mRNA incorporation of C(5)-halogenated pyrimidine ribonucleotides and induced high expression of corresponding protein for the development of mRNA vaccine.

In this report, we present our studies on mRNA, which was modified by introducing various halogen substituents at the C(5) position of the pyrimidine base. Specifically, we synthesized C(5)-halogenated (F, Cl, Br, I) pyrimidine ribonucleoside triphosphates and incorporated them into mRNA during in-vitro transcription. The efficiency of the in-vitro transcription reaction of halogenated pyrimidine was observed to decrease as the size of the halogen substituent increased and the electronegativity thereof decreased (F > Cl > Br) except for iodine. Interestingly, we found that, among the C(5)-halogenated pyrimidine ribonucleotides, mRNA incorporating C(5)-halogenated cytidine (5-F rCTP and 5-Cl rCTP) exhibited more prominent protein expression than mRNA modified with C(5)-halogenated uridine and unmodified mRNA. In particular, in the case of mRNA to which fluorine (5-F rCTP) and chlorine (5-Cl rCTP) were introduced, the protein was dramatically expressed about 4 to 5 times more efficiently than the unmodified mRNA, which was similar to pseudouridine (ψ). More interestingly, when pseudouridine(ψ) and fluorocytidine nucleotides (5-F rCTP), were simultaneously introduced into mRNA for dual incorporation, the protein expression efficiency dramatically increased as much as tenfold. The efficiency of cap-dependent protein expression is much higher than the IRES-dependent (internal ribosome entry site) expression with mRNA incorporating C(5)-halogenated pyrimidine ribonucleotide. We expect these results to contribute meaningfully to the development of therapeutics based on modified mRNA.

Loop-mediated fluorescent probes for selective discrimination of parallel and antiparallel G-Quadruplexes.

Herein we report simple pyridinium (1-3) and quinolinium (4) salts for the selective recognition of G-quadruplexes (G4s). Among them, the probe 1, interestingly, selectively discriminated parallel (c-KIT-1, c-KIT-2, c-MYC) G4s from anti-parallel/hybrid (22AG, HRAS-1, BOM-17, TBA) G4s at pH 7.2, through a switch on response in the far-red window. Significant changes in the absorption (broad 575 nm â†’ sharp 505 nm) and emission of probe 1 at 620 nm, attributed to selective interaction with parallel G4s, resulted in complete disaggregation-induced monomer emission. Symmetrical push/pull molecular confinements across the styryl units in probe 1 enhanced the intramolecular charge transfer (ICT) by restricting the free rotation of CC units in the presence of sterically less hindered and highly accessible G4 surface/bottom tetrads in the parallel G4s, which is relatively lower extent in antiparallel/hybrid G4s. We confirm that the disaggregation of probe 1 was very effective in the presence of parallel G4-forming ODNs, due to the presence of highly available free surface area, resulting in additional π-stacking interactions. The selective sensing capabilities of probe 1 were analyzed using UV-Vis spectroscopy, fluorescence spectroscopy, molecular dynamics (MD)-based simulation studies, and 1H NMR spectroscopy. This study should afford insights for the future design of selective compounds targeting parallel G4s.