Isoguanine and 5-methyl-isocytosine bases, in vitro and in vivo

Abstract

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoC(Me) ) 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-isoC(Me) 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 isoC(Me) misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoC(Me) /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.

Keywords: HNA; XNA plasmid; isoG; nucleosides; polymerase.

Figure 1

Putative base-pair motifs of duplexes with antiparallel strand orientation.

Figure 2

Deoxyribose and hexitol isoG and isoC^Me nucleosides.

Scheme 1

Synthesis of phosphoramidite of protected deoxyribose-isoG.

Scheme 2

Synthesis of phosphoramidite of protected hexitol-isoG. a) 2,6-Diaminopurine, NaH, DMF, 90 °C, 12 h, 79 %; b) CH₃COOH/H₂O 6:4, 60 °C, 4 h, 91 %; c) NaNO₂, CH₃COOH, H₂O, 55 °C, 10 min, 76 %; d) Me₂NCH(OMe)₂, CH₃OH, RT, 12 h, 80 %; e) Ph₂NCOCl, iPr₂NEt, pyridine, RT, 1 h, 76 %, f) (MeO)₂TrCl, pyridine, 0 °C to RT, 12 h, 73 %, g) (iPr₂N)₂POC₂H₄CN, 1H-tetrazole, DCM, 0 °C to RT, 1 h, 75 %.

Scheme 3

Synthesis of phosphoramidite of protected deoxyribose-isoC^Me.

Scheme 5

Synthesis of phosphoramidite of protected hexitol-isoC^Me. a) sat. NH₃ in CH₃OH, 160 °C, 60 h, 70 %; b) Me₂NCH(OMe)₂, CH₃OH, 65 °C, 3 h, 88 %; c) Im₂S, DMF, 12 h, 88 %; d) AIBN, nBu₃SnH, toluene, 80 °C, 3 h, 69 %; e) CH₃COOH/H₂O (6:4), 60 °C, 4 h, 93 %; f) (MeO)₂TrCl, pyridine, 0 °C to RT, 12 h, 88 %; g) (iPr₂N)₂POC₂H₄CN, 1H-tetrazole, dichloromethane, 0 °C to RT, 1 h, 81 %.

Scheme 4

Synthesis of the triphosphates 26 and 28. a) i) TMP, POCl₃, 0 °C, 5 h; ii) Bu₃N, (NBu₄)₃HP₂O₅, 30 min; iii) 25 % NH₃, 2 h; b) i) Ac₂O, N,N-diisopropylethylamine, DMAP, THF, RT, 20 min; ii) dichloroacetic acid, dichloromethane, 0 °C to RT, 30 min; c) i) salicyl phosphorochloridite, pyridine/dioxane, 1 min, ii) (NBu₃)₂H₂P₂O₇, NBu₃, DMF, 30 min; iii) iodine, pyridine/water, 10 min; iv) 25 % NH₃, 2 h.

Figure 3

Phosphorimage of the enzymatic incorporation of 100 μm of 2) d-isoGTP into hybrid P1:T2 (1 d-isoC^Me) and 3) h-isoGTP into hybrid P1:T3 (1 h-isoC^Me) using 0.08 U μL⁻¹ of Pfu (exo-) and KF (exo-). 0.08 U μL⁻¹ of thermostable inorganic pyrophosphatase (TIPP) was included. 1 represents the incorporation of 10 μm dGTP into hybrid P1:T1 (dC) (positive control). Reaction time is 60 min.

Figure 4

Phosphorimage of the enzymatic incorporation of 500 μm of 2) d-isoGTP into hybrid P1:T5 (3 d-isoC^Me) and 3) h-isoGTP into hybrid P1:T6 (3 h-isoC^Me) using 0.08 U μL⁻¹ of Pfu (exo-) and KF (exo-). 0.08 U μL⁻¹ of TIPP was included. 1 represents the incorporation of 20 μm dGTP into hybrid P1:T4 (dC) (positive control). Reaction time is 60 min.

Figure 5

Response of the ligation of h-isoG (O1, O3, O5, O7) and d-isoG (O2, O4, O6, O8) containing oligomers into the gapped vector as a function of restoring the essential thymidylate synthase (thyA) gene. The ratio is taken from the number of thymidine-prototrophic colonies (bla⁺ thyA⁺) within the total number of colonies (bla⁺ thyA⁻ plus bla⁺ thyA⁺) as an average of experimental repeats. The modified part of the oligomer sequence is shown with the position of the h- and d-isoG nucleotides highlighted.

Figure 6

Response of the ligation of h-isoC^Me (O9, O11, O13, O15) and d-isoC^Me (O10, O12, O14, O16) containing oligomers into the gapped vector as a function of restoring the essential thymidylate synthase (thyA) gene. The ratio is taken from the number of thymidine-prototrophic colonies (bla⁺ thyA⁺) against the total number of colonies (bla⁺ thyA⁻ and bla⁺ thyA⁺) as an average of experimental repeats. The modified part of the oligomer sequence is shown with the position of the incorporated h- and d-isoC^Me nucleotide/s highlighted.