Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli

Abstract

A triazole mimic of a DNA phosphodiester linkage has been produced by templated chemical ligation of oligonucleotides functionalized with 5'-azide and 3'-alkyne. The individual azide and alkyne oligonucleotides were synthesized by standard phosphoramidite methods and assembled using a straightforward ligation procedure. This highly efficient chemical equivalent of enzymatic DNA ligation has been used to assemble a 300-mer from three 100-mer oligonucleotides, demonstrating the total chemical synthesis of very long oligonucleotides. The base sequences of the DNA strands containing this artificial linkage were copied during PCR with high fidelity and a gene containing the triazole linker was functional in Escherichia coli.

Fig. 1.

DNA linkage structures. In 1a, canonical DNA; 1b, previous triazole DNA analogue (25); 1c, biocompatible triazole analogue; 1d, click ligation to produce triazole DNA mimic 1c; and 1e polymerases read through 1b using only one of the two thymines as a template base; i.e., T_tT → T (t = triazole). In 1f, PCR copies the base sequence around the unnatural linkage 1c correctly.

Fig. 2.

Synthesis of alkyne/azide oligonucleotides for use in click ligation and cyclization. (A) Assembly of 3′-alkyne oligonucleotide. (B) Conversion to 5′-azide. Oligonucleotides can be made with 5′-azide, 3′-alkyne or both. A dinucleotide is shown for clarity but the reactions have been carried out on oligonucleotides up to 100-mer in length. (C) The 3′-Propargyl dT introduced as final addition in reverse phosphoramidite assembly of DNA.

Fig. 3.

Cyclization and RCA of 5′-azide-3′-alkyne 100-mer. (A) Reversed-phase HPLC (UV abs at 260 nm) and (B) mass spectrum (ES^-) of cyclic 100-mer ODN-31, required; 31.423 kDa, found 31.422 kDa. (C) Schematic of RCA reaction. (D) RCA product from cyclic 100-mers using phi29 DNA polymerase. Lane 1; 50 bp DNA ladder, lanes 2 and 3; RCA of cyclic triazole ODN-31 and cyclic normal ODN-49 respectively.

Fig. 4.

PCR amplification of 210- and 300-mer click-ligated triazole DNA templates. (A) Schematic representation of click ligation of three oligonucleotides. (B) Click ligation reaction: Lane 1; crude reaction mixture to synthesize 210-mer template from three 70-mers, lane 2; starting oligonucleotide ODN-16 (8% polyacrylamide gel). (C) PCR using 210-mer triazole template. Lane 1; 25 bp DNA ladder, lane 2; control PCR without click-ligated template, lane 3; PCR using 210-mer triazole template. (D) PCR using 300-mer triazole template. Lane 1; 25 bp DNA ladder, lanes 2, 3; PCR using short primers ODN-26 and ODN-27, Lanes 4, 5; PCR using long primers ODN-28 and ODN-29 (2% agarose gels with ethidium staining). (E) Sequencing data from 300-mer triazole amplicon showing that the base sequence of the template was replicated faithfully at the two ligation sites.

Fig. 5.

Assembly of a T7-Luciferase control plasmid containing click-DNA within its BLA gene. A region corresponding to the central part of BLA was PCR-amplified using oligonucleotide primers (ODN-39, ODN-41) containing triazole linkage 1c. The PCR product was ligated into the digested plasmid to give an intact construct containing triazole linkages on each strand of its BLA gene.

Fig. 6.

Biocompatability of click DNA in E. coli. (A) The plate on the left is the negative control (no insert), the middle plate contains transformants of plasmids with the triazole DNA insert in its BLA gene (127 colonies), and the plate on the right is the native plasmid (129 colonies). Twenty-one replicates of each plate were performed. (B, C) Sequencing of the BLA gene from colonies in the triazole DNA plates. In C, the is contained on the complementary strand, therefore appearing as GG. (D) Comparison of colony growth in the control (C), native (N), and triazole (T) plates. Triazole plates contained 96.5% of the colonies in the native plates (S.D. = 1.6%) whereas the negative control was 1.1% (S.D. = 1.0%).

Fig. 7.

Taq polymerase primer-template dNTP closed complex (36). (A) Schematic of interactions between the phosphodiester linkages of the DNA template and amino acids of the enzyme. Only template strand is shown. mc = main chain. (B) Canonical DNA; the majority of the interactions with the polymerase involve the branched phosphate oxygen atoms, few if any involve bridging oxygen atoms. (C) Overlay of canonical DNA and triazole linkage 1c. The N2 and N3 atoms of triazoles are good hydrogen bond acceptors (37, 38) and in principle they could substitute for the phosphate oxygen atoms. (D) Triazole linkage 1b showing the trans-configuration of the amide, with N2 and N3 of the triazole facing into the helix. Linkage 1b is significantly more rigid than 1a and 1c.