1. An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5'-Triphosphates
Muthian Shanmugasundaram, Annamalai Senthilvelan, Zejun Xiao, Anilkumar R Kore Nucleosides Nucleotides Nucleic Acids . 2016 Jul 2;35(7):356-62. doi: 10.1080/15257770.2016.1163382.
A simple, reliable, and an efficient "one-pot, three step" chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5'-triphosphate (5-MeCTP), pseudouridine-5'-triphosphate (pseudoUTP) and N(1)-methylpseudouridine-5'-triphosphate (N(1)-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%).
2. Biosynthetic precursors of some modified nucleosides in the transfer ribonucleic acid of Mycoplasma mycoides var. capri
R T Walker J Bacteriol . 1971 Sep;107(3):618-22. doi: 10.1128/jb.107.3.618-622.1971.
The ribosomal and transfer ribonucleic acid (tRNA) from Mycoplasma mycoides var. capri, grown in a medium containing uridine-((14)C)-5'-triphosphate and cytidine-(5-(3)H)-5'-triphosphate, were isolated and separated. The uridine in both species of RNA was shown to contain (14)C and the cytidine to contain both (3)H and (14)C. Comparison of the labeling of 4-thiouridine and pseudouridine, obtained from an enzymatic digest of the RNA, indicates that their biosynthetic precursor is uridine, not cytidine. It is probable that ribothymidine and dihydrouridine have the same derivation.
3. Defining optimized properties of modified mRNA to enhance virus- and DNA- independent protein expression in adult stem cells and fibroblasts
Frauke Hausburg, Silke Na, Anna Skorska, Paula Müller, Robert David, Natalia Voronina, Gustav Steinhoff Cell Physiol Biochem . 2015;35(4):1360-71. doi: 10.1159/000373957.
Background:By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required.Methods:Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA) species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC) and pseudouridine-5'-triphosphate (Ψ). We then investigated their effect on i) protein expression efficiencies and ii) cell viability for human mesenchymal stem cells (hMSCs) and fibroblasts from different origins.Results:Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies.Conclusion:The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.
