Europium-Labeled Oligonucleotide Hybridization Probes: Preparation and Properties

Dahlén, Patrik; Liukkonen, Liisa; Kwiatkowski, Marek; Hurskainen, Pertti; Iitiä, Antti; Siitari, Harri; Ylikoski, Jyrki; Mukkala, Veli-Matti; Lövgren, Timo

doi:10.1021/bc00027a013

Cited by 23 publications

(12 citation statements)

References 25 publications

(35 reference statements)

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“…The europium-labeled hybridization probe was able to detect as few as 5 × 10 6 molecules (8 attomoles) of the artificial telomerase product β-TSTP. Similar sensitivity with europium-labeled probes has been reported for the detection of other oligonucleotides [Dahlén et al, 1994]. The amplification and detection could be conveniently performed in 5 h; alternatively, after PCR plates could be stored and products detected Fig.…”

Section: Resultssupporting

confidence: 56%

Identification of a series of potent telomerase inhibitors using a time-resolved fluorescence-based assay

Bare¹,

Trinh²,

Wu³

et al. 1998

Drug Dev. Res.

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Section: Resultssupporting

confidence: 56%

Identification of a series of potent telomerase inhibitors using a time-resolved fluorescence-based assay

Bare¹,

Trinh²,

Wu³

et al. 1998

Drug Dev. Res.

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“…In this report, we characterized a newly developed lanthanide chelate, the Eu-DTPA-cs124 Selvin, 1995, 1997;Selvin et al, 1996). This and related compounds are not only good potential labels for time-resolved imaging microscopy (Dahlen et al, 1994;de Haas et al, 1996;Seveus et al, 1992) but are also candidates for FRET measurements in the microscope used to estimate molecular proximity (Förster, 1946;Jovin and Arndt-Jovin, 1989;Selvin, 1995;Szöllösi et al, 1984). Although there are examples of LRET determinations using long-lived fluorophores in cuvette (Mathis, 1995;Youn et al, 1995), no microscope-based applications have been reported to date.…”

Section: Discussionmentioning

confidence: 99%

“…Lanthanide chelates are luminescent compounds with lifetimes on the order of 10 -2000 s. These long-lived probes are better suited for time-resolved microscopy than compounds emitting from the triplet state because they have a high quantum yield and are generally insensitive to oxygen. They have been successfully employed in immunocytochemistry (Seveus et al, 1992), in situ hybridization (Dahlen et al, 1994;de Haas et al, 1996;Seveus et al, 1992), and gene analysis . Timeresolved fluoroimmunoassays employing lanthanide chelates as labels have also been developed for in vitro diagnostics (Altamirano-Bustamante et al, 1991;Barnard et al, 1989;Ci et al, 1995; for a review see Dickson et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Temporally and Spectrally Resolved Imaging Microscopy of Lanthanide Chelates

Vereb

Jares‐Erijman

Selvin

et al. 1998

Biophysical Journal

154

152

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The combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal. Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and rotational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope.

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“…For oligonucleotides (approximately 20-mer) end-labeled with about 20 Eu-chelates, DELFIA detection in a hybridizationcapture assay led to a sensitivity of target molecules, or 17 attomoles. 32 Among directly luminescent chelates, Savitsky et al, in a brief report, claimed equal sensitivity to the DELFIA system. 25 The detection limit for the free terpyrdine complex of Saha et al was reported to be moles (presumably in or 0.3 pM) and moles in 100 mL (1.5 pM) when bound to DNA.…”

Section: Sensitivitymentioning

confidence: 99%

Lanthanide-Labeled DNA

Selvin¹

Topics in Fluorescence Spectroscopy

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Lanthanide ions can have highly unusual emission characteristics in aqueous solution, including a long (millisecond) excited-state lifetime, sharply spiked emission spectra (<10 nm width), and a large Stokes shift (>150 nm). These characteristics, when using pulsed excitation in combination with time-delayed and wavelength-filtered detection, are advantageous for discriminating against background fluorescence, which tends to be short lived (primarily nanosecond) and broadly spread in wavelength. For this reason, lanthanide ions are of significant interest as alternatives to conventional fluorophores, particularly when autofluorescence is a problem. This is particularly true in high-throughput screening assays for drug development where autofluorescence commonly limits sensitivity with conventional probes and radioactivity has undesirable environmental, health, and cost considerations. Detection sensitivity of M can be achieved with lanthanides, exceeding sensitivity achievable with conventional fluorophores and approaching or equalling radioactivity. A number of companies have commercially available lanthanide-based assays although availability of the chelates remains an issue for many university researchers.A second area of practical and fundamental interest is the use of lanthanide ions as donors in resonance energy transfer studies for the detection of binding between biomolecules or the measurement of nanometer-scale distances within and between biomolecules. It has recently been realized that lanthanide ions make excellent donors in energy transfer experiments, enabling distances up to 100 Å feasible with greatly improved accuracy compared to conventional fluorescent probes. Lanthanide-based resonance energy transfer (LRET) has been applied in both basic and applied studies, including DNA and DNA-protein complexes. Very recently, it has been realized that LRET can lead to new classes of DNA-dyes with tuneable excited-state lifetimes in the 10-500 msec time regime and tunable color

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Europium-Labeled Oligonucleotide Hybridization Probes: Preparation and Properties

Cited by 23 publications

References 25 publications

Identification of a series of potent telomerase inhibitors using a time-resolved fluorescence-based assay

Identification of a series of potent telomerase inhibitors using a time-resolved fluorescence-based assay

Temporally and Spectrally Resolved Imaging Microscopy of Lanthanide Chelates

Lanthanide-Labeled DNA

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