Peptide Nucleic Acids: Protocols and Applications (Second Edition) Chapter Abstracts
Chapter 1
An Introduction to PNA
Peter E. Nielsen and Michael Egholm
Peptide nucleic acids (PNA) are DNA mimics with a pseudopeptide backbone. PNA is an extremely good structural mimic of DNA (or RNA), and PNA oligomers are able to form very stable duplex structures with Watson-Crick complementary DNA, RNA (or PNA) oligomers, and they can also bind to targets in duplex DNA by helix invasion. Therefore, these molecules are of interest in many areas of chemistry, biology, and medicine including drug discovery, genetic diagnostics, molecular recognition and the origin of life. A brief overview of the history, properties and applications of PNA in drug discovery and DNA detection is presented.
Chapter 2
PNA Synthesis by Boc Chemistry
Troels Koch
In this chapter PNA synthesis following the Boc/Z strategy is presented with both detailed manual and automated synthesis protocols. The automated protocol is designed to the ABI 433A peptide synthesiser. Common side reactions in PNA synthesis are described along with procedures for reducing their impact on PNA synthesis.
Chapter 3
Synthesis of PNA Oligomers by Fmoc Chemistry
Ralph Casale, Ivar S. Jensen and Michael Egholm
PNA synthesis using Fmoc/Bhoc protected monomers is described. The milder chemistry of this synthesis scheme provides for PNAs carrying sensitive reporter groups and the preparation of PNA-conjugates. Procedures for analysis and purification of PNA are also detailed.
Chapter 4
Thermodynamics of PNA Interactions
Tommi Ratilainen, Anders Holmén and Bengt Nordén
A theoretical framework and a practical guidance are given here for characterizing thermodynamic parameters of PNA interacting with nucleic acids or itself. The van't Hoff analysis based on absorbance melting curves is concluded generally reliable for many purposes, but involves assumptions that may cause problems. Here, calorimetric measurements provide an independent check of H: the isothermal titration (ITC) and differential scanning calorimetry (DSC) methods are reviewed and their respective advantages and disadvantages. By and large, the thermodynamics of PNA resembles that of natural nucleic acids with respect to electrostatic and ionic contributions to enthalpy and entropy as well as effects of mismatch and neighboring bases. However, more detailed studies may reveal subtle differences due to variations in solvent structure, and of hydrogen bonding of backbone.
Chapter 5
Peptide Nucleic Acid Targeting of Double-Stranded DNA
Thomas Bentin, H. Jakob Larsen and Peter E. Nielsen
Peptide nucleic acid (PNA) recognition of nucleic acids occurs by a variety of mechanisms depending on the PNA sequence and nucleic acid target composition. Targeted binding to double-stranded DNA is primarily accomplished by strand-invasion, a mechanism that involves the local dissociation of the two complementary DNA strands and binding of the sequence complementary PNA oligomer(s) to one or both of the exposed strands. The resulting "invasion" complexes display a number of extraordinary qualities that have inspired a number of applications including gene targeting. Various chemical and enzymatic techniques exist to characterize such PNA complexes and these are presented in the chapter.
Chapter 6
PNA Beacons and Fluoroprobes
Bhaskar Datta, Miaomiao Wang and Bruce A. Armitage
The use of PNA probes to detect specific sequences of DNA has benefited from the discovery that cationic cyanine dyes can bind tightly to PNA-DNA hybrids and yield an instantaneous, visible color change, independent of base sequence. Alternatively, an anionic cyanine dye is bound to a PNA hairpin in a manner that results in quenching of the dye fluorescence. Upon hybridization of the PNA probe to its DNA complement, the dye is released into solution, giving a significant increase in fluorescence. This chapter provides procedures for two simple assays based on colorimetric and fluorimetric detection of PNA-DNA hybrids using cyanine dyes.
Chapter 7
Vector Tagging via PNA
Alex G. Rebuffat, Alessio G. Bernasconi and Brigitte M. Frey
We describe a novel non-viral approach named steroid-mediated gene delivery (SMGD) with the aim to facilitate the nuclear import of transfected DNA with the help of steroid receptors, which are natural cytoplasm-nuclear shuttles. In this study the SMGD strategy was modeled with the Glucocorticoid Receptor (GR) system. The initial step was to chemically derivatize steroids to allow their interaction with plasmid DNA while maintaining their affinity and accessibility for the GR. With this aim, we have synthesized and tested several bifunctional steroid derivatives* and selected finally the compound named DEX-bisPNA. This molecule consists of a dexamethasone backbone linked to a peptide nucleic acid clamp (bisPNA) moiety with a 9 C-atom chemical spacer.
Dex-bisPNA is shown to bind to GR efficiently and to reporter plasmids containing PNA-binding sites only. Most importantly, the expression of Dex-bisPNA reporter plasmid transiently transfected into dividing cells was reproducibly enhanced. This enhanced expression occurred in E8.2T4/GR cells stably transfected with GR only, but not in GR-negative E8.2T4 cells. Such an enhancement was also observed in aphidicolin treated growth arrested E8.2T4/GR cells.
Chapter 8
PCR Clamping
Henrik Ørum
An efficient, PCR based method for the selective amplification of DNA target sequences that differs by as little as a single base pair is described. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and the fact that PNA cannot function as primers for DNA polymerases.
