Abstract
The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoCMe) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)–DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoCMe bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoCMe misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoCMe/isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.
Highlights
Encoding heritable information in polymers other than DNA and RNA in vivo will result in the creation of XNA (Xeno nucleic acid) modified organisms (XMO’s) unable to communicate this [b] Dr D
We have developed an efficient route for the synthesis of oligonucleotides containing h-isoG and h-isoCMe nucleotides synthesised on solid-phase support by using phosphoramidite building blocks 13 and 25, respectively
The amidite of h-isoCMe 25 has been synthesised from commercially available 1,5:2,3-dianhydro-4,6-O-benzylidene-d-allitol, through the formation of an intermediate anhydro sugar 18 that was regioselectively opened with ammonia as a key step
Summary
Encoding heritable information in polymers other than DNA and RNA in vivo will result in the creation of XNA (Xeno nucleic acid) modified organisms (XMO’s) unable to communicate this [b] Dr D. To investigate the selectivity of base pairing of h-isoG and h-isoCMe, the four canonical nucleosides (dA, dT, dC, and dG) were placed opposite to the modification site, and Tm values were measured (Table 2). We wondered what the influence would be on duplex stability when a natural nucleoside is incorporated opposite a hexitol nucleoside with an isoG or isoCMe base.
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