Stereospecific synthesis and characterization of oligodeoxyribonucleotides containing an N2-(1-carboxyethyl)-2'-deoxyguanosine

Abstract

Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.

Figure 1

CD spectra of standard S-N²-CEdG and R-N²-CEdG (50 μM).

Figure 2

HPLC traces for the separation of the synthesized 12mer ODNs: (a) d(GAGTAGYATGAG), Y=O⁶-tse-S-N²-CEdG; (b) d(GAGTAGXATGAG), X= S-N²-CEdG.

Figure 3

HPLC traces for the separation of the synthesized 20mer ODNs: (a) d(ATGGAYCACTATGATCCTAG), Y=O⁶-tse-S-N²-CEdG; (b) d(ATGGAXCACTATGATCCTAG), X= S-N²-CEdG.

Figure 4

ESI-MS and MS/MS characterizations of d(GAGTAGXATGAG), X=S-N²-CEdG: (a) Negative-ion ESI-MS; (b) product-ion spectrum of the [M-4H]⁴⁻ ion (m/z 960.6).

Figure 5

HPLC traces for the separation of: (a) the mixture of dG, dT and dA; (b) R-N²-CEdG; (c) S-N²-CEdG; (d) the enzymatic digestion mixture of ODN2; and (e) the enzymatic digestion mixture of ODN1. A UV detector was set at 260 nm for monitoring the effluents. The presence of residual adenosine deaminase in the commercial enzymes caused the deamination of some dA to dI.

Figure 6

Plots of 1/T_m vs. ln(C_t/4) for the duplex ODNs containing dG and N2-CEdG adducts. The duplex is d(GAGTAGXATGAG)/d(CTCATCCTACTC), where X represents dG(■), S-N²-CEdG(●) and R-N²-CEdG(▲).

Figure 7

In vitro replication studies of N²-CEdG-bearing and control undamaged substrates with Klenow fragment of E. coli polymerase I (X represents S-N²-CEdG, R-N²-CEdG or unmodified dG). The concentrations of the primer and template were 5 and 10 nM, respectively, and the reaction volume was 20 μL. (a) The primer extension was carried out in the presence of all four dNTPs at a concentration of 200 μM each, and the amounts Kf⁻ were as indicated in the figure. The reactions were continued at 37°C for 60 min. (b) The reaction was performed in the presence of one type of dNTP at a time ([dNTP] =1.0 mM) at 37°C for 10 min, and 0.1 unit of Kf⁻ was used.

Scheme 1

Formation of N²-CEdG.

Scheme 2

Postoligomerization method for the incorporation of N²-CEdG into ODNs (“*” indicates the chiral carbon).