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and placed on a fluorescent TLC plate and illuminated with a UV lamp (254nm). The bands were
quickly excised, and the gel pieces were crushed and incubated in 10 mL of sterile water at 55 oC
for 16 hours. Samples were then dried to 1.5 mL, desalted using size exclusion chromatography
(Sephadex G-25), and quantified (OD260) using UV-vis spectroscopy.
After quantification, a denaturing PAGE was performed to analyze the purity of the modified
DNA strands site-specifically modified with 3. As is seen in Figure S1a, the 6 strands doubly
modified with 3 (1a-c and 2a-c) appear as single bands with no byproducts. Similar analysis was
performed on the linking strands (LSa-c) and rigidifying strands (RSa and RSb), as seen in
Figure S1b.
1 2 3 4 5 6a
1 2 3 4 5b
Figure S1 24% denaturing PAGE characterization of the purified oligonucleotides required for T1 and T2 assembly and for 3D assembly of TP a, lane 1-1a, lane 2-1b, lane 3-1c, lane 4-2a, lane 5-2b, and lane 6-2c. b, lane 1-LSa, lane 2-LSb, lane 3-LSc, lane 4-RSa, and lane 5-RSb
To ensure that the connectivity of T1 and T2 is indeed
cyclic, 2D starting materials were subjected to
Exonuclease VII (ExoVII) digestion. ExoVII is selective
for the digestion of single-stranded open DNA, over that of
cyclic closed DNA.S2 In a typical experiment 0.05 nmoles
of DNA (total), whether linear, 2D or 3D, was placed in 10
uL of TAMg buffer. According to the ExoVII optimization
experiments performed on linear single-stranded open-
form DNA shown in Figure S2, 5 U of ExoVII is required
to completely digest this mixture at 15 °C for 2 hrs.
Using these above outlined conditions, T1 was
subjected to ExoVII digestion. Figure S3 indicates that T1
(lane 1) is not digested by ExoVII (lane 2) while an open-
form single-stranded analogue of triangle T1 (lane 3) is
indeed almost completely digested (lane 4). Similar results
were obtained for T2 and revealed that the connectivity of
the triangles was indeed closed and cyclic.
1 2 3 4
Figure S2 8% non-denaturing PAGE characterization of 0.05 nmole of linear DNA in TAMg buffer after digestion with various units (U) of ExoVII. Lane 1-0 U, lane 2-1U, lane 3-3 U and lane 4-5 U.
1 2 3 4
T1
Figure S3 8% non-denaturing PAGE characterization of 0.05 nmole of T1 (lanes 1 and 2) and a single-stranded open-form intermediate of T1 (lanes 3-4). Lanes 2 and 4 are after digestion with 5U of ExoVII, 2hr, 15 °C.
Figure S5. Absorbance due to the dpp ligands at ca.
350 nm is partially obscurred due to the large peak
observed for DNA at 260 nm. Peaks can be seen,
though, in the cases of metalated T1.M3, especially
for T1.CuI3 (_), that are indicative of Metal to
Figure S5 UV/Vis spectrum recorded in TAMg buffer for non-metallated T1 ( - ), T1.CuI
3 ( - ), and T1.AgI
3 ( - ).
a
b
T1 CuI AgI ZnII CoII CdII AuI EuII
1 2 3 4 5 6 7 8
1 2 3 4 5 6
1a-c
T1
Figure S4 Metalation of T1 and analysis by PAGE a, 12% non-denaturing PAGE characterization of T1 before and after site-specific metalation with a variety of transition metals as indicated in the top of the gel. b, 8% non-denaturing PAGE characterization of T1.M3.
for the digestion of single-stranded DNA over double-stranded DNA by a factor of 30,000.S3,S4
Again, prior to MBN digestions of the 3D cage and various intermediates, experiments were
performed using ssDNA and dsDNA systems to optimize conditions. MBN digestion conditions
were again optimized at 15 °C for 2 hr in a 1xTAMg buffer system. The single stranded DNA is
almost completely degraded upon addition of 20U of the MBN enzyme (Figure S8 a, lane 5)
while the dsDNA control is not degraded at this enzyme concentration (Figure S8 b, lane 5).
