Multiplexed DNA detection based on positional encoding/decoding with self-assembled DNA nanostructures† †Electronic supplementary information (ESI) available: Experimental details, additional DNA detection figures, and DNA sequence information. See DOI: 10.1039/c4sc02696a Click here for additional data file.

A multiplexed DNA detection strategy with fast hybridization kinetics based on positional encoding/decoding with self-assembled DNA nanostructures has been developed.


Materials
All DNA stands were purchased from Sangon Biotech (Shanghai) Co. Ltd and DL2000 DNA molecular weight marker was from TaKaRa Biotechnology (Dalian) Co. Ltd. Freeze 'N Squeeze column was purchased from Bio-Rad Laboratories, Inc. and uranyl formate was from Polysciences, Inc. Gel electrophoresis was performed on a Bio-Rad system using 2% agarose gel. UV-vis absorption values were obtained using a Eppendorf Biophotometer Plus facility. Transmission electron microscopy (TEM) imaging was performed using a JEOL JEM-1011 facility.

Design of Self-Assembled DNA Nanostructures
DNA sequences for the nanostructures were generated by program Sequin. S1 To reduce undesired interaction patterns, criton size is set to 7, which means any continuous sequence of 7 or more nucleotides (nt) appears at most once. DNA nanostructures were designed by following Peng Yin's LEGO-like model (for all the DNA sequence information, please see Section 14 of the Supporting Information). S2,S3 In our design of the core cuboid part of CU, DNA double helices were arranged as square lattice bundles, as shown in Figure S1. First, we used Sequin to generate sequences for 36 DNA double helices, each of which is 128 BP (base pairs) in length. Then a sequence of 8Ts (eight continuous thymidines) were added to both ends of one strand of each helix (5'-3' strand for odd helices, 3'-5' strand for even helices, refer to Figure S1 for the odd and even numbering of helices) to prevent non-specific blunt-end stacking. Second, a nick site was created for each helix every 16 nt. Because of the different numbers of nt for the two strands of each helix, the nick sites are staggered by 8 nt. Third, the nick sites of each odd helix were linked with the corresponding sites of neighboring even helix clockwise. Take H9 (helix 9) as an example, its protruding 8Ts on the 5' end is linked to the protruding 8Ts on the 3' end of H14, then the first nick site of H9 should be linked to the first one of H10, the second site of H9 to the second one of H2, the third site of H9 to the third one of H8, and the fourth site of H9 to the fourth one of H14. After the linkage, the remaining 16 nt strands should be merged to 32 nt ones to improve the stability of the nanostructures. The registry marker of CU is direct extension of the 9 helices, each of which is 64 BP in length, at one corner of the core cuboid. Figure S1. The design of CU viewed from the cross-section of the end of core cuboid opposite to the registry marker. This view gives a top left corner location for the registry marker.
The locations of 14 capture probe strands at the top and bottom surfaces of CU are shown in Figure S2. The locations of 15 detection probe strands of DU are shown in Figure S3 and S4. Figure S2. The locations of 14 capture probe strands at the top and bottom surfaces of CU, with base numbers and helix numbers specified (circle-marked sites at right: for TT1 and TB1; circle-marked sites at left: for TT2 and TB2). Figure S3. The locations of 15 detection probe strands at DUT1 (identical for DUT2), with the base number (64 BP) and helix numbers (circle-marked sites) specified. Figure S4. The locations of 15 detection probe strands at DUB2 (identical for DUB1), with the base number (64 BP) and helix numbers (circle-marked sites) specified.

Experimental Procedures
Preparation of nanostructures: Hundreds of single-strand DNA (ssDNA) were mixed and then freeze-dried. After the dissolution of ssDNA in 0.5×TE buffer (5 mM Tris, 1 mM EDTA, pH=8.0, supplemented with 40 mM MgCl 2 ) to a final concentration of 200 nM per strand, the solution was annealed from 90 o C to 60 o C at a cooling rate of 5 min/ o C and from 60 o C to 24 o C at a rate of 2 h/ o C.

Purification of nanostructures:
The annealed samples were loaded to a native 2% agarose gel with 0.5 μg/mL ethidium bromide (running buffer: 0.5×TBE buffer, containing 44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA, supplemented with 11 mM MgCl 2 ) and gel electrophoresis was performed at 80 volts for 2 h in an ice bath. Target bands were excised and cut into small pieces. The gel pieces were placed into Freeze 'N Squeeze columns, frozen at -20 o C for 5 min and then centrifuged at 7000g at 4 o C for 5 min.
Hybridization assay: DNA Nanostructures were quantified by measurements of UV-vis absorption values at 260 nm. Different DNA nanostructures were mixed in a molar ratio of 1:1 and target DNA (3 μL) was then added to the mixture. The solution was diluted with 0.5×TBE buffer to a final volume of 10 μL (5 nM for the final concentration of each nanostructure). The hybridization was allowed to proceed at 30 o C.
TEM imaging: A 2.1 μL of hybridization solution was mixed with 0.3 μL of ssDNA (with a sequence of 5'-GCCTGAAGTCTGGTGCTTAGGCCTTGAAATCA -3' for the generation of a hydrophilic TEM grid surface) and the whole solution was loaded onto a glass slide. On top of the solution was covered with a carbon-coated TEM grid and the contact between the solution and grid was allowed to proceed for 2 min. The TEM grid was then stained with a 2% uranyl formate aqueous solution (containing 25 mM NaOH) for 2 min followed by twice wash with water. TEM imaging was performed at 100 kV. Figure S7. Gel electrophoresis bands for purified CU and DUT1. Lane M: DL2000 molecular weight marker (same for all the following gel electrophoresis images); Lane 1: CU; Lane 2: DUT1. Figure S8. Gel electrophoresis bands for CU, DUT1, and TT1 in the presence of different concentrations of MgCl 2 . Lane 1: 11 mM MgCl 2 ; Lane 2: 20 mM MgCl 2 ; Lane 3: 30 mM MgCl 2 . The concentration of TT1 is 300 nM. The non-penetrating material in the gel is the non-specific aggregation of the hybridization product, which probably appears after storage at 4 o C and could be reduced by pre-heating the sample at 30 o C before gel electrophoresis (same for all the following gel electrophoresis images).