Synthesis of DNA Duplexes Containing Site-Specific Interstrand Cross-Links via Sequential Reductive Amination Reactions Involving Diamine Linkers and Abasic Sites on Complementary Oligodeoxynucleotides

Abstract

Interstrand DNA cross-links are important in biology, medicinal chemistry, and materials science. Accordingly, methods for the targeted installation of interstrand cross-links in DNA duplexes may be useful in diverse fields. Here, a simple procedure is reported for the preparation of DNA duplexes containing site-specific, chemically defined interstrand cross-links. The approach involves sequential reductive amination reactions between diamine linkers and two abasic (apurinic/apyrimidinic, AP) sites on complementary oligodeoxynucleotides. Use of the symmetrical triamine, tris(2-aminoethyl)amine, in this reaction sequence enabled the preparation of a cross-linked DNA duplex bearing a derivatizable aminoethyl group.

Figure 1.

Synthesis of a DNA duplex containing a site-specific interstrand cross-link via sequential reductive amination reactions involving a 1,4-diaminobutane linker and complementary AP-containing oligodeoxynucleotides. The 5’-³²P-labeled oligonucleotides were resolved by electrophoresis on a 20% denaturing polyacrylamide gel and the radioactivity in each band visualized and quantitatively measured by storage phosphor autoradiography. Lane 1: 5’-³²P-labeled AP-containing oligodeoxynucleotide 1 as a size marker. Lane 2: Treatment of the AP-containing oligodeoxynucleotide 1 with piperidine (1 M, 95 °C, 25 min) to generate the strand cleavage product containing a 3’-phosphoryl group.^, Lane 3: Reaction of the 5’-³²P-labeled AP-containing oligodeoxynucleotide 1 with 1,4-diaminobutane and NaBH₃CN (250 mM) at 37 °C for 12 h in HEPES buffer (50 mM, pH 7.4) containing NaCl (100 mM). Lane 4: Generation of the cross-linked duplex 1/2 by reaction of the 5’-³²P-labeled oligodeoxynucleotide bearing the pendent amino group (the product from lane 3) and the complementary AP-containing oligodeoxynucleotide 2 with NaBH₃CN (250 mM) at 37 °C for 24 h in HEPES buffer (50 mM, pH 7.4) containing NaCl (100 mM). The asterisk (*) indicates a 5’-³²P-phosphoryl group. The minor band marked by the “#” symbol was assigned to a small amount of a lower molecular weight ICL (3) resulting from cross-linking of the pendent amine produced in the first step to a 3’-α,β-unsaturated sugar remnant generated by a small amount of strand cleavage of the unlabeled AP-containing oligonucleotide 2.

Figure 2.

Effects of AP/AP spacing in duplex DNA on the yields of AP-AP ICL formation with the 1,4-diaminobutane linker. X = AP in the sequences shown. The 16 nucleotide, AP-containing sequences shown above are part of 35 nucleotide duplexes with the same flanking sequences shown for duplex 1/2 at the bottom of Figure 1. The error bars represent the standard deviation calculated from at least three separate measurements.

Figure 3.

Various linkers function effectively in the AP/AP cross-linking protocol using duplex 1/2 (shown above in Figure 1). The linkers from top to bottom are 1,4-diaminobutane (A), 1,5-diaminopentane (B), 1,6-diaminohexane (C), and tris(2-aminoethyl)amine (D). The error bars represent the standard deviation calculated from at least three separate measurements.

Figure 4.

Conjugation of biotin to the derivatizable NH₂-ICL. In these experiments, the 5’-³²P-labeled NH₂-ICL (prepared using the tris(2-aminoethyl)amine linker) was analyzed by electrophoresis on a 20% denaturing polyacrylamide gel and the radioactivity in each band visualized and quantitatively measured by storage phosphor autoradiography. The schematic diagram simplifies the chemical structure of the biotin-linker conjugate. The actual structure of the biotin-NHS reagent 12 showing the complete linker is shown below. The asterisk (*) indicates a 5’-³²P-phosphoryl group. Lane 1: 5’-³²P-labeled NH₂-ICL as a marker lane. Lane 2: Control experiment in which NH₂-ICL was incubated with the biotin-binding protein streptavidin for 12 h at 37 °C. Lane 3: Reaction of NH₂-ICL with the biotin-NHS reagent 12 in sodium bicarbonate buffer (12.5 mM, pH 8) for 12 h at 4 °C. Lane 4: Incubation of the biotin-NHS-treated NH₂-ICL with streptavidin for 12 h at 37 °C, to generate the gel-shifted NH₂-ICL-biotin•streptavidin complex. The minor band marked by the “#” symbol was assigned to a small amount of a lower molecular weight ICL resulting from cross-linking of the pendent diamine produced in the first step to the 3’-α,β-unsaturated sugar remnant generated by a small amount of strand cleavage of the unlabeled AP-containing oligonucleotide 2.

Figure 5.

Structures discussed in this work.

Scheme 1.

Reductive amination reactions involving an amine and a DNA abasic (AP) site yield a chemically stable alkylamine attachment to the deoxyribose backbone.

Scheme 2.

Sequential reductive amination reactions involving 1,4-diaminobutane and complementary AP-containing oligodeoxyribonucleotides produce high yields of a site-specific AP-AP ICL in duplex DNA. cDNA = complementary AP-containing strand.