A microarray-based method to perform nucleic acid selections.

Nucleic acids that are selected to bind to a small molecule (1–3), protein (4–7), or whole cells (8) have a variety of applications that range from diagnosing (9,10) and treating disease (11,12) to deciphering the origins of life (13,14). Many methods have been developed to select nucleic acids that bind a small molecule of interest; Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is most frequently used. All of these methods require the isolation of active nucleic acids from inactive ones. These separations are most commonly completed by using affinity chromatography in which a ligand is conjugated onto a resin (4). Capillary electrophoresis (CE) (15) has also been used to perform selections and does not require immobilization of a ligand onto a matrix. CE requires, however, that the mobility of the bound nucleic acid be different from that of the unbound. Though powerful, both methods have disadvantages. For example, in resin-based selections, kinetic biases can be introduced because a high concentration of the bound ligand is washed over a resin to elute the binders (16). This, the resin-based selections, can select against the highest affinity binders since they are the most difficult to compete off. CE SELEX is not affected by kinetic biases but does require specialized equipment. Neither method allows for multiple selections to be completed in parallel. In an effort to complete multiple selections in parallel, a microarray-based method was developed. Microarrays are advantageous platforms for screening and performing selections because of the small amounts of ligand and analyte required, and the ability of microarrays to probe thousands of interactions in parallel. These advantages are best illustrated in the widespread uses of microarrays, which include studying gene expression (17), and protein–protein (18), carbohydrate–protein (19), cell–ligand (20,21), and small molecule–protein interactions (22–24). In order to develop a microarray-based method to complete selections, a microarray surface that is robust enough for ligand screening and allows bound RNAs to be harvested directly from the array surface is required. We found that the optimal surface is an agarose-coated microarray (25,26). Bound RNAs can be harvested from the array surface by simple excision of the agarose from ligand-functionalized positions (27,28). Additionally, agarose provides an inexpensive and three dimensional surface for high ligand loading. It can also be functionalized to provide a chemical handle for immobilization of a variety of reactive groups. Most importantly, by harvesting all ligand-bound RNAs via gel excision, kinetic biases found in resin-based selections may be mitigated (27). Not only does completing multiple selections in parallel on a microarray surface increase throughput, but it also allows for the identification of the highest affinity and specific RNA-ligand interactions. Higher affinity RNA-ligand interactions are selected at lower ligand loadings that captured the members of an RNA library (27). In contrast, separate experiments would be required for each ligand loading to identify higher affinity interactions using resin-based SELEX. Specific RNA-ligand interactions are identified using the microarray selection procedure because it may set up a competition experiment between the arrayed ligands for the members of the RNA library (28). In this protocol, the steps for completion of a microarray-based selection are described (Fig. 1). We specifically focus on ligands that are immobilized onto an agarose array surface using a Huisgen cycloaddition reaction in which the array displays either azide or alkyne groups (29,30), however, other immobilization chemistries are compatible with this platform, including immobilization of amine-containing ligands via reductive amination (25). This approach should be applicable to any nucleic acid-based selection. Open in a separate window Fig. 1 Schematic of the microarray-based selection process: (a) immobilization of small molecules on an activated agarose microarray via a Huisgen 1,3-dipolar cycloaddition reaction. (b) An image of the slide after hybridization with radiolabeled RNA. (c) An image of the same slide after the mechanical removal of bound RNA. (d) A schematic of an agar plate after transformation with a vector containing selected members of the library. (e) Colonies that are white from blue/white screening should be grown in culture and submitted for sequencing to deconvolute the selected members of the library.