Peptide Nucleic Acids: Protocols and Applications Chapter Abstracts
Chapter 1
An Introduction to PNA
Peter E. Nielsen and Michael Egholm
This section provides a complete overview of the scientific theory and applications of PNA as well as comprehensive background information.
Chapter 2.1
PNA oligomer 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 2.2
Synthesis of PNA oligomers by Fmoc Chemistry
Ralph Casale, Ivar S. Jensen and Michael Egholm
PNA synthesis using Fmoc/Bhoc protected monomers on a common DNA synthesizer, Expedite 8909, is described. The milder chemistry of this synthesis scheme provides for PNAs carrying sensitive reporter groups and the preparation of PNA-conjugates. Procedures for labeling, analysis, and purification of PNA are also detailed.
Chapter 2.3
PNA/DNA Chimeras
Eugen Uhlmann, Beate Greiner and Gerhard Breipohl
A convenient method for the solid-support synthesis of PNA/DNA chimeras is described which makes use of monomethoxytrityl/acyl-protected monomeric building blocks. The acid-labile monomethoxytrityl (Mmt) group is employed for the temporary protection of the amino function of aminoethylglycine, while the exocyclic amino functions of the nucleobases are protected with ammonia-cleavable acyl protecting groups. This orthogonal protecting-group strategy is fully compatible with the standard phosphoramidite DNA synthesis method. The resulting PNA/DNA chimeras obey the Watson-Crick rules on binding to complementary DNA and RNA. Binding affinity of the PNA-DNA chimeras strongly depends on the PNA:DNA ratio. The PNA/DNA chimeras bind with higher affinity to RNA than to DNA, and the type of linking moiety between PNA and DNA could be adjusted to obtain optimal binding affinity. In addition to their promising binding properties, PNA-DNA chimeras can also assume biological functions, such as a primer function for DNA polymerases. Pure PNAs cannot induce RNase H cleavage of target RNA, which often supports the biological efficacy of antisense agents. In contrast, the DNA-PNA chimeras are able to stimulate cleavage of the target RNA by RNase H on formation of a RNA·chimera duplex.
Chapter 2.4
Synthesis of PNA-DNA chimera by MMT Chemistry
Ravi Vinayak
Automated chemical synthesis of PNA, 5'DNA3'-PNA and PNA-5'DNA3'chimera using the monomethoxytrityl / acyl protecting group strategy is described. A base-labile solid support and appropriately protected PNA building blocks allow a synthetic strategy for construction of PNA similar to standard DNA synthesis conditions.
Chapter 2.5
Labeling of PNA
Henrik Ørum, Ralph Casale and Michael Egholm
Procedures for non-radioctive labeling of PNAs with biotin, fluorescein, rhodamine a.o. are given both using on-resin solid phase coupling and solution phase post-modification. Furthermore, a method for 32P-labeling of PNA-peptide chimeras containing the Kemptide sequence motif (H-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH2) using protein kinase A (PKA) and g-32P is described.
Chapter 3.1
Thermodynamics of PNA-nucleic acid interactions
Anders Holmén and Bengt Nordén
An introduction to the practical use of absorbance melting curves for determination of thermodynamic parameters of PNA hybridisation is given. A number of potential complications are considered, including how they should be identified and potentially eliminated. The method of using isothermal titration calorimetry for determining enthalpy of hybridisation is also illustrated.
Chapter 3.2
Application of Peptide Nucleic Acid Probes for in situ Hybridization
M. Thisted, T. Just, K.-J. Pluzek, J.J. Hyldig-Nielsen, K.V. Nielsen, T. A. Mollerup, H. Stender, O.F. Rasmussen, K. Adelhorst, and S.E. Godtfredsen
In situ hybridization based techniques are gaining increasing importance in a wide variety of areas such as molecular biology, microbiology, histochemistry, cytogenetics, cytochemistry and others. Over the latest years the use of PNA probes for in situ hybridization in these areas have been explored by several groups. These investigations consistently show that PNA probes are superior to traditional oligonucleotide probes. The unique physico-chemical properties of PNA probes translate into unique behaviour in in situ hybridization that enable design of sensitive, robust and user-friendly protecols.
