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Controlling codes for enhanced proteins

GenScript’s programmable semiconductor technology platform precisely synthesizes mutant DNAs that can be used to engineer strongly binding antibodies.

For protein engineers, a new DNA synthesis platform provides precise control over codon usage, enabling the construction of custom-designed mutant libraries.Credit: CIPhotos / iStock / Getty Images

Scientists are increasingly constructing libraries of mutant DNA sequences that can be used to search for and generate new proteins with enhanced functions. Generally, this involves starting out with a protein that scientists want to change or improve. When the knowledge of the protein’s structure and function, together with computational predictions, is available, mutations are introduced into hotspot locations to influence the protein’s function by designing DNA templates containing the codes for specific changes in the protein’s amino acid sequences. These ‘mutant’ DNAs are then transferred into a host organism, where their codes are translated into proteins that are screened for a desired function.

Current approaches for synthesizing mutant DNA libraries, however, have several disadvantages. Most importantly, they offer little control over the amino acid codes, called codons, introduced into the DNA fragments. This results in a low-quality mutant library containing unwanted ‘stop’ codons that signal the termination of protein synthesis.

To address this issue, the global biotechnology firm GenScript developed a high-quality, cost-effective approach. Its arrayed, semiconductor-based DNA synthesis platform provides precise control over codon usage, which enables the construction of custom-designed, mutant libraries. The platform is made of programmable semiconductor chips containing thousands of arrays of platinum electrodes. Each electrode can synthesize a DNA fragment with a specific nucleotide sequence. The platform also allows regulation of process temperature and humidity as well as minimizes exposure to external elements, thereby improving the quality of the synthesized DNA.

In a recent application note, GenScript scientists demonstrated their platform’s versatility by constructing a DNA library that allowed them to identify antibody variants that bound more strongly to a target antigen than the original antibody. This is important because strongly binding antibodies can be used in therapeutics and diagnostic kits.

DNA can only code for 20 different amino acids, which come together in a huge variety of combinations to form proteins. GenScript scientists constructed their precision mutant library by exchanging each of 63 amino acids on six antigen-binding sites of an antibody with all possible 19 amino acid substitutions, resulting in the synthesis of approximately 1,200 high- quality DNA mutants confirmed by next-generation sequencing. The DNA fragments were then transferred to a host organism to generate their corresponding antibodies. Among them, several were found to bind more strongly to their target antigen than the original antibody. One antibody’s affinity was four orders of magnitude stronger than the original.

In the GenScript workflow, oligonucleotides are synthesized using semiconductor-based DNA synthesis to create individual mutants. Those are PCR amplified or cloned into a vector to produce mutant libraries, which are sequenced with NGS. Libraries can be delivered as double-stranded DNA fragments or cloned plasmids, which can be pooled, sub-pooled, or individually arrayed. Credit: GenScript

The scientists were also able to use their technology to construct a ‘combinatorial mutant library’ in which simultaneous mutations were made in the strongly binding antibody mutants. This led to the generation of new mutant antibodies that bound more strongly to the antigen than their parent mutants.

GenScript’s arrayed, semiconductor-based DNA synthesis platform saves scientists time and money, as it prevents the presence of unwanted codons and ensures the variants are designed as intended. Also, since different organisms have different codon preferences, it allows scientists to use amino acid-coding codons that best suit the intended host organism – another advantage offered by the technology platform

To learn more, explore GenScript’s recent application note.