22 - Oligonucleotide Ligands That Discriminate between Theophylline and Caffeine

Abstract

This chapter describes properties of oligonucleotides selected to bind with high affinity to theophylline and that have low affinity for caffeine. The alkaloid theophylline is a bronchodilator that is employed to treat asthma. Increased levels of theophylline can have serious side effects, so serum levels of theophylline in asthma patients must be carefully monitored. Many diagnostic assays are currently available that use antibodies that recognize theophylline. An important feature of such antibodies is their ability to discriminate between theophylline and closely related xanthine derivatives in the diet, particularly caffeine. Theophylline and caffeine differ only by a methyl group at the N-7 position, and thus provide a stringent test of specificity for binding. The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure uses large repertoire libraries of single-stranded oligonucleotides of random sequence to serve as a source of conformational complexity. The ensemble of random sequence oligonucleotides is allowed to interact with the target, and the subset of sequences with affinity for the target is separated from the sequences that have little or no affinity for the target. The bound sequences are separated from the target, converted to cDNA, and amplified to double-stranded DNA by a polymerase chain reaction (PCR). The DNA sequences contain a promoter for T7 RNA polymerase that permits transcription of the DNA population. Cycles of interaction of the RNA transcripts with target are repeated, imposing selection for those transcripts with high affinity for the target. While exploring the capabilities of SELEX, molecular discrimination of oligonucleotides between chemically similar low-molecular-weight targets is also important.