High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler

Darby, Richard A. J.

doi:10.1093/nar/30.9.e39

Cited by 152 publications

(118 citation statements)

References 27 publications

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“…This second transition is less pronounced for the more stable triplexes (those containing C + GC triplets) and is not observed with the intermolecular triplexes (see below). We have suggested previously that this second transition arises from melting of the underlying duplex [34], since the time-averaged distance between the fluorophore and quencher is greater for the partially melted triplex than for the fully melted random coil. When the third strand dissociates the remaining duplex will be relatively rigid and hold the fluorophore and quencher apart.…”

Section: Fluorescence Melting Studiesmentioning

confidence: 89%

See 1 more Smart Citation

DNA sequence specificity of triplex‐binding ligands

Keppler

James

Neidle

et al. 2003

European Journal of Biochemistry

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We have examined the ability of naphthylquinoline, a 2,7-disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. 1Fluorescence melting studies, with both inter-and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC) n or (CCT) n , but stabilizes triplexes that contain (TTC) n . In contrast, BePI slightly destabilizes the triplexes that are formed at (TC) n (CCT) n and (TTC) n . 2,7-Anthraquinone stabilizes (TC) n (CCT) n and (TTC) n , although it has the greatest effect on the latter. DNase I footprinting studies confirm that triplexes formed with (CCT) n are stabilized by the 2,7-disubstituted amidoanthraquinone but not by naphthylquinoline. Both ligands stabilize the triplex formed with (CCTT) n and neither affects the complex with (CT) n . We suggest that BePI and naphthylquinoline can only bind between adjacent TAT triplets, while the anthraquinone has a broader sequence of selectivity. These differences may be attributed to the presence (naphthylquinoline and BePI) or absence (anthraquinone) of a positive charge on the aromatic portion of the ligand, which prevents intercalation adjacent to C + GC triplets. The most stable structures are formed when the stacked rings (bases or ligand) alternate between charged and uncharged species. Triplexes containing alternating C + GC and TAT triplets are not stabilized by ligands as they would interrupt the alternating pattern of charged and uncharged residues.Keywords: anthraquinone; molecular beacon; naphthylquinoline; triple helix; triplex-binding ligand.The formation of intermolecular DNA triple helices offers a means for designing agents which can bind to specific DNA sequences [1][2][3][4][5][6]. In this approach, a third strand oligonucleotide binds in the major groove of duplex DNA, forming specific hydrogen bond contacts with substituents on the purines of the duplex base pairs. In these structures the third strand can run either parallel or antiparallel to the duplex purine strand. Parallel triplexes, which have been most widely studied, consist of TAT and C + GC triplets, while the antiparallel motif contains GGC and AAT or TAT triplets. Although triplex-forming oligonucleotides bind to their duplex targets with considerable sequence selectivity, their binding may not be strong. Several strategies have therefore been used to improve their affinity, including the use of base and backbone analogues [7][8][9][10], and tethering DNA binding agents such as acridine [11,12] or psoralen [13] to the end of the oligonucleotide. An alternative strategy uses ligands which bind selectively to triplex (not duplex) DNA and which therefore perturb the equilibrium in the direction of triplex formation. Several such ligands have been described (reviewed in [14]) including 3-methoxy-7H-8-methyl-11 [(3¢-amino) [27,28]. Although most studies with the...

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Section: Fluorescence Melting Studiesmentioning

confidence: 89%

“…Fluorescence melting profiles were determined by using a Roche LightCycler as described previously [34]. The principle of these experiments is that when a triplex is formed the fluorophore and quencher are in close proximity and the fluorescence is quenched.…”

Section: Fluorescence Melting Studiesmentioning

confidence: 99%

DNA sequence specificity of triplex‐binding ligands

Keppler

James

Neidle

et al. 2003

European Journal of Biochemistry

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“…Triplex-based fluorescent probes which exhibit changes in fluorescence intensities or wavelengths upon hybridization with a double-stranded DNA have been reported to detect a DNA triplex formation or facilitate the analysis of the thermal stability of DNA triplexes. [6][7][8][9][10][11][12][13][14] Although the probes utilizing Förster resonance energy transfer (FRET) such as molecular beacons could control the emission of the fluorescence, they normally require two kinds of dyes, a fluorophore and quencher, and an extra structure in addition to the sequencerecognition moiety. [6][7][8][9] In contrast, the probes labeling with a single kind of dye have advantages in the facile synthesis and design.…”

