title: SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.
abstract: Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, CB2 1GA Cambridge, UK.
authors.forename: Miriam
authors.initials: M
authors.lastname: Kaltenbach
authors.forename: Viktor
authors.initials: V
authors.lastname: Stein
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D050091:Dendrimers / Q000737:chemistry*; D019020:Directed Molecular Evolution / Q000379:methods*; D006147:Guanine / Q000737:chemistry; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D050091:Dendrimers; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D006147:Guanine; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase