Synthesis of a Fluorescent Cytidine TNA Triphosphate Analogue
Threose nucleic acid (TNA) is refractory to nuclease digestion and capable of undergoing Darwinian evolution, which together make it a promising system for diagnostic and therapeutic applications that require high biological stability. Expanding the seque
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Introduction Threose nucleic acid (TNA) is an artificial genetic polymer that has a backbone structure composed of repeating units of α-L-threose sugars that are vicinally linked by 20 ,30 -phosphodiester bonds (Fig. 1a) [1]. Despite a backbone repeat unit that is one atom (or bond) shorter than that of DNA or RNA, TNA is capable of forming stable antiparallel Watson-Crick duplex structures with itself and with complementary strands of DNA and RNA [2, 3]. Similar to DNA/RNA, TNA is also able to undergo Darwinian evolution in vitro to produce affinity reagents with strong ligand-binding activity [4]. This property, coupled with a backbone structure that is refractory to nuclease digestion [5], makes TNA an excellent candidate for therapeutic and diagnostic applications [6].
Nathaniel Shank (ed.), Non-Natural Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 1973, https://doi.org/10.1007/978-1-4939-9216-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Hui Mei and John Chaput
Fig. 1 Molecular structures, (a) constitutional structure for the linearized backbone of DNA and TNA, (b) Watson-Crick base pairs for C:G and Cf:G. Cf is the cytosine analogue, 1,3-diaza-2-oxo-phenothiazine
Recently, we have developed an engineered polymerase from the thermophilic species Thermococcus kodakarensis, termed Kod-RI, which can copy DNA templates into TNA in just 3 h [7]. However, despite the enhanced activity of Kod-RI over previous TNA polymerases, the synthesis of completely unbiased TNA libraries remains limited by chain termination events that occur when the polymerase encounters sequential G-nucleotides in the DNA template [8]. As a possible solution to this problem, we sought to develop analogues of TNA cytidine triphosphate (tCTP) that would stabilize the tC:dG base pair in the enzyme active site. We found that the cytidine triphosphate analogue 1,3-diaza-2-oxo-phenothiazine (tCfTP), a fluorescent tricyclic aromatic ring system that maintains Watson-Crick base pairing with guanine (Fig. 1b) [9, 10], can efficiently read through sequential G-nucleotides in a polymerase-mediated TNA synthesis reaction [11]. TNA templates synthesized with tCfTP replicate with 98.4% overall fidelity, indicating that in vitro selection experiments could be performed using fluorescent 1,3-diaza-2-oxo-phenothiazine as a modified base [11]. Together, these results provide a platform for synthesizing unbiased TNA libraries with enhanced hydrophobic and fluorescent properties. In this chapter, we provide the protocol for chemical synthesis of 1,3-diaza-2-oxo-phenothiazine TNA nucleoside 30 -triphosphate (tCfTP). To this end, protected α-L-threofuranosyl-1,3-diaza-2oxo-phenothiazine nucleoside was prepared from the universal glycosyl donor and 1,3-diaza-2-oxo-phenothiazine in a Vorbru¨ggen glycosylation reaction [12]. After deprotection, nucleoside was subsequently converted to 30 -monophosphate, which was then converted to 30 -phosphoro-(2-methyl)-imidazolide [13]. Subsequent displacement of
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