Precision Polymers and 3D DNA Nanostructures: Emergent Assemblies from New Parameter Space - Supporting Information

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1 Precision Polymers and 3D DNA Nanostructures: Emergent Assemblies from New Parameter Space - Supporting Information Christopher J. Serpell, Thomas G. W. Edwardson, Pongphak Chidchob, Karina M. M. Carneiro, Hanadi F. Sleiman* Department of Chemistry and Centre for Self-assembled Chemical Structures, McGill University, 801 Sherbrooke Street West, Montreal, QC H3A 2K6, Canada. * hanadi.sleiman@mcgill.ca General Considerations Acetic acid, tris(hydroxymethyl)-aminomethane (Tris), formamide, urea, and polypropylene oxide (PPO) were used as purchased from Aldrich. Acetic acid and boric acid were purchased from Fisher Scientific and used without further purification. Nucleoside (da, dc, dg and T) derivatized 1000Å LCAACPG supports with loading densities between μmol/g and reagents used for automated DNA synthesis were purchased from Bioautomation Corporated. The hexa(ethylene glycol) (HEG) and dodecane-diol (HE) phosphoramidites were purchased from ChemGenes Corporated. Sephadex G-25 (super fine, DNA grade) was purchased from Glen research. 1xTBE buffer is composed of 0.09M Tris and boric acid (TB) and 2 mm EDTA with a ph ~8.3. 1xTAMg buffer is composed of 45 mm Tris, and 12.5 mm Mg(OAc) 2 6H 2O. The ph was adjusted to 8 using glacial acetic acid. Gels were stained using GelRed and imaged by BioRad ChemiDoc MP, with BioRad Image Lab 5.0 software. Synthesis and purification of DNA strands DNA strands were synthesised using automated oligonucleotide solid-phase synthesis performed on a Mermade MM6 synthesizer from Bioautomation. Optimal yields for the HEG insertion could be obtained by using an extended five minute coupling time. Washing, capping, and oxidation were then achieved using the synthesiser, followed by appendage of the next DNA tract. Coupling efficiency was monitored after removal of the dimethoxytrityl (DMT) 5ʹ-OH protecting groups. Strands were synthesised on a 1 µmol scale, after which cleavage and deprotection was achieved by treatment with concentrated NH 4OH (1 hour at room temperature, followed by at least 4 hours at 60 C). Crude clip strands were purified on 12 % polyacrylamide/8m urea polyacrylamide gels (PAGE; up to 20 OD 260 of crude DNA per gel) at constant current of 30 ma and set voltage of 250V for 30 minutes followed by a set voltage of 500V for 1 hour using 1x TBE buffer. Following electrophoresis, the gels were wrapped in plastic and placed on a fluorescent TLC plate and illuminated with a UV lamp (254 nm). The bands were quickly excised, and the gel pieces were crushed and incubated in 12 ml of sterile water at 55 C for 16 hours. Samples were then dried to ca. 1.5 ml, and desalted using size exclusion chromatography (Sephadex G-25), and then quantified (OD 260) using a NanoDrop Lite spectrophotometer from Thermo Scientific. The protocol for fluorescently labelled strands depended upon the dye in question. Strand