Section: Introductionmentioning

confidence: 99%

“…[6][7][8][9][10][11][12][13][14] Although the probes utilizing Förster resonance energy transfer (FRET) such as molecular beacons could control the emission of the fluorescence, they normally require two kinds of dyes, a fluorophore and quencher, and an extra structure in addition to the sequencerecognition moiety. [6][7][8][9] In contrast, the probes labeling with a single kind of dye have advantages in the facile synthesis and design. [10][11][12][13][14][15] Pyrimidine TFOs labeled with thiazole orange (TO) exhibited comparably larger changes in fluorescence intensities before and after triplex formation 14,15 because the fluorescence intensity of TO molecules is low in the presence of poly-pyrimidine singlestranded DNA 16 and high in the presence of DNA triplexes.…”

Section: Introductionmentioning

confidence: 99%

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

Ikeda

Yanagisawa

Yuki

et al. 2013

Artificial DNA: PNA & XNA

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“…[30][31][32] We have included three native G-quadruplex-forming sequences in this study: the human telomeric DNA quadruplex sequence H-telo 5 0 -GGG(TTAGGG) 3 -3 0 , the two c-Kit promoter G-quadruplex sequences of c-Kit 2 17 5 0 -GGG CGG GCG CGA GGG AGG GG-3 0 and c-Kit 1 15 5 0 -GGG AGG GCG CTG GGA GGA GGG-3 0 , all of which are dual labeled (5 0 -FAM and 3 0 -TAMRA). We also included a dual-labeled duplex DNA as a control (see the Experimental Procedures).…”

mentioning

confidence: 99%

Targeting the c-Kit Promoter G-quadruplexes with 6-Substituted Indenoisoquinolines

Bejugam

Gunaratnam

Müller

et al. 2010

ACS Med. Chem. Lett.

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Herein, we demonstrate the design, synthesis, biophysical properties, and preliminary biological evaluation of 6-substituted indenoisoquinolines as a new class of G-quadruplex stabilizing small molecule ligands. We have synthesized 6-substituted indenoisoquinolines 1a-e in two steps from commercially available starting materials with excellent yields. The G-quadruplex stabilization potential of indenoisoquinolines 1a-e was evaluated by fluorescence resonance energy transfer-melting analysis, which showed that indenoisoquinolines show a high level of stabilization of various G-quadruplex DNA structures. Indenoisoquinolines demonstrated potent inhibition of cell growth in the GIST882 patient-derived gastrointestinal stromal tumor cell line, accompanied by inhibition of both c-Kit transcription and KIT oncoprotein levels.KEYWORDS DNA, quadruplex, c-kit regulation, inhibition, ligand G -quadruplexes constitute a structural form of DNA that is distinct from the double helix. 1 They are formed from particular guanine-rich nucleic acid sequences in which guanines form planar quartets stabilized using the Watson-Crick and Hoogsteen edges to form hydrogen bonds. Their occurrence has been demonstrated within ciliate telomeres. 2,3 In addition, G-quadruplex sequence motifs (G 3þ N 1-7 G 3þ N 1-7 G 3þ N 1-7 G 3þ ) are found at many positions within genomes, 4,5 with a notable increase in density in close vicinity to transcriptional start sites, highly suggestive of a biological function in gene regulation. 5,6 NMR spectroscopy and X-ray crystallographical studies have shown that these guanine-rich sequences can fold into G-quadruplex structures in vitro. 7,8 Their unique structural features and possible biological functions make them attractive targets for drug design. 9,10 Genomic locations that are known to possess quadruplex structures include the immunoglobulin switch region and the promoters of numerous proto-oncogenes, such as c-MYC, 11 VEGF, 12 BCL-2, 13 and K-RAS. 14 Two quadruplex-forming motifs have been identified in the c-Kit promoter (c-Kit 1 15,16 and c-Kit 2 17,18 ). Here, we present a new class of quadruplex stabilizing compounds that have been used to explore relationships between quadruplexes and their biological functions.c-Kit is a proto-oncogene that codes for a 145-160 kDa protein belonging to the receptor tyrosine kinase (RTK) family. The KIT protein regulates signal transduction cascades that control cell growth and differentiation. 19 c-Kit expression has been shown to control the growth of various cell lines, including gastrointestinal stromal tumors (GIST), hematopoietic cells, small cell lung cancer, and colorectal cancer. [20][21][22][23] Gain-of-function mutations in the KIT protein are found in various highly malignant human cancers, especially GIST. Gleevec (imatinib mesylate), a small molecule antagonist originally developed to treat chronic myelogenous leukemia (CML), acts by maintaining the bcr-abl kinase in an inactive conformation. Gleevec is also the mainstay of target therapy ...

show abstract

High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler

Cited by 152 publications

References 27 publications

DNA sequence specificity of triplex‐binding ligands

DNA sequence specificity of triplex‐binding ligands

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

Targeting the c-Kit Promoter G-quadruplexes with 6-Substituted Indenoisoquinolines

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