Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds

Chu, Barbara C.F.; Orgel, Leslie E.

doi:10.1093/nar/16.9.3671

Cited by 77 publications

(33 citation statements)

References 18 publications

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“…The single-strand polynucleotide was phosphorylated with T4 polynucleotide kinase, as described by Maniatis et al (9). Cystamine was added to the 5' end of the single-stranded (ss) polynucleotide as described by Chu and Orgel (12), and the product was purified as described by Teare and Wollenzien (13). Typically :20 ,ug of phosphorylated ss DNA in 20 ,ul of water was mixed with 40 1.l of 1 M Abbreviations: ss, single-stranded; ds, double-stranded; CRP, cAMP receptor protein; CPM, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin; 32-bp-CPM, 32-bp fragment of lac promoter DNA labeled with CPM at the 5' end.…”

Section: Methodsmentioning

confidence: 99%

Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.

Heyduk

Lee

1990

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

221

195

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A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe. Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label. Formation of protein-DNA complex was monitored as a function of cAMP concentration. Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP4cAMP)2 to DNA. These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA. These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions. This technique also has the potential of providing information regarding the structure of protein-DNA complexes.Quantitative structural studies on cAMP receptor protein (CRP) in conjunction with ligand-binding studies have shown clearly that CRP from Escherichia coli exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes (1). The binding properties of these two complexes to the lac promoter were investigated by gelretardation technique, and the results showed that the formation of protein-DNA complex is a complicated function of cAMP concentration. At cAMP concentrations that favor the formation of CRP-cAMP, binding of the protein to DNA is favored. At high concentration of cAMP, which favors the formation of CRP-(cAMP)2, a decrease in protein-DNA complex was seen. These results strongly suggest that the CRP-cAMP and CRP-(cAMP)2 complexes have different affinities for the lac promoter (1). These conclusions are not consistent with the report of Takahashi et al. (2). These authors concluded that the CRP-cAMP complex exhibits essentially the same affinity for the lac promoter as that ofthe CRP-(cAMP)2 complex.The differences between the results of these two studies may be attributed to the differences in experimental conditions, as necessitated by the techniques chosen to monitor protein-DNA interaction. The gel-retardation technique (3,4) dictates that the experiments be conducted at low-salt concentration, and because protein-DNA interactions are highly salt dependent (5), possibly the results of DNAbinding study are not applicable to that of the structure and ligand-binding studies, which are conducted at higher salt concentration (1). To acquire detailed valid thermodynamic data to define the linkages among the interactions of cAMP, CRP, and DNA, we looked for a simple and reliable approach that allows collection of a large amount of accurate data under well...

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Section: Methodsmentioning

confidence: 99%

Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.

Heyduk

Lee

1990

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

221

195

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“…Disulfide coupling chemistry has been used in the ligation of peptide to peptide (37), DNA to DNA (37), peptide to DNA (37), DNA to haptens (36), DNA to paramagnetic beads (15), and DNA to controlled porosity glass (CPG) 2 (35). Here, we report a simple, efficient, and specific attachment chemistry utilizing the disulfide bond for the covalent immobilization of presynthesized DNA probes onto a silane-derivatized glass support.…”

mentioning

confidence: 99%

Immobilization of Oligonucleotides onto a Glass Support via Disulfide Bonds: A Method for Preparation of DNA Microarrays

Rogers¹,

Jiang-Baucom²,

Huang³

et al. 1999

Analytical Biochemistry

239

158

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The covalent attachment of disulfide-modified oligonucleotides to a mercaptosilane-modified glass surface is described. This method provides an efficient and specific covalent attachment chemistry for immobilization of DNA probes onto a solid support. Glass slides were derivatized with 3-mercaptopropyl silane for attachment of 5-prime disulfide-modified oligonucleotides via disulfide bonds. An attachment density of approximately 3 ؋ 10 5 oligonucleotides/m 2 was observed. Oligonucleotides attached by this method provided a highly efficient substrate for nucleic acid hybridization and primer extension assays. In addition, we have demonstrated patterning of multiple DNA probes on a glass surface utilizing this attachment chemistry, which allows for array densities of at least 20,000 spots/cm 2 . © 1999 Academic Press Key Words: covalent immobilization; oligonucleotide; glass; disulfide bonds; DNA microarray.In recent years, high-density miniaturized oligonucleotide arrays have emerged as promising tools for assessing genomic data with a lower cost and higher throughput than the traditional gel-based methods. Such oligonucleotide arrays, or DNA chips, have been applied to genetic mutational scanning (1, 2), molecular bar coding (3), gene expression monitoring (4, 5), and sequencing (6 -8). The power of the DNA chips come from the highly parallel, addressable, miniaturized array format that provides significant advantages over traditional gel-based formats in terms of reagent cost, labor, speed, throughput, and operational simplicity. The development of efficient chemistries for the manufacture of spatially resolved, microscale DNA arrays on a solid-support is essential for the realization of the DNA chip technology potential. In most DNA chip applications, the DNA arrays are used to capture or analyze the target sequences and/or detection probes via hybridization reactions alone (1-7) or with subsequent primer extension reactions (8). The reliability and integrity of the hybridization reactions are highly dependent, in addition to the actual base composition of the arrayed oligonucleotides, on the quality and the characteristics of the DNA arrays. In developing a useful and reliable chemistry for producing DNA arrays, the accessibility and functionality of the surface-bound DNA, the density of attachment, the stability of the array, the reproducibility of the attachment chemistry, and the fidelity of the immobilized sequences are all critical.There have been numerous reports regarding immobilization (9 -26) or direct synthesis (27, 28) of oligonucleotides on solid supports, such as glass, silicon, membranes, and polystyrene. Parallel synthesis of oligonucleotides directly onto the solid support by photoactivatable chemistries (27) or standard phosphoramidite chemistries (28) have, thus far, been the most successful approach to manufacturing high-density DNA arrays. Patterning of presynthesized oligonucleotides, however, is preferred for many research applications and low-to moderate-density-array applications requi...

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“…Both the azide and alkyne SAMs are expected to make the slide surface highly hydrophobic, and thus help to prevent nonspecific interactions with the glass surface. Both the click chemistry and the thiol approaches have been previously shown to be compatible with downstream processing 13. 14 We found that the click chemistry approach with the alkyne‐functionalized surface was the less time consuming and required fewer materials.…”

Section: Methodsmentioning

confidence: 83%

Covalent‐Bond‐Based Immobilization Approaches for Single‐Molecule Fluorescence

2009

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INH: We report two new approaches, using click-chemistry and disulfide bond bridges, to surface-immobilize nucleic acids for single-molecule fluorescence experiments using covalent bonds and self-assembled monolayers. Both approaches are specific and yield comparable results to the avidin-biotin linkage, but offer new surface chemical properties that might be advantageous to prevent non-specific binding of biopolymers to the surface and to expand the range of fluorescent probes that can be employed in single-molecule studies.

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Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds

Cited by 77 publications

References 18 publications

Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.

Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.

Immobilization of Oligonucleotides onto a Glass Support via Disulfide Bonds: A Method for Preparation of DNA Microarrays

Covalent‐Bond‐Based Immobilization Approaches for Single‐Molecule Fluorescence

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