In vitro selection of aptamers from RNA libraries.

Abstract

Methods of iterative nucleic acid selection and amplification were enabled by the invention of the polymerase chain reaction (PCR). Thus, the ability to amplify as few as a single DNA or RNA molecule made it possible to diversify sequences and to partition the desirable from the undesirable subset (Fig. 1A) (,). Initially, this approach was practiced in vivo using biological amplification and selection, by diversification of plasmid sequences and iterative growth against a selective marker (). The power of in vitro RNA selection from a randomized combinatorial library was demonstrated by Tuerk and Gold () using T4 DNA polymerase and the R17 phage coat protein. These investigators called their iterative RNA selection procedure “SELEX.” Ellington and Szostak () also derived RNA ligands against organic dyes using iterative in vitro selection. Moreover, the demonstration that RNAs could be selected that bind to proteins and compounds with no known role in RNA-binding in vivo led to the concept of aptamers. An aptamer is a folded RNA that forms a shape that coincidentally fits against another surface, to which it is “apt” to bind. Generally, RNA ligands that bind to naturally occurring RNA-binding domains of proteins are not considered to be aptamers. Unexpectedly, an RNA ligand selected against an antibody generated by immunization with a peptide demonstrated that RNA and protein could crossreact at the level of antibody recognition (). Open image in new window Fig. 1A. (A) A schematic representation of the randomized RNA selection protocol. The sequences of the primers and template are given in (B). The T7 promoter region is labeled T7 pro. DNA and RNA are represented by double lines and single lines, respectively. The number of iterative cycles used varies with the nature of the target molecule and can allow evolution of the target set using error-prone PCR.