No abstract

Modification of the DNA backbone by replacement of a single phosphate with an alkylamine is shown to generate a selective ligation catalyst. Related backbone modifications can be chemically read from a complementary DNA sequence. With this demonstration, it now appears possible to generate a two‐stage replication cycle (see scheme), a simplified version of the biological three‐stage DNA–RNA–protein cycle necessary for biological replication.

The chemical methods available for the ligation of complementary oligomers along a DNA template are complex, requiring transition metal and imidazole catalysis of the condensation reaction. We were interested in simplifying the required conditions by converting the DNA template itself into a better catalyst, and report a modified backbone linkage which greatly simplifies the reaction. As related backbone linkages have been prepared by chemically reading the DNA sequence, this reaction completes a replication cycle, simplifying the ubiquitous three-stage replication cycle, the central dogma of biology, [1] to a two-stage chemical process.Several laboratories have contributed to optimizing the reaction conditions for template-directed phosphodiester formation. [2] In the BrCN, imidazole, NiCl 2 cocktail, BrCN has a half-life of several minutes in the aqueous media. Premixing the cocktail generates N-cyanoimidazole, the reagent required to activate ligation. [2d,e] While more stable than BrCN under the reaction conditions, displaying a halflife of several hours, N-cyanoimidazole will chemoselectivity ligate a nick site along a DNA template. Many aromatic drugs are known to bind tightly within the narrow minor groove of DNA, particularly A/T tracts, [3] including netropsin, distamycin, Hoechst 33258, and the bis-amidine compounds benenil and pentamidine, and do so by displacing specific wellordered H 2 O molecules known to line this cavity. [4] It is therefore possible that N-cyanoimidazole associates with the nick site better than BrCN to facilitate the activation and/or the ligation steps in the condensation.In an attempt to replicate this effect by template modification, five synthetic templates, T N±H , T N±pr , T N±bu , T N±im , and T N±ea , were compared with the native DNA (T P ) under ligation conditions (Figure 1). Each template was prepared N OH O P O O O S 2 S 1 R H N H N NH 2 T N-pr : R = T N-H : R = H Template Substrates 3' 5' 5' 3' T N-im : R = T N-ea : R = T N-bu : R = Figure 1. The ternary complex formed by template and complementary substrates, 8 mer S 1 and 12 mer S 2 . The sequence of the template is HO-dCpCpGpTpTpCpGpTpTpTpTXTpCpTpGpTpCpTpCpG-OH, S 1 is HO-dApCpGpApApCpGpGp-OH, and S 2 is HO-dCpGpApGpApCp-ApGpApApApA-OH. X in the sequence represents the site of insertion of the various linkage structures. In Tp, X represents the native phosphodiester linkage of DNA.with the appropriate synthetic amine thymidine dimer incorporated into standard solid-phase synthesis protocols. [5d] The 20 mer templates were designed such that the complementary DNA substrates, S 1 and S 2 , displayed high binding affinity with the template. Thermal melting analyses indeed established that the melting temperature for all template ± substrate duplexes T/S 1 (35 AE 2 8C) and T/S 2 (45 AE 2 8C) were very similar and > 10 8C above the reaction temperature. A 1:1:1 stoichiometry therefore generated significant and equivalent concentrations of the ternary complexes (T:S 1 :S 2 ) for each reaction.All modified templates...