Gene assembly via one-pot chemical ligation of DNA promoted by DNA nanostructures† †Electronic supplementary information (ESI) available: Experimental procedures, supplementary figures, sequencing data, oligonucleotides sequences. See DOI: 10.1039/c8cc00738a

A gene was obtained from 14 oligonucleotides self-assembled and chemically ligated in a DNA nanostructure.


Design of the 6HB
The design of the 6HB was supported by caDNAno software. After introducing the sequence of the scaffold the staples were added automatically by the software and then manually modified to achieve a hierarchical folding.
In Supplementary Figure 3 a high-resolution blueprint with gene oligonucleotides and staples is shown.

Folding of the 6HB
Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2018 Samples were made mixing staples and in-house synthetized GOs in ratio 1:1 to a final concentration of 500 nM/each oligo in 1X TE buffer with 20 mM MgCl 2 . The sample was folded in a thermocycler using the following program: from 95 ºC to 80 ºC with a ramp of 1º C/min, from 80 ºC to 40 ºC with a ramp of 0.03ºC/min, from 40 ºC to 23 ºC with a ramp of 0.1 ºC/min and final hold at 8 ºC.

Click reactions on the 6HB
-Click reaction (CuAAC) using an heterogeneous catalyst A volume of 15 µl of THPTA 0.1 M was added to the "reactor M", a vial containing the heterogeneous catalyst (baseclick), then 20 µl of folding reaction was added to the "reactor". The sample was incubated at 32ºC with gentle shaking (200 rpm) for 5h.
-Click reaction (CuAAC) using CuSO 4 The Baseclick EdU kit (reaction buffer, catalyst solution and reducing agent/buffer additive) was used for the experiments with CuSO 4 as source of Cu(I). The indications of the producer were used for the ligation assay using the click reaction.
In this case, 40 µl of folding reaction were used in the assays.

-Click reaction (CuAAC) using CuBr
For the experiments with CuBr as source of Cu(I): 1 mg of CuBr was weighted under inert atmosphere and dissolved in 70 µl of "click solution". A total of 5 mg of the ligand TBTA were dissolved in 94 µl click solution and the two solutions were combined in ratio 1:2 (TBTA:CuBr). A volume of 20 µl of the mix were mixed with 4 µl of the folding reaction and incubated at 32ºC with gentle shaking.

AGE/AGE-Mg
AGE/AGE-Mg were prepared dissolving agarose (Ultra-pure, Thermo Scientific) to achieve a 1% gel in 0.5X TBE buffer (and 11 mM MgCl 2 final concentration for AGE-Mg, 1X TAE was used when the bands were extracted from gel). The gel was casted and left solidify at RT for 30 min.

PCR reactions
All the PCR reactions were tested using the following primers (Metabion): Sequencing PCR reactions were cloned into a plasmid using the TOPO PCR cloning kit by Thermo Fisher Scientific and following manufacturer instructions. Plasmids from clones were extracted with Gene Elute HP Miniprep by Sigma-Aldrich and sent to sequencing to GATC Biotech.

AFM imaging
A volume of 2 µl sample was deposited on freshly cleaved mica and incubated for 1 min. Subsequently, 400 μl of imaging buffer (1× TAE-Mg2+ containing 5 mM NiCl2, 1× TAE-Mg-Ni) were added into the cell. Nanostructures were visualized by tapping mode AFM (Agilent AFM series 5100) using silicon nitride cantilevers (Olympus). All recorded AFM images were processed and analyzed by Gwyddion software.

Supplementary Text
Sequencing result analysis of PCR products using Taq polymerase (mediumlow fidelity)   The click region of gene oligonucleotides 9-10 appears to be prone to mutations. We speculate that GO10 might not have enough complementary bases at the level of the staples (see Supplementary Figure 3). This might explain why gene oligonucleotides 10 (represented in red box, Fig Sx) is missing in one of the clones sequenced.

Oligonucleotides
All reagents for chemical synthesis were purchased from Sigma-Aldrich. Unmodified oligonucleotides were acquired from Metabion International AG, while 3'-alkyne and 5'-azide modified gene oligonucleotides were synthesized in-house using a previously reported procedure. 6