Cytosine modification

Fig. 5 Structures of various modifications studied for optimal TLR9-mediated immune response. Cytosine modifications C 2 -dC, Cj 5-hydroxy-2 -dC, C2 5-propyne-2 -dC, C3 furano-2 -dT, C4 pyrrolo-2 -dC, C5 dF, Cg 4-thio-2 -dU, Cy N -methyl-2 -dC, Cs N -ethyl-2 -dC, C9 P-iso-2 -dC, and Cio arabinoC. Guanine modifications G 2 -dG, Gj 7-deaza-2 -dG, Gy 7-deaza-8-aza-2 -dG, G3 9-deaza-2 -dG, G4 6-(9-methyl-2 -dG, G5 N -methyl-2 -dG, Gg N -methyl-2 -dG, Gy 6-thio-2 -dG, Gs 2 -deoxyinosine, G9 8-G-methyl-2 -dG, Gw 8-G-allyl-2 -dG, and Gn arabinoG. Linkers (L) Lj glycerol (1,2,3-propanetriol), Ly (5)-(-)-l,2,4-butanetriol, L3 1,3,5-pentanetriol, L4 cis, cis-l,3,5-cyclohexanetriol, L cis,trans-l,3,5-cyclohexanetriol, Lg l,3,5-tris(2-hydroxyethyl) isocyanurate, Ly tetraethyleneglycol, and Lg hexaethyleneglycol...

Figure 1.43 indicates major sites of reactivity within the ring structures for nucleophilic displacement reactions. Cytosine, thymine, and uracil all react toward nucleophilic attack at the same two sites, the C-4 and C-6 positions. The presence of powerful nucleophiles, even at neutral pH, can lead to significant base modification or cleavage with pyrimidine residues (Debye, 1947). For instance, hydrazine spontaneously adds to the 5,6-double bond, initiating further ring reactions,... 

Figure 1.45 Reaction of bisulfite with cytosine bases is an important route of derivatization. It can lead to uracil formation or, in the presence of an amine (or hydrazide) containing compound, transamination can occur, resulting in covalent modification.

As in the case of pyrimidine bases discussed previously, adenine and guanine are subject to nucleophilic displacement reactions at particular sites on their ring structures (Figure 1.50). Both compounds are reactive with nucleophiles at C-2, C-6, and C-8, with C-8 being the most common target for modification. However, the purines are much less reactive to nucleophiles than the pyrimidines. Hydrazine, hydroxylamine, and bisulfite—all important reactive species with cytosine, thymine, and uracil—are almost unreactive with guanine and adenine. 

Since the site of modification on cytosine bases is at a hydrogen bonding position in double helix formation, the degree of bisulfite derivatization should be carefully controlled. Reaction conditions such as pH, diamine concentration, and incubation time and temperature affect the yield and type of products formed during the transamination process. At low concentrations of diamine, deamination and uracil formation dramatically exceed transamination. At high concentrations of diamine (3M), transamination can approach 100 percent yield (Draper and Gold, 1980). Ideally, only about 30-40 bases should be modified per 1,000 bases to assure hybridization ability after derivatization. 

Bisulfite modification of cytosine residues also can be used to add permanently a sulfone group to the C-6 position. In this scheme, the sulfone functions as a hapten recognizable by specific anti-sulfone antibodies. At high concentrations of bisulfite and in the presence of methyl-hydroxylamine, cytosines are transformed into N4-methoxy-5,6-dihydrocytosine-6-sulphonate derivatives (Herzberg, 1984 Nur et al., 1989). Labeled antibodies can then be used to detect the hybridization of such probes. 

Prepare bisulfite modification solution consisting of 3 M concentration of a diamine (i.e., ethylenediamine), 1M sodium bisulfite, pH 6. The use of the dihydrochloride form of the diamine avoids having to adjust the pH down from the severe alkaline pH of the free-base form. Note The optimum pH for transaminating biotin-hydrazide to cytosine residues using bisulfite is 4.5 (see Section 2.3, this chapter). 

Biotin-dUTP derivatives are formed by modification of the C-5 position of uridine. This location is not involved in hydrogen bonding activity with complementary DNA strands, thus hybridization efficiency is not immediately compromised. By contrast, biotin-dCTP or biotin-dATP derivatives involve modification of the bases at the N-4 position of cytosine and the N-6 position of adenine, locations directly involved in hydrogen bond formation with complementary bases. Thus, DNA biotinylation through the use of modified deoxynucleoside triphosphates to be incorporated into existing DNA strands may result in better activity of the probe if dUTP is used over dATP or dCTP. 

Biotin-Hydrazide Modification of Bisulfite-Activated Cytosine Groups... 

Fig. 9. The transacetylase ribozyme. A Secondary structure of the clone 11 transacylase ribozyme based on the Zuker RNA folding algorithm Mfold. The oligonucleotide substrate is shaded in gray. The 2 -OH group of cytosine 147 (arrow) is the site of modification of the oligonucleotide substrate. B Reaction catalyzed by the clone 11 transacylase ribozyme. Note that the equilibrium of the reaction lies strongly on the side of the Bio-Phe-AMP substrate...

The story of DNA replication is followed by one of DNA modification. The most important chemical modification of DNA is methylation that is, the methyl group, CH3—, is added to two of the bases of DNA, adenine (A) and cytosine (C). Three modified bases are formed. 

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