-chapters are organized uniformly throughout the book, to start with an abstract and introduction to the idea and method; this is followed by a part that gives detailed practical information usually with one example. The chapters end with a short discussion of the presented method including critical remarks, if necessary. Finally, the chapters contain a comprehensive list of references. A useful appendix summarizes how to handle PNA and further gives a few comments on oligonucleotide nomenclature. The index is rather poor, but the organization of the book into five sections (introduction, synthesis, hybridization with oligonucleotides, biomolecular tools, and antisense/antigene technology) is clear enough to make the index dispensable.

What are the physical properties that make PNAs superior in many applications? In contrast to the polyanionic DNA oligomers, PNAs have an uncharged backbone. Due to the lack of electrostatic repulsion PNA binding is not dependent on salt concentration and allows access to environments not readily accessible by oligonucleotides; most importantly, PNA-DNA as well as PNA-RNA double strands are much more stable than oligonucleotide duplexes. The sequence specificity is high and a single base pair mismatch is recognized by PNA with high selectivity. The nonpolar backbone offers a wider range of experimental conditions. Nonspecific interactions based on charge effects are excluded. Furthermore, PNA is stable against enzymatic degradation and cannot function as a primer for DNA polymerases. PNAs can be easily synthesized by using solid-phase peptide synthesis.

Three different methods for PNA synthesis are described, based on Boc-, Fmoc- and MMT-protected nucleo amino acids. A detailed protocol is given for PNA synthesis on the solid support based on Boc chemistry, including the adaptation to the automated procedure. Fmoc chemistry is better qualified for the synthesis of labeled PNA, the attachment of reporter groups, and for the preparation of PNA/peptide chimeras. A protocol based on MMT-protected nucleo amino acids is suitable for the synthesis of PNA/DNA chimeras since these amino acid building units are compatible with the phosphoramidite DNA synthesis protocol. Overall, the synthesis chapter contains a lot of practical information regarding protecting groups, activation, solid support, linkers, purification, and analysis.

In the second part of the book (Chapters 3-5) applications of PNA in biology, biochemistry, biomedicine, and diagnostics are discussed. The major developments up to 1999 are covered using specific examples with their respective protocols. It is obvious that not all facets of PNA chemistry can be highlighted, but the reader gets a very good impression of the possibilities and methods since the applications are also given with experimental details. The tighter hybridization of PNA with oligonucleotides, which is detectable with temperature-dependent UV spectroscopy as well as isothermal titration calorimetry, allows a more sensitive in situ hybridization with more convenient protocols. Therefore, PNA can be used as a diagnostic tool for the identification and visualization of oligonucleotides or even different microorganisms, or it can be used as a biosensor. Further, PNA can serve as a blocker for sequences that are closely related to the target of nucleic acid hybridization. For diagnostic purposes it is advantageous to use complementary PNA for MALDI-TOF MS instead of the appropriate oligonucleotide. Interesting biotechnological applications are based on partial blocking of DNA double strands for methylation by methyltransferase, thereby producing rare cleavage sites for restriction enzymes. Furthermore, PNA can be used for affinity purification of DNA, PCR clamping, and plasmid labeling.

In the early years, PNA oligomers were mainly discussed in respect to antisense or antigene therapy. Despite the high binding affinity, the bioavailability and lack of RNAse H activation are the major drawbacks that explain why PNA has not succeeded as a therapeutic agent so far. Nevertheless, antisense PNA can be used to control cell growth and gene expression in vitro and in vivo. Strand displacement is characteristic of homopyrimidine PNA binding, which might be used to prevent transcription. Finally, PNA complexed to RNA inhibits enzyme activity.

This book gives a very useful overview of synthesis and applications of PNAs. Chances and limitations are discussed. Despite the many review articles on PNAs that have already been published and very active research during the two years since the appearance of this book, it is a valuable contribution for the inexperienced reader, who can easily get an overview of PNA chemistry and its use in biology. The detailed step-by-step protocols and examples of applications also make this book highly valuable for experts as a reference book.