Chapter 3.3
PNA-Arrays for Nucleic Acid Detection
S. Matysiak, S. Würtz, N. C. Hauser, H. Gausepohl and J. D. Hoheisel
In recent years, the use of array technologies for the analysis of nucleic acids has taken a leap forward, with much more still to come. Apart from miniaturisation and other technical advances, also the chemistry of the production of such arrays is a focus of attention. Because of the unique features of PNA-DNA interaction, the use of arrayed PNA oligomers could be superior for such purposes in many respects. Here, the means and procedures for the in situ synthesis of PNA-arrays are described.
Chapter 3.4
PNA Blocker Probes Enhance Specificity in Probe Assays.
Mark J. Fiandaca, Jens J. Hyldig-Nielsen, and James M. Coull
Non-labeled PNA "blocker" probes were used to prevent mismatch hybridization of labeled probes to non-target sequences. The use of PNA blockers significantly decreased unwanted hybridization without a corresponding decrease in the sensitivity of detection of complementary targets. Furthermore, PNA probes and blockers provided higher signal to noise ratios than corresponding probes and blockers made of DNA. As a result, following PCR amplification, it was possible to detect a single base mutation in the K-ras gene at levels of only 1.5 copies per 100 copies of wild type DNA.
Chapter 3.5
MALDI-TOF Mass Spectrometric Detection of PNA Hybrids for Genetic Analysis
Timothy J. Griffin, Wei Tang and Lloyd M. Smith
An approach is described for the detection of single nucleotide polymorphisms in DNA, employing allele-specific, mass-labeled, PNA hybridization probes, and analysis by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). MALDI-TOF MS detection of the PNA probes produces composite mass spectra containing peaks of distinct masses corresponding to each allele present, resulting in a mass spectral "fingerprint" for each DNA sample. PNA oligomers offer unique advantages in their use as allele-specific hybridization probes and for their detection by MALDI-TOF MS. The hybridization characteristics of PNA-DNA duplexes were found to be highly dependent on both base content and sequence. The results presented here show the application of this approach to the analysis of single nucleotide polymorphisms contained in exon 4 of the human tyrosinase gene.
Chapter 3.6
PNA Biosensors for Nucleic Acid Detection
Joseph Wang
Biosensor devices, based on the conversion of nucleic acid recognition reactions into useful electrical signals, offer considerable promise for DNA diagnostics. The unique hybridization properties of solution-phase PNA can be extrapolated onto transducer surfaces in connection with the design of remarkably specific DNA biosensors. The present chapter reviews the development of PNA biosensors, and discusses common PNA-biosensing protocols along with their prospects in DNA biosensor technology.
Chapter 4.1
PNA directed genome rare cutting
Vadim V. Demidov and Maxim D. Frank-Kamenetskii
PNA-assisted rare cleavage (PARC) is based on the general 'Achilles' heel' cleavage strategy. The PARC technique makes it possible to convert usual restriction enzymes into infrequent genome cutters. In this method, a very stable and sequence-specific complex is formed between double-stranded genomic DNA and a cationic pyrimidine bis-PNA. Then the sample is treated with a DNA methyltransferase (methylase), the bis-PNA is removed from the DNA and the sample is treated with a restriction enzyme. The restriction enzyme recognizes the same sites as the methylase did and thus cannot cleave them. The only exceptions are very few non-methylated sites, which were protected against methylation by the bis-PNA overlapping the methylation sites. These rare sites become accessible for enzymatic recognition after PNA is removed. As a result, the restriction enzyme cuts the genomic DNA into a small number of fragments with lengths from several hundreds kbp to several Mbp. A pool of numerous combinations of various bis-PNAs with different methylation/restriction enzymatic pairs generates a new class of genome rare cutters. These cutters cover the range of recognition specificities, where very few, if any, cutters are now available. Biomolecular tools of that kind may find applications for processing chromosomes.
