It has been 15 years since the term "aptamer" and the acronym "SELEX" were coined. With the field of directed molecular evolution now transitioning from its adolescence to young adulthood, it is an appropriate time to take stock of what aptamer science has to offer, both now and for the future. In this monograph, the first ever completely devoted to the subject of aptamers, you will find a well-chosen set of contributions from leading investigators in the field, describing the methods and applications of aptamer technology. This is not a laboratory manual, but neither is it a collection of review articles; it is a handbook that is meant to give you an appreciation for the principles and practice of in vitro selection as applied to functional nucleic acids. Whether you already are or will be a practitioner yourself, or simply want to know what all the fuss is about, this book is something that you will want to attack with a highlighter pen and scratch paper on the side. Evolution is a very powerful process, but it is surprisingly easy to carry out in a modern laboratory. You too can evolve molecules for fun and profit.

The first aptamer, although it was not referred to as such, actually was created almost 40 years ago, before the advent of recombinant DNA technology ("B.C., before cloning", as Sydney Brenner likes to say). In the late 1960s, Sol Spiegel-man realized that the three fundamental processes of Darwinian evolution - amplification, mutation, and selection - could be applied to a population of RNA molecules in vitro. Amplification of RNA was achieved by employing an RNA-depen-dent RNA polymerase, the replicase protein of Qp bacteriophage. Mutation occurred as a result of the intrinsic error rate of the polymerase in copying variants of Qp genomic RNA. Selection was based on the ability of particular RNAs to serve as efficient templates for the production of complementary RNAs and, in turn, for the production of additional copies of themselves. "(Go forth and) multiply, with the biological proviso that (you) do so as rapidly as possible," Spiegelman famously declared. The result, following multiple rounds of selective amplification and mutation, was a population of evolved RNA molecules that were amplified much more efficiently by the replicase compared with their ancestors.

Discussion of Spiegelman's pioneering work usually focuses on the perhaps unsurprising result that the evolved RNAs were truncated variants of Qp genomic

RNA that, by virtue of their smaller size, could be copied more rapidly than the wild type. A more subtle point, however, is that the evolved RNAs also were selected to be efficient ligands for the replicase protein, which recognizes particular features of RNA secondary and tertiary structure in both the positive- and negative-stranded RNA. Thus the evolved RNAs were both an aptamer for the replicase protein and a substrate for the protein, leading to the production of progeny RNAs.

One of the great advances in the history of life on Earth was the transition from an "RNA world," in which both genetic and functional properties resided within RNA, to a DNA and protein world, in which genotype and phenotype were relegated to separate macromolecules. Another critical advance in directed molecular evolution was the development of techniques that decoupled amplification of nucleic acid molecules from selection based on their functional properties. This made it possible to select RNAs that are a ligand for any protein, for example, T4 DNA polymerase, as demonstrated by Craig Tuerk and Larry Gold. RNAs could even be selected that bound to small molecules, as shown by Andrew Ellington and Jack Szostak.

In the early 1980s, following the discovery of catalytic RNA by Thomas Cech and Sidney Altman, one wondered what it would take to coax Qp replicase to amplify RNA molecules that included a ribozyme or some other functional motif. Fred Kramer and colleagues had shown that it was possible to sneak exogenous nucleotides into variants of Qp genomic RNA that could be amplified in vitro. Those familiar with the details of the system knew, however, that it was only a matter a time - and usually not much time - before the insert would be trimmed or spit out entirely, resulting in a more efficient amplicon. What was needed was a general-purpose RNA amplification method that would be indifferent to the sequence being amplified.

Then came polymerase chain reaction (PCR), soon followed by reverse transcriptase PCR (RT-PCR), and everything changed. A population of nucleic acid molecules could be asked to do anything the investigator had the nerve to ask them to do: bind a target molecule, bind a target molecule but not some closely related molecule, catalyze a reaction, catalyze a reaction only after binding to some other target molecule, and so on. In retrospect, most of the early efforts were rather timid, but soon the gloves came off and it seemed that nearly everything was fair game. Literally, of course, the gloves were kept on a bit longer because RNA molecules are highly susceptible to degradation by biological nucleases, limiting their potential applications. This limitation was overcome by carrying out directed evolution with RNA analogs that are nuclease resistant, yet can be amplified by RT-PCR. Particularly intriguing in this regard are "Spiegelmers," which first are selected as natural RNAs that bind the enantiomer of the desired target, then are prepared as the corresponding non-natural enantiomer of RNA for binding to the actual target. These reverse aptamers are aptly named because they are the mirror (Spiegel) of their biological counterparts, and in recognition of Spiegelman's contributions to initiating the practice of in vitro Darwinian evolution.

Aptamer science has now reached maturity, not just as a result of its longevity and accumulated knowledge, but through its growing impact on biology and medicine. In December 2004 the first aptamer compound was approved for clinical use. As discussed in the chapter by Anthony Adamis and colleagues, Macu-gen (pegaptanib) is a chemically modified RNA aptamer that binds tightly and specifically to vascular endothelial growth factor. It has become a preferred treatment for the neovascular form of age-related macular degeneration. Other chapters describe aptamers that are being developed for various therapeutic applications, medical imaging, clinical diagnostics, drug target validation, biosensor applications, and process chemistry. All this and more awaits you on the pages that follow.

Darwinian evolution in nature has provided a bounty of functional macromole-cules. However, just as synthetic organic chemistry has taken us beyond the small molecules that can be harvested as natural products, directed evolution has expanded upon the set of macromolecules to include compounds that have been tailored for our own purposes. This is not intelligent design - quite the opposite in fact - but in this book you will see how the vision and skill of the experimenter, combined with the power of an evolutionary search, can lead to some remarkable discoveries.

Gerald F. Joyce

Departments of Chemistry and Molecular Biology and The Skaggs Institute for Chemical Biology The Scripps Research Institute La Jolla, California, USA

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