Novel 3'-O-fluorescently modified nucleotides for reversible termination of DNA synthesis

Novel 3'-O-fluorescently modified nucleotides for reversible termination of DNA synthesis
DNA; fluorescence; next-generation sequencing; nucleotides; reversible terminator; sequencing-by-synthesis
Issue Date
Chembiochem : a European journal of chemical biology
VOL 11, NO 1, 75-78
In summary, we synthesized and characterized 3’-O-modified nucleoside triphosphates, wherein the modification is both fluorescent and chemically cleavable to regenerate a free 3’-OH. Two allyl-based linkages of different lengths were tested between the 3’-hydroxyl and the fluorophore. The MRT 9, with an appropriate linker, effectively terminated DNA synthesis after incorporation and was efficiently utilized by a commercially available polymerase, Therminator II. The coumarin moiety, a fluorescent group at the 3’-O-position, could easily be removed under aqueous conditions to restore the 3’-OH group for incorporation of the next base. In addition, fluorescence emission of the coumarin moiety could be utilized to detect incorporation of the nucleotide; this provides the proof-of-concept for use in SBS. To the best of our knowledge, this is the first successful demonstration of cyclic reversible termination by using an MRT containing a fluorophore at the 3’-O-position. The MRTbased SBS should not leave molecular scars on nucleobases, and therefore can potentially address the read-length problems that currently compromise SBS technology. As further improvements, including optimization of linkers that are easily cleavable under milder conditions and development of MRTs for all four bases, are needed to eventually integrate the strategy into a commercially viable SBS system, efforts are underway to extrapolate the synthesis of our model nucleotide to the remaining nucleobases for production of a novel, fourcolor MRT set.
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