Evolution of Diversely Functionalized Nucleic Acid Polymers

Darwinian evolution gave rise to biopolymers with a myriad of folded structures and functions, as well as all the diverse life forms on earth that are supported by those molecules. Amazingly, the essence of molecular evolution can be recapitulated in a test tube with purely biochemical manipulations outside of living cells. For nearly three decades, researchers have been evolving biopolymers – and even biomimetic polymers that are not produced in any living system – by subjecting diverse libraries of polymers to iterated cycles of templated synthesis, functional selection, and amplification. In principle, any class of sequence-defined polymer whose synthesis can be templated by an amplifiable genetic material (such as DNA) is evolvable in this manner. To date, however, the evolution of polymers made of building blocks beyond those compatible with polymerase enzymes or the ribosome has not been demonstrated. To explore new kinds of evolvable sequence-defined polymers and discover new classes of receptors, catalysts, and materials, we used a ligase-mediated DNA-templated polymerization system and in vitro selection to evolve highly functionalized nucleic acid polymers (HFNAPs) made from 32 building blocks containing eight chemically diverse side-chains on a DNA backbone. Through iterated cycles of polymer translation, selection, and reverse translation, we discovered HFNAPs that bind PCSK9 and IL-6, two protein targets implicated in human diseases. Mutation and reselection of an active PCSK9-binding polymer yielded evolved polymers with high affinity (KD = 3 nM). This evolved polymer potently inhibited binding between PCSK9 and the LDL receptor. Structure-activity relationship studies revealed that specific side-chains at defined positions in the polymers are required for binding to their respective targets. Our findings expand the chemical space of evolvable polymers to include densely functionalized nucleic acids with diverse, researcher-defined chemical repertoires. SELEX; Sequence-defined synthetic polymer