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. 2001 Jun 15;29(12):2456-63.
doi: 10.1093/nar/29.12.2456.

Synthesis and properties of oligonucleotides containing 5-formyl-2'-deoxycytidine: in vitro DNA polymerase reactions on DNA templates containing 5-formyl-2'-deoxycytidine

N Karino  1 , Y Ueno, A Matsuda
Affiliations

Synthesis and properties of oligonucleotides containing 5-formyl-2'-deoxycytidine: in vitro DNA polymerase reactions on DNA templates containing 5-formyl-2'-deoxycytidine

N Karino et al. Nucleic Acids Res. .

Abstract

Oligodeoxynucleotides (ODNs) containing 5-formyl-2'-deoxycytidine (fC) were synthesized by the phosphoramidite method and subsequent oxidation with sodium periodate. The stabilities of duplexes containing A, G, C or T opposite fC were studied by thermal denaturation. It was found that fC:A, fC:C or fC:T base pairs significantly reduce the thermal stabilities of duplexes. Next, single nucleotide insertion reactions were performed using ODNs containing fC as templates and the Klenow fragment of Escherichia coli DNA polymerase I. It was found that: (i) insertion of dGMP opposite fC appears to be less efficient relative to insertion opposite 5-methyl-2'-deoxycytidine (mC); (ii) dAMP is misincorporated more frequently opposite fC than mC, although the frequency of misincorporation seems to be dependent on the sequence; (iii) TMP is misincorporated more frequently opposite fC than mC. These results suggest that fC may induce the transition mutation C.G-->T.A and the transversion mutation C.G-->A.T during DNA synthesis.

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Figures

Figure 1
Figure 1
Structures of the modified nucleosides.
Figure 2
Figure 2
Sequences of ODNs.
Figure 3
Figure 3
Primer extention assay to identify the nucleotide inserted opposite fC by KF (exo–) and KF (exo+). Lanes 2–13, KF (exo–); lanes 15–26, KF (exo+). Lanes 2–5 and 15–18, template 1; lanes 6–9 and 19–22, template 2; lanes 10–13 and 23–26, template 3. Lanes 2, 6, 10, 15, 19 and 23, dCTP; lanes 3, 7, 11, 16, 20 and 24, TTP; lanes 4, 8, 12, 17, 21 and 25, dATP; lanes 5, 9, 13, 18, 22 and 26, dGTP. Lanes 1 and 14, primer 1. Experimental conditions are described in Materials and Methods.
Figure 4
Figure 4
Primer extention assay to identify the nucleotide inserted opposite fC by KF (exo–) and KF (exo+). Lanes 2–13, KF (exo–); lanes 15–26, KF (exo+). Lanes 2–5 and 15–18, template 4; lanes 6–9 and 19–22, template 5; lanes 10–13 and 23–26, template 6. Lanes 2, 6, 10, 15, 19 and 23, dCTP; lanes 3, 7, 11, 16, 20 and 24, TTP; lanes 4, 8, 12, 17, 21 and 25, dATP; lanes 5, 9, 13, 18, 22 and 26, dGTP. Lanes 1 and 14, primer 2. Experimental conditions are described in Materials and Methods.
None
Scheme 1. Conditions: (a) Bu3SnCH=CH2, (Ph3P)2PdCl2, DMF, 80°C, 1.5 h; (b) (1) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt, 28 h; (2) conc. NH4OH, 0°C–rt, 1 h, 67% (three steps); (c) OsO4, 4-methylmorpholine-N-oxide, acetone-H2O-t-BuOH (4:1:1), rt, 4 h, 77%; (d) Ac2O, DMAP, py, rt, 20 h, 93%; (e) TBAF, THF, 0°C–rt, 6 h, 87%; (f) i-Pr2NP(Cl)O(CH2)2CN, i-Pr2NEt, CH2Cl2, 0°C–rt, 0.5 h, 76%.
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