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. 1998 May 12;95(10):5462-7.
doi: 10.1073/pnas.95.10.5462.

DNA ligands that bind tightly and selectively to cellobiose

Affiliations

DNA ligands that bind tightly and selectively to cellobiose

Q Yang et al. Proc Natl Acad Sci U S A. .

Abstract

Cell surface oligosaccharides have been shown to play essential biological roles in such diverse biological phenomena as cellular adhesion, molecular recognition, and inflammatory response. The development of high-affinity ligands capable of selectively recognizing a variety of small motifs in different oligosaccharides would be of significant interest as experimental and diagnostic tools. As a step toward this goal we have developed DNA ligands that recognize the disaccharide cellobiose, whether in soluble form or as the repeating unit of the polymer, cellulose. These DNA "aptamers" bind with high selectivity to cellobiose with little or no affinity for the related disaccharides lactose, maltose, and gentiobiose. Thus, the DNA ligands can discriminate sugar epimers, anomers, and disaccharide linkages.

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Figures

Figure 1
Figure 1
Selection of anticellulose DNA aptamers. Selection began with a library of 1015 DNA molecules containing known 5′ and 3′ sequences (for PCR priming) and 40 central nucleotide positions at which the sequence had been randomized during synthesis. In the first round, the library was bound to cellulose, washed extensively, and eluted first with method A (50 mM cellobiose competitor, 2.5 mM EDTA) and then with method B (50% formamide). DNA eluted from the first round of binding under these two conditions was kept separate in the subsequent 13 rounds of binding selection and amplification by PCR, eluting only with the original method. Pools of DNA aptamers from the 14th round of selection by each method were converted to double-stranded DNA, ligated into plasmid vectors to produce individual clones. DNA from more than 200 clones was reamplified to produce “monoclonal” aptamers, of which 80% were found to bind tightly to cellulose.
Figure 2
Figure 2
Blocking cellulose binding with cellobiose. The ability of the soluble repeating unit of cellulose, cellobiose, to inhibit cellulose binding was tested. Less than 1 nM 5′-32P DNA aptamer was incubated in 200 μl with 10 mg of insoluble cellulose in the presence (+) or absence (−) of 50 mM cellobiose. Equal percentages of the input DNA, unbound DNA, and the DNA eluted from the cellulose (with method A or method B, depending on aptamer) were visualized by using a PhosphorImager after denaturing PAGE. (A) Three aptamers (Cel#18, Cel#75, and Cel#106) not competed by cellobiose. (B) Three aptamers (Cel#16, Cel#183, and Cel#202) for which preincubation with cellobiose prevented most subsequent binding to cellulose. (C) Binding by the original random-sequence DNA library. Low levels of library DNA reproducibly bound to cellulose and could be eluted with method B (shown).
Figure 3
Figure 3
Specificity of aptamer binding. Aptamers Cel#16, Cel#183, and Cel#202 were tested for the ability to bind four similar biose sugars by titrating the concentration of sugars and detecting DNA binding by measuring the resulting small decrease in absorbance of the solution at 260 nm (hypochromicity). The DNA concentration was constant within each experiment and was 0.6 to 0.7 μM. (AC) For each aptamer, the cellobiose dimer and cellotetraose tetramer produced hypochromic shifts at similar concentrations (apparent Kd 10−7 to 10−5 M), but the related biose sugars did not produce significant hypochromicity. (D) A control 41-mer DNA oligonucleotide did not give a hypochromic response to any of the sugars tested.
Figure 4
Figure 4
Structure comparison of cellobiose, lactose, maltose, and gentiobiose.

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