Frayne Consultants


Phosphate analogues that block nuclease acitivity in and out of cells. 
mRNA accumulator for the rapid increase in mRNA levels in bacteria
TLR9 agonist via modified single-stranded phage DNA
        

VivoSyn

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Technology Background

VivoSyn┬« products and services involve the use of patented phosphate analogues, namely mono or di-substituted phosphates. The monosubstituted form Phos-1S appears to be universally accepted as a phosphate analogue in vivo where it is readily taken up by cells and incorporated into nucleic acids. The incorporation of the analogue occurs readily even in the presence of normal phosphate. Incorporation of the analogue creates nuclease resistant linkages in nucleic acids, including but not limited to genomic DNA, mRNA, rRNA, plasmid DNA, phage DNA etc. Organisms tested include bacteria, yeast, fish, human tissue culture cells, and human white blood cells. The analogue is even incorporated into complex organisms such as goldfish, where it is easily incorporated into the cells of the gut epithelium that rapidly turn over. Cells are fully viable for and appear to undergo normal spicing as demonstrated by the analysis of the ACT 1 gene in yeast and the general viability of the various cells examined. Theoretically the analogue can be incorporated into ATP either 1) during glycolysis or 2) during mitochondrial oxidation. During glycolysis Glyceradehyde-3- phosphate dehydrogenase incorporates inorganic phosphate to form bis-phosphoglycerate. In the next step the phosphate group added is transferred to ADP with ATP formation by Phosphoglycerate kinase. Experiments in yeast demonstrate that the analogue can be incorporated under anaerobic conditions favoring the glycolysis pathway for incorporation. Experiments in bacteria indicate greater than 90% of plasmid DNA can be modified with the Phos-1S analogue. Note in vitro modification is stable in single-strand DNA or RNA but not double stranded DNA.


A variety of media can used, such as minimal media prepared by substituting the Phos-1S analogue for normal phosphate. Note, the minimal media needs to be adapted for the particular organism used in terms of salts and overall ionic strength. Nutrient based medias can also be used as long as the normal phosphate levels are low. Several commonly used medias work fine when the analogue is simply added. For mammalian cell culture the analogue can be added to media or media can be specially prepared using the analogue instead of normal phosphate. 


Apart from generating novel nucleic acids, the Phos-1S analogue can be used to stabilize and accumulate many mRNAs relative to rRNA in vivo. Over time transcriptional changes occur which lead to global alterations in the distribution of proteins, allowing useful manipulations for the production of proteins, enzymes, metabolites, or differentiated cells. In yeast a 2-4X fold increase in the total amount of protein secreted has been observed, at both low and high concentrations of the analogue.


References:

Frayne, E. (2021). Time Course of Thio-phosphate Induced Metabolic Changes in E coli. SEED Synbioconference, AIChE (virtual).

Frayne, E.G. (2020).  Metabolic Regulation of Cellular Differentiation. Advanced Chemicobiology Research.

http://ojs.wiserpub.com/index.php/ACBR/article/view/543/389

Frayne, E. G. (2020). RNA Stabilization via Thio-phosphate and Gene Regulation. Cell & Experimental Biology Virtual Conference. United Scientific Group.

Frayne, E.G. (2020). Global profile changes in transcripts induced with a phosphate analogue: implications for gene regulation. Mol Cell Biochem. https://doi.org/10.1007/s11010-020-03715-9

Frayne, E. (2018). Exploring Applications of Thio-phosphate in Drug Discovery. Annual Congress of International Drug Discovery, Science, and Technology.      IDDST, Boston, MA.

Frayne, E. (2018). Metabolic Engineering with Thio-phosphate. BIO World Congress on Industrial Biotechnology, Philadelphia, PA.

Frayne, E. (2018). Increase in the Synthetic Pathways for RNA, Tryptophan, Fatty Acids, and Isoprenoids in Thio-phosphate Treated E. coli. Proceedings of the Annual Meeting of the American Institute of Chemical Engineers, Scottsdale, Az.

Frayne, E. (2017). In Vivo Modification of RNA with Phosphorothioate Linkages. RNA Biology -Salk Symposium on Biological Complexity, San Diego, CA.

Frayne, E. (2016). In Vivo Engineering of mRNA Stability with Applications for Protein Synthesis. Bio World Congress, San Diego, CA.

Frayne, E. (2016). Conserved mechanism of action of a phosphate analogue. Mol. & Cell. Biochemistry, 415:111-117. doi:10.1007/s11010-016-2681-6.

Frayne, E. (2016). A Novel Differentiation Factor. AAAS Meeting, Washington, DC.

Frayne, E. (2015). Induction of Microbial Defense Mechanisms. Proceedings of the Annual Meeting of the American Institute of Chemical Engineers, Salt Lake City, UT.

Frayne, E. (2013). Global Alterations in the Secretomes of Bacillus subtilis and S. cerevisiae. Proceedings of the Annual Meeting of the American Institute of Chemical Engineers, San Francisco, CA.