Assessing the biocompatibility of click-linked DNA in Escherichia coli

Abstract

The biocompatibility of a triazole mimic of the DNA phosphodiester linkage in Escherichia coli has been evaluated. The requirement for selective pressure on the click-containing gene was probed via a plasmid containing click DNA backbone linkages in each strand of the gene encoding the fluorescent protein mCherry. The effect of proximity of the click linkers on their biocompatibility was also probed by placing two click DNA linkers 4-bp apart at the region encoding the fluorophore of the fluorescent protein. The resulting click-containing plasmid was found to encode mCherry in E. coli at a similar level to the canonical equivalent. The ability of the cellular machinery to read through click-linked DNA was further probed by using the above click-linked plasmid to express mCherry using an in vitro transcription/translation system, and found to also be similar to that from canonical DNA. The yield and fluorescence of recombinant mCherry expressed from the click-linked plasmid was also compared to that from the canonical equivalent, and found to be the same. The biocompatibility of click DNA ligation sites at close proximity in a non-essential gene demonstrated in E. coli suggests the possibility of using click DNA ligation for the enzyme-free assembly of chemically modified genes and genomes.

Chemical Morphing of DNA Containing Four Noncanonical Bases

“…More recently the chemical diversification of nucleic acids has been attempted in vivo to propagate additional types of nucleic acids (XNA) as templates for DNA synthesis in E. coli and for transcription into RNA in mammalian cells . In addition, DNA with a triazole linker instead of a phosphodiester bound have been accepted by E. coli , and human cells. It has also been reported that a semisynthetic organism is able to replicate with a single unnatural base pair in vivo .…”

Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

“…Recent reports from Brown, El-Sagheer and Tavassolli have demonstrated that oligonucleotides containing a triazole linkage in place of a phosphodiester are competent substrates for PCR and thus could provide a “readable” encoding sequence 16 17 18 19 . Based on these results, and our prior experience with Cu-catalyzed alkyne-azide cycloaddition (CuAAC) of oligos 20 , we wondered whether a readable chemical ligation strategy might offer some advantages over the current enzymatic methods.…”

A triazole linkage that mimics the DNA phosphodiester group in living systems

“…However, there possibility that the success of these experiments was a consequence of selective pressure on the essential antibiotic resistant clickcontaining gene. To resolve this we repeated the work with a plasmid containing click DNA backbone linkages in the gene encoding the fluorescent protein mCherry which is clearly not required for survival of the bacteria (Sanzone et al 2012). The effect of the proximity of the click linkers on biocompatibility was also probed by placing two click DNA linkers 4-bp apart in the region encoding the mCherry fluorophore.…”