chapter  20
14 Pages

Globular Oligonucleotide Screening via the SELEX Process: Aptamers as High-Affinity, High-Specificity Compounds for Drug Development and Proteomic Diagnostics Larry Gold

I. Introduction ........................................................................................491 II. The Status of Aptamers-An Example ..............................................493 III. The SELEX Process-A Brief Description ........................................495 IV. Aptamers-Some Generalizations ......................................................497 V. Proteomics-The Use of Aptamers for Diagnostics...........................501 VI. Summary and Future Prospects..........................................................502 Acknowledgments ........................................................................................503 References ....................................................................................................503

Craig Tuerk and I published a paper in Science in August 1990 entitled ‘‘Selection of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase’’ [1]. We were studying the regulation of translation of bacteriophage T4 DNA polymerase, a protein that represses

its own translation by binding to an RNA site that overlaps the ribosomebinding site in its own mRNA [2]. We showed that randomizing the eight nucleotides in a hairpin loop in that domain (so as to provide 65,536 different octamer sequences in the loop), and then selecting for tight binders with purified T4 DNA polymerase, led to the isolation of two octamers-the wildtype sequence and a quadruple mutation of that sequence (that is, four changes within eight nucleotides). We used amplification (RT-PCR) of the binding subset between rounds, thus creating a winnowing or culling of winners from the bulk of the RNA sequences as the SELEX process proceeded. In the abstract of the paper we wrote, ‘‘These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.’’ That bold statement regarding the potential of the SELEX process (the products of the SELEX process are called ‘‘aptamers,’’ based on a suggestion made by Ellington and Szostak in a paper published just after ours [3]) was reiterated in the paper to make the point: we believed that we had found something as powerful as antibodies for measuring or inactivating/ activating therapeutically interesting proteins [4]. Craig and I said that the ‘‘products of SELEX can affect the activity of the protein to which they have been fit.’’ We implied that aptamers could be used to agonize or antagonize protein targets, and thus might become therapeutic and/or diagnostic agents. This review article and many lovely scientific papers from academic labs and NeXagen/NeXstar Pharmaceuticals suggest strongly that Craig Tuerk and I had seen correctly the power and potential of oligonucleotide aptamers.