Additive‐Free Enzymatic Phosphorylation and Ligation of Artificial Oligonucleotides with C‐Nucleosides at the Reaction Points

Abstract

We report enzymatic phosphorylation and additive‐free ligation of DNAs containing unnatural C‐nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D*, T*, G*, and C*, corresponding—from a viewpoint of hydrogen‐bonding patterns—to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5′‐end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5′‐end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3′‐natural⋅5′‐artificial, 3′‐artificial⋅5′‐natural, and 3′‐artificial⋅5′‐artificial terminal combination at the nicks.

Template-dependent DNA ligation for the synthesis of modified oligonucleotides

“…Despite impressive catalytic efficiencies 33 of around 10 µM -1 •s -1 (equating to the insertion of up to a few hundreds of nucleotides per second 34 ), DNA polymerases struggle with substrates consisting of altered sugar motifs which are not only found in most therapeutic oligonucleotides but also commonly used in chemoenzymatic approaches for de novo synthesis of DNA. On the other hand, DNA ligases have been reported to be rather tolerant to the presence of both base- [35][36][37][38][39] and sugar-modifications even on short sequences [40][41][42] which constitutes the basis of our approach. Nonetheless, existing ligation methods rely on using scarecly modified oligonucleotides of moderate size (10 -12 nt) which are already impinged by some of the aforementioned limitations.…”

Recent progress in non-native nucleic acid modifications

“…The adenylated DNA then undergoes a second nucleophilic attack by the ligase-activated 3′-OH of the acceptor sequence to form the phosphodiester bond and release AMP 256. T4 DNA ligase is capable of efficiently joining short pieces of DNA or RNA containing C-nucleotides257 and nucleobase258 as well as XNA sugar modifications such as FANA or 2′-OMe259 especially when the modified building blocks are located in the acceptor sequence. The T4 DNA ligase, however, has a limited substrate tolerance, and other ligases such as the T3 or the T7 DNA ligase can be employed instead, as was reported in the case of TNA (threose nucleic acid) containing oligonucleotides.…”

2-Aminopyridine as a Nucleobase Substitute for Adenine in DNA-like Architectures: Synthesis of Alkynyl C-Nucleotides and Their Hybridization Characteristics

“…This fact might be one of the reasons why our DNA-like oligomer closely resembles natural DNA in terms of structural and physicochemical aspects. Based on this similarity, we recently reported successful enzymatic applications of the artificial DNA with terminal deoxynucleotidyl transferase (TdT), T4 phage polynucleotide kinase (T4 PNK), and T4 DNA ligase. , …”

“…Based on this similarity, we recently reported successful enzymatic applications of the artificial DNA with terminal deoxynucleotidyl transferase (TdT), T4 phage polynucleotide kinase (T4 PNK), and T4 DNA ligase. 20,21 Various heterocycles can be introduced into our nucleoside skeleton by means of acetylene chemistry with organometallic strategy. 22−25 In this context, further modification of nonnatural bases has been carried out in order to imitate natural DNA more closely.…”