Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases - SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal.
View Article and Find Full Text PDFReactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7-deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiol-containing molecules as well as with cysteine- or histidine-containing peptides to form stable covalent products.
View Article and Find Full Text PDF2-Formyl-2'-deoxyadenosine triphosphate (d ATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of d ATP was based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The d ATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX).
View Article and Find Full Text PDFThe synthesis of novel sphingoid base-like compounds with a quaternary stereocentre was achieved in a sequence featuring [3,3]-sigmatropic rearrangements and olefin cross-metathesis transformation as the key reaction steps, which were accompanied by the rational selection of suitable functional group transformations. The stereochemistry of the desired tetra-substituted carbon bearing nitrogen functionality was determined via NOESY experiments of the advanced oxazolidine-2-thiones. Cell viability experiments revealed significant antiproliferative/cytotoxic activity of the target compounds 7, ent-7 and 29 against the Jurkat cell line, with the IC values of 6.
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