HPLC chromatogramof click-ligation reaction

Figure S1. HPLC purification chromatogram of the clickDNA ligation reaction. The HPLC traces show two peaks for the click product and the splint. X-axis is time (min), Y-axis is UV absorbance at 254 nm. A) Purification of ODN-7, B) purification of ODN-8. Sequences in Figure 2A of main paper and table S1

Table S1. Oligonucleotide mass spectra

Code / Oligonucleotide sequences (5’-3’) / Calc. mass / Found mass
ODN-1 / AGTTCATGTACGGATMeCk / 4949 / 4947
ODN-2 / CGTAGGCCTTGGATMeCk / 4637 / 4635
ODN-3 / zCAAGGCCTACG / 3351 / 3350
ODN-4 / zCGTACATGAACT / 3654 / 3653
ODN-5 / CATGTACGGATCCAAG splint / 4890 / 4890
ODN-6 / AGGCCTTGGATCCGTA splint / 4897 / 4896
ODN-7 / CGTAGGCCTTGGATMeCtCGTACATGAACT / 8291 / 8289
ODN-8 / AGTTCATGTACGGATMeCtCAAGGCCTACG / 8300 / 8298

zC = 5’-azide dC, MeCk = 3’-propargyl-5-methyl dC, t = triazole linkage. Mass spectra were recorded on a Bruker micrOTOF™ II focus ESI-TOF MS instrument in ES− mode.

Optimization of SDM protocol

The SDM protocol was optimized with canonicaland click-linked mutagenic primers incorporated and extended over the entire length of the plasmidpRSET-mCherry using Pfu DNA polymerase. The annealing temperature of PCR reaction was optimized as shown in figure s2a. Gel electrophoresis of the product showed bands of the expected size (3577 bp) but in poor yield (Figure s2a).

Figure S2.Optimizing the SDM reaction. a) PCR product amplified with Pfu Polymerase. Lane 1 shows the DNA ladder in Kb; PCR product amplified with Pfu Polymerase, Lane 2, 3, 4, 5, 6, 7, 8 and 9 PCR carried out using normal primers, annealing temperature at 59°C, 58.6 °C, 57.6 °C, 56.6 °C, 55.1 °C, 53.8 °C, 52.9 °C and 52.5 °C respectively; Lane 10, 11, 12, 13, 14, 15, 16, 17 PCR carried out using click primers, annealing temperature at 59°C, 58.6 °C, 57.6 °C, 56.6 °C, 55.1 °C, 53.8 °C, 52.9 °C and 52.5 °C respectively.b)PCR product amplified with KOD Polymerase, Lane 2, 3, 4, 5, 6, 7, 8 and 9 PCR carried out using normal primers, annealing temperature at 59°C, 58.6 °C, 57.6 °C, 56.6 °C, 55.1 °C, 53.8 °C, 52.9 °C and 52.5 °C respectively; Lane 10, 11, 12, 13, 14, 15, 16, 17 PCR carried out using click primers, annealing temperature at 59°C, 58.6 °C, 57.6 °C, 56.6 °C, 55.1 °C, 53.8 °C, 52.9 °C and 52.5 °C respectively.

The low yield of the reaction with pfu polymerase prompted us to investigate KOD DNA polymerase, which was reported to be the best candidate because of its high yield, fidelity and speed. Electrophoresis analysis of mutagenesis PCR product (Figure s2b) showed higher yields with KODpolymerase, with little difference in yield observed over the range of annealing temperatures examined (52.5 -59 °C). No difference was observed in the yield of the reaction using normal primers (Figure s2b, lanes 2-9) and click-linkedprimers(Figure s2b, lanes 10-18).

Optimization of incubation time with DpnI restriction endonuclease.

DpnI restriction digestion was performed to cleave the methylated parental plasmid, leaving only the product of the SDM reaction.Incubation time with DpnI was varied, with the reaction carried out over 1, 2, 3, 6, 8 hr and overnight (Figure s3). Each reaction was repeated for the product of SDM with click-linked primers, canonical primers (positive control), and water (negative control). With the negative control reaction, the amount of template plasmid will be quantified. The product of each restriction digestion was transformed into DH5α, and the number of colonies present the next day on LB-agar plates counted. The6hr, 8hr and overnight incubation time showeda similar ratio of colonies on the click, positive and negative plates (Figure s3). Shorter incubation timesresulted in colonies on the negative control plates, the number of colonies decreasing with increased incubation time (Figure s3).

Figure S3.Optimization of the incubation time with DpnIrestriction endonuclease.

Data for the analysis of the biocompatability of click DNA in E. coli.

Table S2.Number of colonies after transformation of the SDM products into DH5α (data for Figure 3B of main paper)

Set number / Negative Control / Normal primers / Click primers
1 / 3 / 165 / 159
2 / 6 / 220 / 212
3 / 1 / 172 / 166

Table S3.Number of colonies after transformation of the SDM products intoKRXE. coli(data for Figure 3B of main paper)

Set number / Negative control / Normal primers / Click primers
1 / 0 / 38 / 38
2 / 0 / 40 / 50
3 / 0 / 34 / 31
4 / 0 / 58 / 54
5 / 0 / 71 / 64
6 / 0 / 45 / 35
7 / 0 / 54 / 40
8 / 0 / 60 / 57
9 / 0 / 42 / 32
10 / 0 / 34 / 31

Transformation of KRX cells with plasmids isolated from colonies transformed with click-SDM product

Figure S4: Representative plates showing mCherry expression in all colonies transformed with the plasmid isolated from a single colony transformed with the click SDM product. The absence of white colonies indicates that every copy of the plasmid contained within a single colony was correctly replicated from the original click-containing plasmid.

T4 DNA ligase treatment of the SDM product is not necessary for in vitro mCherry expression

Figure S5: Western blot of the product of in vitro transcription/translation with the canonical and click SDM product, with and without T4 DNA ligase treatment. The blot shows higher mcherry levels when the SDM product is not treated with T4 DNA ligase.

Protein expression timecourse showing comparable mCherry expression from click and normal template.

Figure S6: Time course protein expression. The protocol outlined in the manuscript was repeated, except that the expression mixture was shaken at 37 °C. 1 mL samples were taken at 1, 2, 3, 4 and 12 hr after IPTG addition. The results show comparable levels of mCherry (highlighted) produced from the normal, and click plasmids at various intervals. For loading control, the intensity of the band at 45 kDa may be used.