Click Chemistry to Construct Fluorescent Oligonucleotides for DNA Sequencing

Seo, Tae Seok; Li, Zengmin; Ruparel, Hameer; Ju, Jingyue

doi:10.1021/jo026615r

Cited by 232 publications

(146 citation statements)

References 32 publications

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“…Current methods for DNA bioconjugation, however, are inefficient. The procedure must be able to tolerate aqueous conditions, give high yields, and the resulting linkage must be stable in biological conditions (124). This is the perfect situation for click chemistry.…”

Section: Labeling Dnamentioning

confidence: 99%

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Click Chemistry, A Powerful Tool for Pharmaceutical Sciences

2008

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Abstract. Click chemistry refers to a group of reactions that are fast, simple to use, easy to purify, versatile, regiospecific, and give high product yields. While there are a number of reactions that fulfill the criteria, the Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes has emerged as the frontrunner. It has found applications in a wide variety of research areas, including materials sciences, polymer chemistry, and pharmaceutical sciences. In this manuscript, important aspects of the Huisgen cycloaddition will be reviewed, along with some of its many pharmaceutical applications. Bioconjugation, nanoparticle surface modification, and pharmaceutical-related polymer chemistry will all be covered. Limitations of the reaction will also be discussed.

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Section: Labeling Dnamentioning

confidence: 99%

“…Utilizing the HDC reaction, Seo et al were able to tag a fluorophore to the 5′-end of single-stranded DNA (124). The oligonucleotide was modified, through several reactions, to display a terminal alkyne at its 5′-end and the fluorophore contained an azido functional group.…”

Section: Labeling Dnamentioning

confidence: 99%

Click Chemistry, A Powerful Tool for Pharmaceutical Sciences

2008

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“…Azido-labeling of this DNA molecule was achieved by reacting the above-described 5Ј-amino-modified DNA with succinimidyl 5-azidovalerate in 0.25 M Na 2 CO 3 ͞NaHCO 3 buffer (pH 9.0) for 12 h at room temperature. The resulting azido-labeled DNA was purified by size-exclusion chromatography and desalted with an oligonucleotide purification cartridge (24). The DNA product was analyzed by MALDI-TOF MS by using the amino-modified DNA as an internal standard.…”

Section: Synthesis Of the Crosslinker Succinimidyl N-propargyl Glumentioning

confidence: 99%

“…One ideal property required for the functional groups to be coupled (one from the DNA and the other from the surface) is the stability of the groups in aqueous conditions, which are typically needed to perform the coupling. We previously explored the use of the 1,3-dipolar cycloaddition click chemistry between an azide and alkyne for coupling a f luorophore [5-carboxyf luorescein (FAM)] to single-stranded DNA (24). This chemistry was shown to chemoselectively produce FAM-labeled DNA in a quantitative yield under aqueous conditions at 80°C.…”

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Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry

Seo

Bai

Ruparel

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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125

105

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DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3␣,4␣-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to sitespecifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence.1,3-dipolar azide-alkyne cycloaddition ͉ DNA chip ͉ DNA sequencing by synthesis ͉ 2-nitrobenzyl linker ͉ photocleavage T he completion of the Human Genome Project has set the stage for screening genetic mutations to identify disease genes on a genome-wide scale (1). Accurate high-throughput methods for resequencing the intron͞exon regions of candidate genes are needed to explore the complete human genome sequence for disease-gene discovery. Recent studies also have demonstrated that an important route for identifying functional elements in the human genome is to sequence the genomes of many species that represent a wide sampling of the evolutionary tree (2). To overcome the limitations of the current sequencing technology based on electrophoresis using laser-induced fluorescence detection (3-5), a variety of new DNA-sequencing methods have been explored. Such techniques include pyrosequencing (6), MS sequencing (7-9), sequencing by hybridization (10), sequence-specific detection of singlestranded DNA using engineered nanopores (11), and sequencing of single DNA molecules (12) and polymerase colonies (13).Recently, new DNA-sequencing approaches based on sequencing by syn...

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“…-The copper(I)-catalyzed Huisgen -Sharpless dipolar [2 þ 3] cycloaddition of azide and alkyne is a powerful direct method to prepare 1,4-disubstituted 1H-1,2,3-triazoles [1]. This reaction has been used increasingly in modular drug development, in the preparation of small-molecule radiopharmaceuticals, in DNA sequencing [2], and also for the conjugation of various label molecules to nanomaterials [3] and biomolecules [4]. The reaction has also been exploited in the preparation of peptidomimetics [5], oligonucleotide -carbohydrate conjugates [6], and various organic molecules [7].…”

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confidence: 99%

Synthesis of Lanthanide(III) Chelates by Using ‘Click’ Chemistry

Ketola

Katajisto

Hakala

et al. 2007

Helvetica Chimica Acta

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The copper(I)-catalyzed dipolar [2 þ 3] cycloaddition reaction of an azide and a terminal alkyne is exploited in the preparation of various europium(III), terbium(III), and dysprosium(III) chelates (Schemes 1 -3). By changing the nature of the alkyne and the azide, a wide range of chelates and biomolecule-labeling reactants were obtained. The photophysical properties (Table) of the synthesized chelates are also discussed.Introduction. -The copper(I)-catalyzed Huisgen -Sharpless dipolar [2 þ 3] cycloaddition of azide and alkyne is a powerful direct method to prepare 1,4-disubstituted 1H-1,2,3-triazoles [1]. This reaction has been used increasingly in modular drug development, in the preparation of small-molecule radiopharmaceuticals, in DNA sequencing [2], and also for the conjugation of various label molecules to nanomaterials [3] and biomolecules [4]. The reaction has also been exploited in the preparation of peptidomimetics [5], oligonucleotide -carbohydrate conjugates [6], and various organic molecules [7].The labels disclosed in the literature of click chemistry are organic dyes. However, the organic fluorophores suffer from several drawbacks, such as Raman and Rayleigh scattering, low water solubility and concentration quenching. Thus, multilabeling of biomolecules with organic fluorophores may not enhance the detection sensitivity to the degree required in several applications. Furthermore, this type of labels may decrease the water solubility of the target molecule dramatically.Lanthanide(III) chelates have several special properties which make them excellent alternatives in bioaffinity assays [8]. Their large Stokes shift has a decreasing effect on scattering phenomena. The long fluorescence decay after excitation of these molecules allows time-delayed signal detection, which eliminates completely the background luminescence originating, e.g., from buffer components, plastics, and biomaterials. The very narrow emission lines allow the use of effective filters which diminish the background. Since the lanthanide(III) chelates do not suffer from concentration quenching, it is possible to have several luminescent chelates in close proximity. Furthermore, the chelates are most commonly readily soluble in water. The different photophysical properties of europium, samarium, terbium, and dysprosium chelates enable even development of multiparametric homogeneous assays; with conventional fluorescent compounds, this is much more difficult because of the strong overlapping of the emission spectra.

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Click Chemistry to Construct Fluorescent Oligonucleotides for DNA Sequencing

Cited by 232 publications

References 32 publications

Click Chemistry, A Powerful Tool for Pharmaceutical Sciences

Click Chemistry, A Powerful Tool for Pharmaceutical Sciences

Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry

Synthesis of Lanthanide(III) Chelates by Using ‘Click’ Chemistry

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