Combinatorial Chemistry

In addition to its use in drug development, combinatorial chemistry can be applied to other areas of biomedical research, such as the design of molecules for diagnosing medical conditions. Compounds for these applications can be larger than pharmaceutical compounds, and do not have to be designed to enter the body. Using a novel combinatorial chemistry method called in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment), a short DNA or RNA molecule (termed an oligonucleotide) with a desired property, such as the ability to specifically recognize and bind a molecule associated with a particular disease, can be selected in a single experiment from a library containing approximately 10¹⁵ different compounds. First, a library of oligonucleotides is created in a machine called an oligonucleotide synthesizer. This apparatus can make oligonucleotides with either a defined or random sequence.

Oligonucleotides for SELEX are designed to have a central region containing random sequence and outer, flanking regions with defined sequences. These defined sequences will be used as primer-binding sites for the polymerase chain reaction (PCR). The oligonucleotide library is prepared as a mixture, usually containing about 10¹⁴ to 10¹⁵ different sequences. These specialized oligonucleotides, termed aptamers, are then exposed to target ligand molecules, which are typically attached to a solid support, such as a filter membrane. The unbound aptamers are then washed away, leaving only the rare aptamers that can bind the ligand adhering to the filter. These aptamers can then be recovered from the filter by washing it with a solution that disrupts the binding.

These binding candidate aptamers represent a minuscule fraction of the original library. Some may bind the target ligand tightly, but others may bind weakly. Since all the aptamers have defined primer-binding sites on the ends, this much-reduced population can now be amplified exponentially by PCR. After amplification, the aptamers can be subjected to another round of ligand binding, now using more stringent washing conditions, in which only the tightest-binding molecules will stay bound. These high-affinity binders can be recovered again subjected to still more cycles of PCR amplification, binding, washing, and recovery, until the population of aptamers consists exclusively of very tightly binding molecules.

For some applications, these molecules are useful directly. They can also be studied to design non-DNA molecules that have similar shapes but that will have more potential as drugs.