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Characterization of a K+-induced conformational switch in a human telomeric DNA oligonucleotide using 2-aminopurine fluorescence.

ABSTRACT: Human telomeric DNA consists of tandem repeats of the DNA sequence d(GGGTTA). Oligodeoxynucleotide telomere models such as d[A(GGGTTA)(3)GGG] (Tel22) fold in a cation-dependent manner into quadruplex structures consisting of stacked G-quartets linked by d(TTA) loops. NMR has shown that in Na(+) solutions Tel22 forms a "basket" topology of four antiparallel strands; in contrast, Tel22 in K(+) solutions consists of a mixture of unknown topologies. Our previous studies on the mechanism of folding of Tel22 and similar telomere analogues utilized changes in UV absorption between 270 and 325 nm that report primarily on G-quartet formation and stacking showed that quadruplex formation occurs within milliseconds upon mixing with an appropriate cation. In this study, we assess the dynamics and equilibria of folding of specific loops by using Tel22 derivatives in which the dA residues were serially substituted with the fluorescent reporter base, 2-aminopurine (2-AP). Tel22 folding induced by Na(+) or K(+) assessed by changes in 2-AP fluorescence consists of at least three kinetic steps with time constants spanning a range from milliseconds to several hundred seconds. Na(+)-dependent equilibrium titrations of Tel22 folding could be approximated as a cooperative two-state process. In contrast, K(+)-dependent folding curves were biphasic, revealing that different conformational ensembles are present in 1 and 30 mM K(+). This conclusion was confirmed by (1)H NMR. Molecular dynamics simulations revealed a K(+) binding pocket in Tel22 located near dA1 that is specific for the so-called hybrid-1 conformation in which strand 1 is in a parallel arrangement. The possible presence of this topologically specific binding site suggests that K(+) may play an allosteric role in regulating telomere conformation and function by modulating quadruplex tertiary structure.

SUBMITTER: Gray RD

PROVIDER: S-EPMC2803354 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Characterization of a K+-induced conformational switch in a human telomeric DNA oligonucleotide using 2-aminopurine fluorescence.

Gray Robert D RD Petraccone Luigi L Trent John O JO Chaires Jonathan B JB

Biochemistry 20100101 1

Human telomeric DNA consists of tandem repeats of the DNA sequence d(GGGTTA). Oligodeoxynucleotide telomere models such as d[A(GGGTTA)(3)GGG] (Tel22) fold in a cation-dependent manner into quadruplex structures consisting of stacked G-quartets linked by d(TTA) loops. NMR has shown that in Na(+) solutions Tel22 forms a "basket" topology of four antiparallel strands; in contrast, Tel22 in K(+) solutions consists of a mixture of unknown topologies. Our previous studies on the mechanism of folding o ...[more]

PMID: 19961180

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Project description:BackgroundRapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.Methods and resultsHere we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.ConclusionOverall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.

Dataset Information

Characterization of a K+-induced conformational switch in a human telomeric DNA oligonucleotide using 2-aminopurine fluorescence.

Publications

Characterization of a K+-induced conformational switch in a human telomeric DNA oligonucleotide using 2-aminopurine fluorescence.

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