Chapter 9
PNA Directed Genome Rare Cutting: New Developments
Maxim D. Frank-Kamenetskii and Vadim V. Demidov
This chapter deals with the PNA-assisted rare cleavage (PARC) of duplex DNA. The technique is a variety of the general 'Achilles' heel cleavage strategy and uses PNA oligomers to protect very few sites on genomic DNA against enzymatic methylation. As a result, the PARC technique makes it possible to convert common restriction enzymes into a pool of infrequent genome cutters. These artificial genome cutting systems cover the range of recognition specificities, where very few, if any, cutters are now available. Here, we present the PARC-based method for robust purification of yeast artificial chromosomes (YACs) from host chromosomes with similar lengths and demonstrate the PARC potential for rare fragmentation of human DNA. Further progress in the PARC approach also includes the use of pseudocomplementary PNAs (pcPNAs) as sequence-unrestricted duplex DNA-binding ligands.
Chapter 10
PNA Openers for Duplex DNA: Basic Facts, Fine Tuning and Emerging Applications
Vadim V. Demidov
This chapter describes the concept and exemplary applications of PNA 'openers', i.e. short pyrimidine bis-PNAs employed for local exposing of a chosen site on double-stranded (ds) DNA. Use of these biomolecular tools forms the basis of the emerging PD-loop technology, which deals with so-called PD-loops and related structures. The methods discussed include the rolling-circle amplification (RCA) of 'topological' DNA probes, hybridization of molecular beacons to dsDNA and duplex DNA capture.
Chapter 11
Combined Use of PNA and DNA for Fluorescence In Situ Hybridization (FISH)
Kirsten Vang Nielsen, Sven Müller, Tim Svenstrup Poulsen, Susanne Gabs, and Andreas Schønau
The successful use of large genomic probes for fluorescence in situ hybridization (FISH) is dependent on blocking of the undesired background staining derived from repetitive sequences that are present throughout the genome. Traditionally, this has been achieved by using a component of the total genomic DNA, Cot-1 DNA, enriched with repetitive sequences. Within and around the genes, the most frequent repetitive element is the Alu sequence. By selection of specific PNA oligos, directed towards the Alu sequences, an effective suppression of the background has been achieved. The selected Alu PNA Mixture can be used in protocols with a variety of different probes. Also, it can be used in combination with labeled PNA probes targeting other repetitive sequences in the genome, e.g. the centromeres. The suppression method can be used to analyze cells for the occurrence of chromosomes, chromosome fragments, genes, or chromosome aberrations (e.g. translocations, deletions, amplifications, insertions, inversions) associated with a condition or disease. Any method that can detect, identify and/or quantify selected target genomic nucleic acid in metaphase spreads, interphase nuclei, tissue sections, and extracted DNA from these cells can potentially take advantage of the present method as a substitute for the conventional Cot-1 DNA blocking. These methods include CISH (chromogenic in situ hybridization), FISH, multi-color FISH, Fiber-FISH, CGH, chromosome paints and arrays.
Chapter 12
Production of PNA-Arrays for Nucleic Acid Detection
Anette Jacob, Ole Brandt, Simone Würtz, Achim Stephan, Martina Schnölzer and Jörg D. Hoheisel
In recent years, the use of microarray technologies for the analysis of nucleic acids has become routine, and much more is still to come. Because of the special features of PNA-DNA interaction, the use of PNA-oligomers as chip-bound receptors could be advantageous for some applications. In this chapter, procedures for the production of PNA-microarrays are presented. Arrays were produced either by direct in situ synthesis or by a two-step process that consists of a highly parallel, fully automated synthesis of PNA-molecules and their subsequent attachment to the microarray-surface. Currently, the latter technique permits higher densities and allows for sample purification during the spotting. Synthesis occurs in resin-filled micro-well plates followed by a release from the support and spotting of the crude products to glass slides or silicon wafers. Selective binding via the amino-ends provides a means for a simple and effective purification of the synthesis products. Only full-length molecules are bound resulting in high-quality microarrays. Here, detailed synthesis protocols based on Fmoc chemistry are presented for both the in situ and the micro-well synthesis technique. MALDI-TOF mass spectrometry and HPLC were applied for quality control. The utility of the different procedures and the influence of parameters that are critical for production and application of PNA-microarrays are discussed.
Chapter 13
Antisense in Eukaryotic Systems
David R. Corey
In this chapter I describe a simple protocol for introducing PNAs into cultured mammalian cells. I then present a second protocol that exploits intracellular delivery of antisense PNAs to inhibit gene expression. These two protocols enable a wide range of experiments designed to explore the control of biological processes by PNAs.
Chapter 14
Antisense Effects in Escherichia coli
Liam Good, Rikard Dryselius and Peter E. Nielsen
Antisense peptide nucleic acid (PNA) can enter bacteria and control gene expression and even cell growth when targeted against essential genes. Standard PNAs are typically very effective in vitro, however, modifications are needed to obtain potent effects in bacteria. PNAs can be attached to cell wall active peptides to confer improved uptake and antisense properties. Antisense peptide-PNAs provide a new approach to control bacterial gene expression for gene functional studies and anti-infectives development.
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