In vitro selection with expanded genetic alphabets

Abstract

This invention enables processes for extracting from a mixture of oligonucleotide molecules individuals that bind to a preselected target (aptamers) or catalyze a preselected reaction (xNAzymes) that contain one or more non-standard nucleotides. These pair with their complements in a Watson-Crick geometry with a pattern of hydrogen bonds different from that pairing adenine and thymine, and guanine and cytosine. The processes comprise (a) obtaining this mixture containing non-standard nucleotides within preselected regions, (b) contacting the mixture with a preselected target or one or more reactants for the reaction whose catalysis is desired, (c) separating oligonucleotides having a greater affinity to the target or catalytic effectiveness from the remainder that have less affinity or catalytic effectiveness, and (d) amplifying the separated oligonucleotides. The process exploits PCR conditions that amplify oligonucleotides with less than 5% loss of the non-standard nucleotide per cycle, and processes to determine the sequences of the amplified oligonucleotides.