Sequence‐Unrestricted, Watson–Crick Recognition of Double Helical B‐DNA by (<i>R</i>)‐MiniPEG‐γPNAs

Bahal, Raman; Sahu, Bichismita; Rapireddy, Srinivas; Lee, Chong-Min; Ly, Danith H.

doi:10.1002/cbic.201100646

Cited by 79 publications

(86 citation statements)

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“…However, previous studies have shown that γPNA affinity for complementary DNA is sufficiently high to allow direct invasion of double-stranded DNA in order to access target sites. 15 Therefore, we hypothesized that the denaturing step could be omitted when using the γ P 12 probe to stain the metaphase chromosome preparations. To test this, we compared unmodified P 18 and γ P 12 under non-denaturing hybridization conditions, in which formamide was omitted from the hybridization buffer and heating and RNAse A treatment steps were deleted from the protocol.…”

Section: Resultsmentioning

confidence: 99%

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Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis

et al. 2014

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GammaPNA oligomers having one or two repeats of the sequence AATCCC were designed to hybridize to DNA having one or more repeats of the complementary TTAGGG sequence found in the human telomere. UV melting curves and surface plasmon resonance experiments demonstrate high affinity and cooperativity for hybridization of these miniprobes to DNA having multiple complementary repeats. Fluorescence spectroscopy for Cy3-labeled miniprobes demonstrate increases in fluorescence intensity for assembling multiple short probes on a DNA target compared with fewer longer probes. The fluorescent γPNA miniprobes were then used to stain telomeres in metaphase chromosomes derived from U2OS cells possessing heterogeneous long telomeres and Jurkat cells harboring homogenous short telomeres. The miniprobes yielded comparable fluorescence intensity to a commercially available PNA 18mer probe in U2OS cells, but significantly brighter fluorescence was observed for telomeres in Jurkat cells. These results suggest that γPNA miniprobes can be effective telomere-staining reagents with applications toward analysis of critically short telomeres, which have been implicated in a range of human diseases.

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Section: Resultsmentioning

confidence: 99%

“…8, 11 The added affinity extends the strand invasion capability first reported for unmodified PNA 12, 13 to virtually any sequence of double-stranded DNA. 14, 15 …”

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confidence: 99%

Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis

et al. 2014

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“…However, they too require non-physiological salinity for optimal strand invasion. 12 In summary, oligonucleotide-based probes that recognize mixed-sequence dsDNA targets at physiologic conditions remain largely elusive.…”

Section: Introductionmentioning

confidence: 99%

Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2′-alkylated RNA monomers

et al. 2014

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Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and - more recently - engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2′-alkylated uridine monomers X–Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔTm/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics.

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“…In essence, this PNA structure, termed gPNA, is the first PNA oligomer to have sequenceunrestricted invasion capabilities while maintaining single base-pair specificity (Chenna et al, 2008;He, Rapireddy, Bahal, Sahu, & Ly, 2009). In addition, in order to address the issue of solubility using gPNAs, Sahu et al demonstrated that by incorporating, at the g-position of the backbone a diethylene glycol moiety, this results in a significant increase in solubility while not adversely affecting affinity or sequence specificity (Bahal, Sahu, Rapireddy, Lee, & Ly, 2012;Sahu et al, 2011).…”

Section: The Many Flavours Of Artificial Nucleic Acids; Different Strmentioning

confidence: 99%

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

Singer

Tang

2015

Methods in Microbiology

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Sequence‐Unrestricted, Watson–Crick Recognition of Double Helical B‐DNA by (R)‐MiniPEG‐γPNAs

Cited by 79 publications

References 50 publications

Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis

Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis

Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2′-alkylated RNA monomers

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

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