High-efficiency Nucleic Acid Manufacturing Systems to Fulfil the Promise of Genomic Medicine:
Helge Zieler PhD, Founder & President, Primordial Genetics
The Function Generator technology developed by Primordial Genetics is a transformative genetic technology for the development of highly efficient microbes and enzymes. The starting point of the workflow is a microbial genome (yeast genome with 17 chromosomes). Next, they mine all the protein-coding sequences and combinatorialize them by fusion. Eventually, it is possible to find new and improved microbes regarding productivity and robustness, and novel enzymes with improved activity, resistance to inhibitors and substrate specificity.
To explain how the Function Generator works, he first defined multidomain proteins, which have at least two individual moieties as separate folding units. As Prof. Levitt’s paper indicated, there is a limited number of multidomain proteins in nature. Their goal is to build new multidomain protein architectures by fusing entire microbial genomes from their library, which is large in terms of sequence complexity. The Function Generator works in two ways: by creating completely new parts apart from those found in nature or by learning about the relevant genes and their function following a design-build-test-learn cycle.
He began the next part of his talk by comparing DNA sequencing and DNA synthesis, both of which are crucial in the biotech field. While DNA sequencing has rapidly evolved by becoming more affordable and expanding its potential with the advancement of next generation sequencing, DNA synthesis has been relatively stagnant and limited to a single chemical technology. Recognizing this opportunity, the market needs its close familiarity with nucleic acid polymerases, they set out to develop a new approach to address the bottleneck of oligonucleotide synthesis.
Enzymatic oligonucleotide synthesis is advantageous in terms of cost, scalability, racemic purity, and the absence of organic solvents. Enzymatic DNA synthesis involves the cyclic enzymatic addition of 3’-blocked nucleotides followed by cleavage of the protecting group. The challenge is to design an enzyme that can accommodate the protective group and to design the protective group itself. They preferred to avoid this because they are intricately linked and asked themselves whether it is possible to eliminate the protecting group.
Their motivation is to build a synthetic oligo without a protecting group and a processive enzyme that incorporates just a single nucleotide into the oligo at a time. They isolated 1st generation enzymes (DNA polymerases) that could process natural nucleotide triphosphates without protecting the group and incorporate only one per cycle. As a proof of concept, he showed a gel image with lanes loaded with oligos from 20-mer to 24-mer. In each pair of lanes, the one on the left was a standard chemically synthesized oligo (control) and the one on the right represented 1 nt enzymatic additions to the control oligos. They achieved the successful addition of all four bases. Complete characterization of the oligo end base versus the nucleotide being added gives a range of different efficiencies. Some of these are low (e.g. A to A addition, 13%) and should be increased to similar levels close to 100%. So, one of their goals was to develop these enzymes to add anything to anything else, including all the modifications commonly used in pharmaceuticals—ribo- and deoxyribo-nucleotides. To do this, they used a very high-throughput emulsion-based screening system to make a significant amount ofenzyme variants and selected the ones that worked better:
Template-independent DNA polymerases (TIDPs) are diversified using the Function Generator or various methods of mutagenesis.
One template molecule at a time is encapsulated in droplets in an emulsion.
The droplet contains RNA polymerase to transcribe this template and the RNA will be translated; a detection system in the droplet allows the detection of proper activity.
Improved TIDPs are isolated and cycled back into another improvement round.
He briefly mentioned their work on mRNA production, which has four components: enzyme development, process development, mRNA design, and enzyme production, all of which contribute to drug enablement. He also showed a graph comparing the remarkable performance of their enzymes with the standard, T7 polymerase, in terms of yield, translational capacity (a measure of RNA quality), and dsRNA (impurities appear in vitro transcription reactions). Among all, they applied the Function Generator to six of these enzymes that showed significant fold change increases in activity.
In conclusion, he drew an analogy to modifications at the DNA genome level as a command, while the RNA level as a suggestion. He underlined the importance of RNAs in therapeutics by mentioning that RNA modulation is a little less restricted by ethical and regulatory bodies and leaves some room for imagination, design, and testing. He said that the use of creative processes of target discovery or disease targeting to solve problems is basically all made possible using a ‘softer’ way of intervening in an organism that’s represented by RNA.
