A Novel Solid Phase Technology for High-Throughput Gene Synthesis

Beyond the Natural Proteome

“…ProxiMAX randomization has recently been used to target 24 consecutive codons (Frigotto et al, 2015), whilst the maximum number of residues that can be targeted by SlonoMAX™ has not been defined by the inventors (Van den Brulle et al, 2008), but is surely equal to that of ProxiMAX. Both of these techniques are expensive in terms of DNA synthesis, but the oligonucleotides involved may be consistently re-used, because everything except the saturated codons is removed by Type IIS restriction digestion, once the saturation protocol has been completed.…”

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

“…Assembly of the SHM Diversified Libraries-The CDR3-grafted CDR1,2 SHM diversified heavy chain library used for initiation of humanization was synthesized as previously described (19) with the germ line IGHV3-23 nucleic acid sequence serving as its basis (5Ј-ATGGAGTTTGGGCTGAG-CTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCA-GTGTGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCT-TGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG-CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGA-GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG-TGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACA-TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATC-TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA-ATGAACAGCCTGAGAGCCGAGGACACGGCCGTATA-TTACTGTGCGAGA-3Ј. The DNA sequence for H3 was taken directly from the ␣D11 HC CDR3 sequence as published, and the FW4 sequence was taken from the closest human J-region IGHJ6 (5Ј-TGGGGGCAAGGGACCACGGTCACCGTCTC-CTCA-3Ј).…”

Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing

“…Additionally, the number, identity and position of residues favored to make antigen contacts in both VH and VL domains varies both according to the type of antigen (protein, peptide or hapten), thus providing information that can be used to design libraries that have a greater proportion of functional members or even to design target-specific antibody libraries [3]. Previous synthetic methods (TRIM, SlonoMAX [11] and ProxiMAX [7]) have developed approaches to control amino acid identities and frequencies on a per-position basis. These all create DNA fragments in a codonby-codon manner, though via different means.…”

“…TRIM functions by adding three bases at a time, using trinucleotide phosphoramidites, to a growing single strand of synthetic DNA. In contrast, SlonoMAX [11] ligates up to 4096 sticky-ended "anchors" to up to 64 "splinkers" (both are hairpin oligonucleotides with 3-base, single-stranded overhangs) together. The ligated product is then cleaved with a TypeIIS restriction enzyme that leaves a new 3-base overhang on the splinker [11].…”

“…In contrast, SlonoMAX [11] ligates up to 4096 sticky-ended "anchors" to up to 64 "splinkers" (both are hairpin oligonucleotides with 3-base, single-stranded overhangs) together. The ligated product is then cleaved with a TypeIIS restriction enzyme that leaves a new 3-base overhang on the splinker [11]. In this manner, a singlestranded codon is effectively added to the splinker in each cycle.…”