Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

Abstract

Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method—termed SpliMLiB for Split-and-Mix Library on Beads—was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.

“NAD‐display”: Ultrahigh‐Throughput in Vitro Screening of NAD(H) Dehydrogenases Using Bead Display and Flow Cytometry

“…NAD-display uses water-in-oil emulsion droplets to generate aphenotype-genotype linkage by compartmentalization of single genes.I ne ach droplet compartment one protein variant was expressed (by IVTT) from agene library member attached to aSpliMLiB bead [16] and catalysis took place with an immobilised co-factor analogue as co-substrate.O nce the substrate is oxidised, the redox state of the co-factor indicates whether catalysis is complete,w hich is monitored using an immobilised fluorescent protein-based sensor.Then beads are screened for product cofactor state,a tu ltrahigh-throughput, and selection was achieved using flow cytometric sorting ( Figure 1).…”

“…[For clarity only asingle dsDNA DNA molecule is drawn, although > 10 6 identical DNA molecules exist per bead. [16] ]( iii)B eads are singly encapsulatedinwater-in-oil emulsion droplets, together with in vitro transcription & translation mixture (IVTT) and the enzymatic substrate. Upon expression of functionald ehydrogenase enzyme, catalytict urnover is permanentlyr ecorded in the form of reduction of the immobilisedco-factor to NADH.…”

“…To generate beads densely coated with DNAe ncoding single variants of FDH (i.e.m onoclonal beads), SpliMLiB libraries were generated. In this recent library approach [16] beads are split into separate tubes,aDNA fragment carrying adifferent codon at atargeted site is immobilised, and beads are pooled and split again. Solid phase ligation is used to further extend the DNAand, at the same time,for introducing am utation at another targeted site,with the split &p ool process repeated.…”

“…Solid phase ligation is used to further extend the DNAand, at the same time,for introducing am utation at another targeted site,with the split &p ool process repeated. [16] We limited the number of residues per site to 12, keeping the library to am oderate size of 12 4 = 20 736 variants,b yl imiting replacement in saturation mutagenesis to those amino acids with side chains possessing similar physical and/or chemical properties to the wild-type amino acidsside chain (Table S5). Adetailed, sequence-level overview of the library construction design is provided ( Supplementary Figure S2).…”

“…To avoid interference of NAD(H) redox state detection with cellular processes other than the desired transformation, we completely avoid cells by using in vitro transcription &t ranslation (IVTT) to effect expression of NADH dehydrogenase variants,e nabling interrogation of large libraries of enzymes without endogenous biological interference.I nl ieu of the genotype-phenotype linkage normally provided by cells, [15] we link the redox-phenotype to the DNA-encoded genotype on micrometre-sized paramagnetic beads.W edemonstrate our approach, which we call NAD-display,byultrahigh throughput selection of aformate dehydrogenase site saturation library,g enerated using our recently developed SpliMLiB method. [16] NAD-display is straightforward to implement in molecular biology settings, relying on am odular assembly of functional molecular components.T his approach enables ready access to ultra-high-throughput screening in droplets, [2,3] obviating the need to install microfluidic technologies to create compartments.…”