Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures

Abstract

Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into two groups based on their consensus sequences and the aptamers from both groups showed strong binding to Sephadex G-100. One of the highest affinity aptamers, D8, was chosen for further characterization. Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but not to isomaltose, isomaltotriose and isomaltotetraose, suggesting that its optimal binding site might consist of more than four glucose residues linked via alpha-1,6 linkages. The aptamer was very specific to the Sephadex matrix and did not bind appreciably to other supporting matrices, such as Sepharose, Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer could be purified from a complex mixture of cellular RNA, giving an enrichment of at least 60 000-fold, compared with a non-specific control RNA. These RNA aptamers can be used as affinity tags for RNAs or RNA subunits of ribonucleoproteins to allow rapid purification from complex mixtures of RNA using only Sephadex.

Figure 1

Sequences of Sephadex-binding aptamers. Only the sequences corresponding to the randomized region in the starting library are shown. The aptamers were classified as group 1 or group 2 based on their consensus sequences (underlined). All of them were unique, except aptamer D8, which appeared twice during screening.

Figure 2

Competitive binding assays showing the ability of various sugars (isomaltose, isomaltotriose, isomaltotetraose and dextran B512) to compete with Sephadex G-100 for binding to aptamer D8. The aptamer was incubated with Sephadex G-100 in the presence of various sugar concentrations ranging from 0.1 to 20 mM. The ratios of the percentage of aptamer binding to Sephadex in the presence of various competitor concentrations over binding in the absence of competitors are shown in the graph.

Figure 3

The binding of aptamer D8 to various supporting matrices, compared with that of the control RNA. Radiolabeled RNA of either aptamer D8 or the control RNA was incubated with several matrices, then washed and eluted and the percentage of bound RNA was determined.

Figure 4

Purification of aptamer D8 from a complex mixture of human cellular RNA using Sephadex G-100. Radiolabeled aptamer D8 RNA and the control RNA (5 × 10⁴ c.p.m. or 30 ng each) were added to 10 µg unlabeled cellular RNA. The sample was incubated with 200 µl of Sephadex beads in 500 µl total volume, washed and eluted as detailed in Materials and Methods. (A) Samples from each fraction, i.e. input, unbound, wash and eluate, were analyzed for total cellular RNA by electrophoresis on a native 2% agarose gel and stained with ethidium bromide. (B) The radiolabeled RNA in the same fractions was analyzed by electrophoresis in a 10% denaturing polyacrylamide gel and visualized with a PhosphorImager system. The ratios of aptamer D8 to the control RNA were calculated to show the enrichment of purification and are shown below each lane.

Figure 5

Sephadex structural components. The drawing shows the structure of dextran B512, a homopolysaccharide consisting of d-glucopyranose mainly linked via α-1,6 linkages (95%) and, to a lesser extent, via α-1,3 linkages (5%) at the branch points. Sephadex is prepared by cross-linking dextran B512 with epichlorohydrin. Isomaltose, isomaltotriose and isomaltotetraose are oligosaccharides consisting of two, three and four residues of d-glucopyranose linked via α-1,6 linkages, respectively.