Recently, aptamer selection has been involved in linking this magnetic bead-based strategy to the SELEX technique

Recently, aptamer selection has been involved in linking this magnetic bead-based strategy to the SELEX technique. from each other in sequence and folding pattern, S(-)-Propranolol HCl although they bind to the same target. The concept of joining nucleic acids with proteins began to emerge in the 1980s from research on human immunodeficiency virus (HIV) and adenovirus. It indicated that these viruses encode a number of small structured RNAs that bind to viral or cellular proteins with high affinity and specificity [1]. In the case of HIV, a short RNA ligand called the trans-activation response (TAR) S(-)-Propranolol HCl element promotes trans-activation and virus replication by binding with the viral Tat protein [2]. The adenovirus also has a short RNA aptamer, virus-associated (VA)-RNA, that regulates translation [3,4]. Substantive studies on aptamers have progressed since the selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) was first reported by both Golds group and Szostaks group in 1990 [5,6]. Due to the development of SELEX, which is now a basic technique for the isolation of aptamers, many aptamers could be directly selected against various targets, from small biomolecules to proteins and even cells [7]. Aptamers have been studied as a bio-material in numerous investigations concerning their use as a diagnostic and therapeutic tool and biosensing probe, and in the development of new drugs, drug delivery systems, Selection 2.1. General As mentioned above, SELEX or selection is a technique used to isolate aptamers with high affinity for a given target from approximately 1012C1015 combinatorial oligonucleotide libraries. In general, the SELEX process is comprised of three steps that are repeated in order to search for nucleotides that are better able to bind to the target (Figure 2) [16]. In the first step (library generation), a library, except the initial compound in the library, is converted into single-strand nucleotides that consist of random sequence regions, usually 30C40 mers, flanked by the primer binding site. In the second step (binding and separation), the target-bound library components are separated from the unbound components. This step is generally coupled with several other methods to make selection of the target or the library easy and rapid. Finally, in the third step (amplification), the target-bound library component is amplified by the PCR to create a new library to be used in the next round. Aptamers are continuously developed through this on-going process, and their characteristics are identified using various biological assays. Open in a separate window Figure 2. The general SELEX strategy. Starting with combinatorial libraries (first S(-)-Propranolol HCl step), the specific binders are isolated by an iterative process of ligand binding, elution (second step), and amplification (third step). 2.2. Nitrocellulose Membrane Filtration-Based SELEX A nitrocellulose membrane is often used to immobilize proteins in Western blots and atomic force microscopy (AFM) because it provides simple and rapid protein immobilization by its non-specific affinity for Rabbit Polyclonal to HUCE1 amino acids. In 1968, a method using nitrocellulose membranes was employed by Kramlovas group to easily and quickly separate a protein from RNA molecules [17]. This method has been developed as a research tool for separating proteins from many other components and for immobilizing proteins, which can then react with other biomolecules. When the SELEX method was initially established by Golds group, the aptamer against the T4 DNA polymerase was obtained using a strategy that was based on the use of a nitrocellulose membrane [6]. Because the targets were primarily proteins in the early stage of SELEX, the use of a.