- Efficient Automated Solid-Phase Synthesis of DNA and RNA 5′-Triphosphates
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A fast, high-yielding and reliable method for the synthesis of DNA- and RNA 5′-triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5-chloro-saligenyl-N,N-diisopropylphosphoramidite was coupled to the 5′-end. Oxidation of the formed 5′-phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5′-cycloSal-oligonucleotides. Reaction of the support-bonded 5′-cycloSal-oligonucleotide with pyrophosphate yielded the corresponding 5′-triphosphates. The 5′-triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.
- Sarac, Ivo,Meier, Chris
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- Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery
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Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.
- Baladi, Tom,Nilsson, Jesper R.,Gallud, Audrey,Celauro, Emanuele,Gasse, Cécile,Levi-Acobas, Fabienne,Sarac, Ivo,Hollenstein, Marcel R.,Dahlén, Anders,Esbj?rner, Elin K.,Wilhelmsson, L. Marcus
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- Operationally simple regioselective 59-phosphorylation of unprotected 5-ethynyl-29-deoxyuridine analogues
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Here, we present the development of a straightforward methodology to regioselectively phosphorylate the 50-OH group of unprotected nucleosides. We employ cyclosaligenyl phosphoramidite reagents together with pyridinium trifluoroacetate as activator, followed by in situ oxidation to prepare a panel of novel nucleoside-based chemical probes, ProLabel compounds 4-15. Alternative procedures for this transformation are available, but are limited in number and scope. Furthermore, the benefits of the new methodology include milder reaction conditions, wider solvent applicability, and, by avoiding sensitive reagents, a more straightforward handling of reagents, reactions, and workup processes. The panel of novel cyclosaligenyl phosphotriester uridine ProLabels have variable 20 substituents (H, F, Cl, Br, I) as well as four different cyclosaligenyl groups that would span a range of half-lives for in vitro applications.
- Hilko, David H.,Bornaghi, Laurent F.,Poulsen, Sally-Ann
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p. 1010 - 1019
(2020/05/26)
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