In vitro selection with artificial expanded genetic information systems
- 30 December 2013
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 111 (4), 1449-1454
- https://doi.org/10.1073/pnas.1311778111
Abstract
Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson–Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS–SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS–SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS–SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS–SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins. Significance Many chemicals are valuable because they bind to other molecules. Chemical theory cannot directly design “binders.” However, we might recreate in the laboratory the Darwinian processes that nature uses to create binders. This in vitro evolution uses nucleic acids as binders, libraries of DNA/RNA to survive a selection challenge before they can have “children” (systematic evolution of ligands by exponential enrichment, SELEX). Unfortunately, with only four nucleotides, natural DNA/RNA often yields only poor binders, perhaps because they are built from only four building blocks. Synthetic biology has increased the number of DNA/RNA building blocks, with tools to sequence, PCR amplify, and clone artificially expanded genetic information systems (AEGISs). We report here the first example of a SELEX using AEGIS, producing a molecule that binds to cancer cells.Keywords
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