Detection of Hybrid Formation between Peptide Nucleic Acids and DNA by Fluorescence Polarization in the Presence of Polylysine

Nikiforov, Theo T.; Jeong, Sang Hoon

doi:10.1006/abio.1999.4338

Cited by 17 publications

(10 citation statements)

References 25 publications

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“…PNA is a hydrophobic species and the formation of self-aggregates has been suggested (17). PNA interactions with complementary DNA have recently been studied with fluorescence polarization (28). While complementary PNA-DNA duplexes show little change in fluorescence polarization, the addition of a high molecular weight polymer like polylysine will interact with the nucleic acid and significantly alter the rotational correlation time (28).…”

Section: Pna Forms Non-watson-crick Interactions With Ssdnamentioning

confidence: 99%

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Tackett

Corey²,

Raney

2002

Nucleic Acids Research

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Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson-Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications.

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Section: Pna Forms Non-watson-crick Interactions With Ssdnamentioning

confidence: 99%

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Tackett

Corey²,

Raney

2002

Nucleic Acids Research

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“…27,28 For this reason, PNA has found a number of applications in molecular biology where there is a need to design probes that bind strongly to a complementary DNA target whilst keeping the probe as short as possible. 29,30 The properties of PNA mean that it has received extensive attention as a probe molecule in DNA detection assays, including those using electrochemical methods. [31][32][33] Despite widespread use, there are few studies of the underlying electrochemistry of PNA, 31,[34][35][36] and, to the best of our knowledge, electrochemical unwinding and denaturation of PNA/PNA duplexes and DNA/PNA hybrids under an applied potential has not previously been observed.…”

Section: Introductionmentioning

confidence: 99%

Denaturation of dsDNA immobilised at a negatively charged gold electrode is not caused by electrostatic repulsion

et al. 2013

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Double-stranded DNA immobilised through a thiol anchor at a gold electrode surface can be unwound and denatured by applying a negative potential. One proposed mechanism for this electrochemical denaturation is that electrostatic field effects are responsible for the destabilisation of the dsDNA through repulsion of the DNA sugar-phosphate backbone away from the electrode surface. Herein, we demonstrate conclusively that electrochemical melting at gold electrodes cannot be explained solely as a simple repulsion mechanism by showing that immobilised DNA denatures at high ionic strengths, where the DNA base-pairs are situated outside of the electrochemical double-layer (and outside the influence of the electric field), and further, that oligomers comprised of the mimic peptide nucleic acid (PNA) can also be denatured at negative potentials, despite the absence of a negatively charged backbone

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“…They are easily manufactured and modified and have good hybridization properties compared with DNA oligos. PNA has been used in a handful of SNP genotyping assays (13)(14)(15)(16)(17)(18)(19) and as RNA capture probe coupled to the surface of microspheres (25).…”

Section: Discussionmentioning

confidence: 99%

“…We recently reported that short PNA beacons can successfully replace DNA-based probes in two real-time polymerase chain reaction (PCR) genotyping assays (13). Other groups have reported PNA-based allele discrimination using mass spectroscopy (14 -16), quartz crystal microbalance biosensors (17), surface plasmon resonance (18), or fluorescence polarization (19).…”

mentioning

confidence: 99%

SNP genotyping using microsphere‐linked PNA and flow cytometric detection

Rockenbauer

Petersen²,

Vogel

et al. 2005

Cytometry Pt A

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Background: Single nucleotide polymorphisms (SNPs) represent the most frequent form of genetic variations. Some of the most sensitive methods for SNP genotyping employ synthetic oligonucleotides, such as the peptide nucleic acid (PNA). We introduce a new method combining allele-specific hybridization, PNA technology, and flow cytometric detection. We tested the design by genotyping a Danish basal cell carcinoma cohort of 80 individuals for an A/C SNP in exon 6 of the XPD gene. Methods: Genomic DNA was amplified by a two-step polymerase chain reaction (PCR) in the presence of fluorescein-dyed primers and fluorescein-12-dUTP. The allelespecific PNA molecules were covalently coupled to carboxylated microspheres with and without rhodamine. Allele-specific hybridization between PCR products and

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Detection of Hybrid Formation between Peptide Nucleic Acids and DNA by Fluorescence Polarization in the Presence of Polylysine

Cited by 17 publications

References 25 publications

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Denaturation of dsDNA immobilised at a negatively charged gold electrode is not caused by electrostatic repulsion

SNP genotyping using microsphere‐linked PNA and flow cytometric detection

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