Multiple Applications of Deoxyxanthosine Modified Oligonucleotides

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Deoxyxanthosine (dX) is the product of oxidative deamination of deoxyguanosine and as such has been incorporated into oligonucleotides using the amidite below (2164) in order to study the effects of this type of mutation.1

dX(2164)

It is known that the deamination reaction is a result of the reaction of nitric oxide with dG hence is often used in the study of the role of dX in nitric oxide mediated mutations. It is notable that where dX is paired with dC no mutation arises since the dX residue is removed via base exclusion repair mechanism and is converted back to dG. However if dX is paired with dT this results in a transition from dG to dA.

dX mutations in DNA are also repaired by E.Coli. endonuclease V where the second phosphodiester bond 3’ with respect to the dX residue is cleaved resulting in a 3’-OH and a 5’-phosphate at the nick site.3

Deoxyxanthosine is well known as a universal base3 with the order of stability in terms of base pairing being T>C>A=G which slightly differs from the stability with respect to xanthosine; T>G>A=C. The possibility of extending the genetic alphabet using dX when paired with 2,4-diaminopyrimidine has also been shown.4 The ability of deoxyxanthosine to act as a universal base has proved useful in terms of triplex stabilisation and has been shown to form stable triplexes when opposite all four natural bases.5

Incorporation of this modifier into an oligonucleotide is straightforward: 0.1M amidite in MeCN with a coupling time of 3min. Deprotection is a two-step process where the oligonucleotide is cleaved from the support and deprotection of the nucleobases and phosphate backbone takes place using ammonium hydroxide, RT, 24h. It is therefore important to use fast deprotection on the standard bases (e.g. dCAc, dABz, dGdmf). The second deprotection step removes the NPE groups by treatment with 0.3M tetramethylguanidinium 2-nitrobenzaldoximate in water/dioxane (1:1) at 70°C for 48h. Although other protecting groups have been used, NPE protection remains the most effective6,7.

While the use of allyl6 has been shown to be an excellent protecting group, the practicalities of using Pd(0) to remove these outweighs the benefit.

  1. Biochemistry, 2003, 42(12), 3608-3616.
  2. Mut. Res., 2000, 459(2), 109-114.
  3. Nucleic Acids Res. 1986, 14, 8135-8153.
  4. Nucleic Acids Res. 1996, 24, 1308-1313.
  5. Bio. Org. Med. Chem., 2004, 12(24), 6581-6586.
  6. Helv. Chim. Acta, 2006, 89(11), 2974-2814.
  7. Helv. Chim. Acta, 2000, 83, 1517.

 

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