VIII. Metal Coordination of DNA Prisms
a 1 2 3 4 5 6 7 8 9 10
Figure S8 Optimization of MBN enzymatic assay conditions. a, 8% non-denaturing PAGE characterization of 0.05 nmole of linear open form single-stranded DNA in TAMg buffer after digestion with increasing units (U) of MBN b, A similar analysis done for double stranded DNA. The lane assignments in both both a and b are the same: lane 1-0 U, lane 2- 5 U, lane 4-10 U, lane 5- 15 U, lane 6-20 U, lane 7- 25 U, lane 8- 30 U, lane 9- 35 U, lane 10- 40 U.
metalated species. Figures S11a and S11b indeed show the presence of the tris-, bis-,
mono and non-metalated triangles at intermediate ratios, with the proportion of tris-
metalated triangle increasing and that of the non-metalated triangle decreasing as more
copper is added.
Figure S11 Analysis of 2D and 3D metalation products under various equivalents of added transition metal. a, T1 before metal addition (lane 1) and after the addition of 1, 2, and 3 equiv. Cu(I) (lanes 2-4, respectively). Proposed intermediates are represented to the right of the gel. b, TP before metal addition (lane 1) and after the addition of 1-6 equiv. Cu(I) (lanes 2-7, respectively). Intermediates are again shown schematically to the right of gel.
In order to address this problem, a commercially available triethylene glycol (EG)
derivative was inserted in place of the T residues (shown in Scheme S3). As is observed in Figure
S13a, the sequential assembly of T1 and T2 using the EG modified linking strands LSEGa-c leads
to the quantitative formation of a 3D product TPEG which was indeed stable under both ExoVII
and MBN digestion conditions (data not shown). Although the LS modification led to the desired
product, problems were encountered upon metalation. As evidenced by the CD data shown in
Figure S13b, a large excess of copper was required to promote saturation of the signal. This large
molar excess was indeed rectified upon insertion of the C6 1,6-hexanediol derivative which led to
both excellent assembly, stability, and transition metal binding properties of TP.
a b 1 2 3 4 5 1 2 3
Figure S12 PAGE analysis of the 3D construct TPT a, 7% non-denaturing PAGE characterization of 3D structure TPT prepared by starting from T1 (lane 1) and sequentially adding LSTa (lane 2), LSTb (lane 3), LSTc (lane 4) and finally RSa and RSb (lane 5). b, 7% non-denaturing PAGE analysis of TPT (lane 1) and the results of digestion with MBN (lane 2) and ExoVII (lane 3).
Figure S14 Approximate dimensions of TP based on modeling. a, A top-view of TP with the triangular face labeled with height (h 5 nm) and base (a 5 nm ) markers. b, Side-view of TP showing one of the rectangular sides labeled with height (c 3 nm ) and width (b 4 nm) markers.
a b
a b
Figure S13 PAGE analysis of the 3D construct TPEG a, 7% non-denaturing PAGE characterization of 3D structure TPEG prepared by starting from T1 (lane 1) and sequentially adding LSEGa (lane 2), LSEGb (lane 3), LSEGc (lane 4) and finally RSa and RSb (lane 5). b, CD titration experiment for TPEG which reveals a disruption of site-specific metal binding for this 3D architecture.
Figure S15 TEM images of negatively stained TP cages. a, A typical field of view obtained for TP (40 nM) deposited onto a 400 mesh carbon coated copper grid subsequently stained with a 2% solution of uranyl acetate. Imaging was performed at a nominal magnification of 50K, on a Tecnai G2 F20 microscope operating in low dose conditions at an accelerating voltage of 200 keV. Images were recorded on a Gatan Ultrascan 4k x 4k CCD camera. Scale bar shown is 100 nm. b, Representative particles obtained from the negative-staining TEM data for TP. Dimensions of the box used to select each particle are ca. 22 x 22 nm.
XII. References S1. Yang, H. & Sleiman, H.F. Templated synthesis of highly stable, electroactive, and