Chapter 4.2
Duplex DNA capture
Vadim V. Demidov, Nikolay O. Bukanov and Maxim D. Frank-Kamenetskii
This chapter describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC). The OPAC assay is based on selective tagging of a DNA duplex by biotinylated oligodeoxyribonucleotide (ODN) through formation of a so-called PD-loop. The PD-loop is assembled with the aid of a pair of PNA "openers", which allow sequence-specific targeting with a Watson-Crick complementary ODN probe in the exposed region of the dsDNA. The protocol involves three steps. First, two cationic bis-PNAs locally pry the DNA duplex apart at a predetermined site. Then, the exposed DNA single strand is targeted by a complementary biotinylated ODN to selectively form a stable PD-loop complex. Finally, the capture of dsDNA is performed using streptavidin covered magnetic beads. The OPAC procedure has many advantages in the isolation of highly purified native DNA over other affinity capture and amplification techniques.
Chapter 4.3
Purification of nucleic acids by hybridisation to affinity tagged PNA probes
Henrik Ørum
he use of affinity tagged PNA capture probes offers an efficient means for the purification of nucleic acids by hybridisation. Two different approaches are described. A sequence specific method and a generic method. The sequence specific method requires sequence information on the target and synthesis of a dedicated PNA. It can be used to selectively purify the nucleic acid containing the target from non-related nucleic acids and other cellular components. The generic method uses a "universal" triplex forming PNA and requires no sequence information on the target. It can be used in the bulk purification of large nucleic acids.
Chapter 4.4
PCR Clamping
Henrik Ørum
An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
Chapter 4.5
Plasmid Labeling using PNA
Xiaowu Liang, Olivier Zelphati, Cuong Nguyen, and Philip Felgner
In this chapter, we describe an effective approach using a peptide nucleic acid (PNA) 'clamp' to directly and essentially irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. As an example, we demonstrate the generation of a highly fluorescent preparation of plasmid DNA by hybridizing fluorescently labeled PNA to the plasmid. Fluorescent plasmid prepared in this way is neither functionally nor conformationally altered. The PNA clamp binding is sequencespecific, saturable, extremely stable, and does not influence the nucleic acid intracellular distribution. This method can be utilized to study the biodistribution of conformationally intact plasmid DNA in living cells after cationic lipid mediated transfection. A Rhodaminelabeled fluorescent plasmid expressing green fluorescent protein (GFP) enables simultaneous colocalization of both plasmid and expressed protein in living cells and in realtime.
Chapter 5.1
Antisense effects in Escherichia coli
Liam Good and Peter E. Nielsen
Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacterium Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can specifically limit gene expression, with gene and sequence specificity. For in vitro experiments, efficient inhibition is observed when using PNA concentrations in the nanomolar range, and for in vivo experiments the concentrations required are in the micromolar range. A mutant strain of E. coli that is more permeable to antibiotics is more susceptible to antisense PNAs than wildtype cells. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of specific antisense inhibition, we show the effects of an anti-ß-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake, antisense PNAs may find applications as antimicrobial agents and as a tool for microbial functional genomics.
Chapter 5.2
Characterization of PNA-dsDNA strand displacement complexes
H. Jakob Larsen and Peter E. Nielsen
This chapter describes the background, the protocols, and the limitations of the methods commonly used to examine PNA/DNA strand displacement complexes. The methods discussed include gel-mobility shift assay, chemical and enzymatic probing, and enzyme inhibition assays.
Chapter 5.3
Interactions of Peptide Nucleic Acids with DNA Processing Enzymes
Kevin D. Raney, Susan E. Hamilton, and David R. Corey
We describe two protocols for using PNAs to examine the action of enzymes that interact with DNA. In the first, we show how PNAs complementary to the RNA template of telomerase can be used to inhibit addition of telomeric repeats. In the second, we describe how PNAs can be used to probe substrate recognition by helicases. These protocols and the results obtained through their use support the conclusion that PNAs have important advantages for studying enzymatic activity.
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