A selective and atom-economic rearrangement of uridine by cascade biocatalysis for production of pseudouridine.

As a crucial factor of their therapeutic efficacy, the currently marketed mRNA vaccines feature uniform substitution of uridine (U) by the corresponding C-nucleoside, pseudouridine (Ψ), in 1-N-methylated form. Synthetic supply of the mRNA building block (1-N-Me-Ψ-5'-triphosphate) involves expedient access to Ψ as the principal challenge. Here, we show selective and atom-economic 1N-5C rearrangement of ß-D-ribosyl on uracil to obtain Ψ from unprotected U in quantitative yield. One-pot cascade transformation of U in four enzyme-catalyzed steps, via D-ribose (Rib)-1-phosphate, Rib-5-phosphate (Rib5P) and Ψ-5'-phosphate (ΨMP), gives Ψ. Coordinated function of the coupled enzymes in the overall rearrangement necessitates specific release of phosphate from the ΨMP, but not from the intermediary ribose phosphates. Discovery of Yjjg as ΨMP-specific phosphatase enables internally controlled regeneration of phosphate as catalytic reagent. With driving force provided from the net N-C rearrangement, the optimized U reaction yields a supersaturated product solution (∼250 g/L) from which the pure Ψ crystallizes (90% recovery). Scale up to 25 g isolated product at enzyme turnovers of ∼105 mol/mol demonstrates a robust process technology, promising for Ψ production. Our study identifies a multistep rearrangement reaction, realized by cascade biocatalysis, for C-nucleoside synthesis in high efficiency.

Assessment by differential display-RT-PCR of mRNA transcript transitions and alpha-amanitin sensitivity during bovine preattachment development.

The objectives of this study were to compare patterns of mRNA expression, investigate the onset of transcription, and isolate stage-specific and alpha-amanitin-sensitive mRNAs during early bovine development by differential-display-reverse transcription-polymerase chain reaction (DD-RT-PCR). Embryos representing a preattachment developmental series from the 1-cell to the expanded/hatched blastocyst stage were cultured in synthetic oviduct fluid medium + citrate and amino acids (cSOFMaa) with and without alpha-amanitin (100 microg/mL) for 4 and 12 hr. mRNA profiles were displayed by DD-RT-PCR using 5' primers A and N. Total conserved cDNA banding patterns varied according to embryo stage with cDNA band numbers declining during early cleavage stages compared to oocyte values and then increasing in total number from the 6-8-cell stage through to the blastocyst stage. A cDNA banding pattern was established at the 8-16-cell stage that was largely unchanged through to the blastocyst stage. These findings with respect to cDNA banding patterns were conserved between oligo primer sets and experimental replicates. alpha-Amanitin sensitivity was first detected at the 2-5-cell stage but became predominant following the 6-8-cell stage of development to eventually affect the appearance of up to 40% of all cDNA bands by the blastocyst stage. A 12 hr alpha-amanitin treatment was required to effectively block (3)H-uridine incorporation into mRNA in blastocyst stage embryos. Several stage-specific and alpha-amanitin-sensitive cDNAs were isolated and they will be a focus for future studies. In conclusion, DD-RT-PCR is an effective tool for contrasting gene expression patterns and isolating uncharacterized mRNA transcripts during bovine early development. Mol. Reprod. Dev. 55:152-163, 2000.

In vitro assessment of salvage pathways for pyrimidine bases in rat liver and brain.

In this paper we extend our previous observation on the mobilization of the ribose moiety from guanosine to xanthine catalyzed by rat liver extracts (Giorgelli et al., Biochim. Biophys. Acta 1335 (1997) 16-22). The data show that in rat liver and brain extracts the activated ribose, stemming from inosine and guanosine phosphorolysis as ribose 1-phosphate, can be used to salvage uracil to uracil nucleotides. Uridine is an intermediate. The salvage process occurs even in the presence of excess inorganic phosphate suggesting that uridine phosphorylase may function in vivo as an anabolic enzyme. Ribose 5-phosphate cannot substitute for inosine, guanosine or ribose 1-phosphate as ribose donor. When inorganic phosphate was substituted with arsenate, hindering the formation of ribose 1-phosphate, no ribose transfer could be observed. A similar pathway occurs at the deoxy level. The deoxyribose moiety of deoxyinosine can be used to salvage thymine to thymine nucleotides, again in the presence of excess inorganic phosphate. Our results introduce a novel aspect of the salvage pathway, in which ribose 1-phosphate seems to play a pivotal role.

An autoradiographic assessment of the incorporation of H3-uridine into DNA and RNA of osteoblasts of aging mice.

Intracellular labeling of DNA and RNA after H3-uridine administration was investigated autoradiographically during aging. Five to 78 weeks old mice were injected with 5 muCi of H3-uridine/gm of body weight and were killed from 15 minutes to 30 days later. Five mum decalcified sagittal sections of femora were treated with RNAase, DNAase or appropriate buffers. Autoradiographs were prepared and grain counts were made over diaphyseal periosteal osteoblasts. Both DNA and RNA incorporated H3-uridine. RNA label was 88 to 95% of the total cell label. DNA labeling ranged from 2 to 5%. DNA labeling appeared slightly increased after longer exposure to H3-uridine, whereas, RNA labeling remained relatively unaltered. With increasing age, incorporation into DNA decreased, whereas, RNA label showed a slight increase. A variable amount of non-specific label was undigested by either enzyme and may reflect insoluble conversion and/or degradation products. Apparently, some conversion of uridine into a DNA precursor occurs without loss of tritium label, thus rendering uridine less than totally specific for RNA. Nevertheless, the uptake of H3-uridine is largely indicative of RNA biosynthesis especially in skeletal cells that are not normally highly proliferative.

An assessment of the effects of hormones on short term organ cultures of human breast carcinomata.

Twenty-eight mammary carcinomata were maintained in organ culture in the presence of various hormones. The effects of the hormones have been assessed histologically by estimation of total dehydrogenases activity of the pentose glycolytic pathway and by the incorporation of tritiated thymidine or uridine into DNA or RNA. No significant effects on tumour cell activity due to hormones have been observed.