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.

Figure 1.

(A) Click DNA ligation. The CuAAC reaction is used in combination with alkyne and azide-modified DNA bases to join two oligonucleotides together. (B) The structure of the equivalent canonical DNA for comparison of the triazole backbone with the natural phosphodiester.

Figure 2.

(A) The click-oligonucleotides used for site directed mutagenesis contained a silent C to A mutation (shown in blue), that introduces a BamHI restriction site not present in the native mCherry gene. The click-linked bases are shown in red. (B) Assembly of the click-linked pRSET-mCherry plasmid by site directed mutagenesis, introducing a BamHI watermark. (C) Gel electrophoresis (0.8% agarose gel) of SDM products (expected size 3577 bp); Lane 1, 2-log DNA ladder (New England Biolabs); lane 2, pRSET-mCherry SDM with normal primers; lane 3, pRSET-mCherry SDM with normal primers followed by DpnI digestion; lane 4, pRSET-mCherry SDM using click primers; lane 5, pRSET-mCherry SDM using click primers followed by DpnI digestion; lane 6, negative control (pRSET-mCherry SDM using water instead of primers); lane 7, negative control followed by DpnI digestion; lane 8, pRSET-mCherry template plasmid.

Figure 3.

Biocompatability of click DNA in E. coli. (A) The plate on the left contains transformants that have undergone SDM with native primers, and the plate on the right shows transformants of SDM with the triazole-containing primers. Ten replicates of the experiment were performed, with all colonies showing the expected mCherry fluorescence when viewed under a UV light source. (B) Comparison of the number of colonies in the water control (W), native (N) and triazole (T) plates. The triazole plates contained 92.7 ± 8.3% of the colonies on the native plates, whereas the negative control plate contained <1%.

Figure 4.

(A) and (B) Representative sequencing data of the mCherry gene from colonies on the triazole DNA plates. The forward strand is shown in (A), and the complementary strand is shown in (B), with the location of the triazole backbone linkers shown on each strand. (C) Lane 1 is 2-Log DNA ladder (New England Biolabs), lane 2 is BamHI restriction digestion of native pRSET mCherry plasmid, and lane 3 is the progeny of the click-modified variant. The presence of the additional BamHI watermark incorporated by the click-SDM primers results in the highlighted 229-bp fragment in lane 3.

Figure 5.

(A) Western blot of the product of in vitro transcription/translation using the SDM products with water (lane 1), canonical primers (lane 2) and click-linked primers (lane 3), and as template. The intensity of each band is quantified below the blot (average of three repeats). (B) Fluorescent emission of mCherry (at 610 nm) from in vitro transcription/translation of pRSET-mCherry constructed by SDM from click-linked primers (red line), normal primers (blue line); no fluorescence is observed from the negative control (black line).

Figure 6.

(A) SDS–PAGE of Ni²⁺ affinity purified mCherry (30 kDa) from colonies transformed with the SDM product with water (negative control, lane 2), canonical (positive control, lane 3), and click-linked primers (lane 4). Lane 1 is NEB ColorPlus Prestained Protein Ladder and insert shows the readily visible mCherry protein product at 30 kDa when using normal (left) or click-linked primers (right) in white light. (B) Fluorescent emission of recombinant mCherry (610 nm) expressed from pRSET-mCherry constructed by SDM from click-linked primers (red line), normal primers (blue line) and water control (black line). Insert shows purified mCherry from pRSET-mCherry constructed by SDM from normal (left) and click-linked (right) primers, under UV light at 365 nm.