ProxiMAX randomization: a new technology for non-degenerate saturation mutagenesis of contiguous codons

Abstract

Back in 2003, we published ‘MAX’ randomization, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomization saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an α-helix, as in zinc-finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomization, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomization uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialized chemistry, reagents or equipment, and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in predefined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomization is particularly relevant to antibody engineering.

Beyond the Natural Proteome

“…nondegenerate methods a may be created via various methodologies as described in section 3. a) Diversity was calculated using the formula d=1/(N∑kpk 2 ) (Makowski & Soares, 2003) and is in agreement for a 12-mer peptide saturated with codon NNN ( Ashraf, M., Frigotto, L., Smith, M.E., Patel, S., Hughes, M.D., Poole, A.J., et al (2013) end only). The donors can take the form of partially double-stranded DNA, fully doublestranded DNA or hairpin oligonucleotides (as shown).…”

“…Adapted from Ashraf, M., Frigotto, L., Smith, M.E., Patel, S., Hughes, M.D., Poole, A.J., et al (2013) Adapted from Odegrip et al, 2004. Double-standed DNA is generated consisting of (in 5'  3' order) a promoter, the saturated library fused in-frame to repA, the CIS element and the ori sequence.…”

“…Like other nondegenerate techniques, ProxiMAX is a nondegenerate saturation technology that uses one codon only per amino acid (Ashraf, Frigotto, Smith, Patel, Hughes, Poole et al, 2013). In common with MAX randomization, ProxiMAX does not require any specialized reagents, but rather relies on conventional oligonucleotides, a Type IIS restriction enzyme and blunt-ended ligation.…”

“…The relative proportions of codons at each saturated position may also be user-defined. ProxiMAX can be achieved manually with good results (Poole, 2015) or via automation, which gives excellent compliance with library design (Ashraf et al, 2013). Automation also permits the addition of hexameric, two-codon units rather than one codon per cycle, though this modification requires automation, owing to the sheer Nondegenerate Saturation Mutagenesis 14 number (400) of oligonucleotides (codon donors) involved (Frigotto, Smith, Brankin, Sedani, Cooper, Kanwar et al, 2015).…”

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

“…Such saturation necessarily encodes all 20 amino acids in ratios dictated by the genetic code. Moreover, this saturation severely limits the functional diversity of such libraries by encoding amino acid composition that is unable to fold natively, and leads to premature termination through the encoding of a stop codon, which in turn can lead to non-functional proteins [5][6][7]. Several solutions to reduce redundancy in the code and exclusion of termination signals has led to inventions such as the 22c trick (all 20 amino acids are encoded by 22 codons through degenerate oligonucleotides), offering near non-degenerate randomization [8].…”