B-A488 was generated using a commercially available amine-modified CPG to give a 3 -amine DNA strand, which was then coupled with Alexa488-NHS ester (Life Technologies) according to the manufacturer s instructions. Cy dyes were appended to strands at the 5 ends using commercial phosphoramidite reagents (Glen Research), performing the reaction manually in a glove box with a coupling time of 15 minutes. The Eclipse Quencher strand was generated using a functionalised CPG (Glen Research), resulting in a 3 modification. Whether the strands were modified at the 3 or 5 ends, a 5T spacer was inserted between the dye and the 14mer binding region. After the synthesis, the strands were purified according the protocol described for clip strands, using 15% denaturing PAGE. Extinction coefficients at 260 nm for the dyes were taken into consideration in the quantification (Alexa488 = , Cy3 = , Cy5 = , EQ = M 1 cm 1 ). S1

2 DNA-polymer strands were synthesised and characterised exactly as previously reported. 1 Sequence Design Clips are designated with a number, representing their position in the prism, and two letters indicating the sequences of the single-stranded binding regions presented in the assembled object. A prefix is appended to the special strands required for non-cubic prisms (TP or PP). X designates a hexa(ethylene glycol) insertion. The A sequence is complementary to the DNA-polymer and is drawn red in the figures. Table S1. DNA sequences. Strand Sequence 1-AA 2-AA 3-AA 4-AA 1-AB 2-AB 3-AB 4-AB 1-TT 3-TT 4-TT 2-AC 3-AD 4-AE TP3-AB PP4-AB PP5-AB HE x-a (HE) xtttttcagttgaccatata HE x-b (HE) xtttttccatctggtattac B-A488 CCATCTGGTATTACTTTTT-A488 C-Cy3 Cy3-TTTTTTCTTACGGCAGAGT D-Cy5 Cy5-TTTTTATGGACCAAGGCCA E-EQ CTCTGCTAATCCTGTTTTT-EQ A20 GAGCAGTTGACCATATAGGA B20 ACTCCATCTGGTATTACTAC XL1 TTTAATGCGCGAGCAGTTGACCATATAGGA XL2 GAGCAGTTGACCATATAGGAACATAGAGCG XLX GCGCATTAAACGCTCTATGT +30 GTAAATGACGACTCCATCTGGTATTACTAC +60 GGATTGCACTCGCGTCCTGAAATTCGATCATGTTTCGCCGACTCCATCTGGTATTACTAC +100 ACTCCATCTGGTATTACTACGCCATTAAGTTAGGCCGGTTGGATTGCACT CGCGTCCTGAAATTCGATCATGTTTCGCCGACTCCATCTGGTATTACTAC TCGCTGAGTAXTCCTATATGGTCAACTGCTCXGCAAGTGTGGGCACGCACACXTCCTATATGGTCAACTGCTCXCACAAATCTG CTATCGGTAGXTCCTATATGGTCAACTGCTCXTACTCAGCGACAGATTTGTGXTCCTATATGGTCAACTGCTCXCAACTAGCGG CACTGGTCAGXTCCTATATGGTCAACTGCTCXCTACCGATAGCCGCTAGTTGXTCCTATATGGTCAACTGCTCXGGTTTGCTGA CCACACTTGCXTCCTATATGGTCAACTGCTCXCTGACCAGTGTCAGCAAACCXTCCTATATGGTCAACTGCTCXGTGTGCGTGC TCGCTGAGTAXTCCTATATGGTCAACTGCTCXGCAAGTGTGGGCACGCACACXGTAGTAATACCAGATGGAGTXCACAAATCTG CTATCGGTAGXTCCTATATGGTCAACTGCTCXTACTCAGCGACAGATTTGTGXGTAGTAATACCAGATGGAGTXCAACTAGCGG CACTGGTCAGXTCCTATATGGTCAACTGCTCXCTACCGATAGCCGCTAGTTGXGTAGTAATACCAGATGGAGTXGGTTTGCTGA CCACACTTGCXTCCTATATGGTCAACTGCTCXCTGACCAGTGTCAGCAAACCXGTAGTAATACCAGATGGAGTXGTGTGCGTGC TCGCTGAGTAXTCCTTTTTTTTTTTTTTCTCXGCAAGTGTGGGCACGCACACXGTATTTTTTTTTTTTTTAGTXCACAAATCTG CACTGGTCAGXTCCTTTTTTTTTTTTTTCTCXCTACCGATAGCCGCTAGTTGXGTATTTTTTTTTTTTTTAGTXGGTTTGCTGA CCACACTTGCXTCCTTTTTTTTTTTTTTCTCXCTGACCAGTGTCAGCAAACCXGTATTTTTTTTTTTTTTAGTXGTGTGCGTGC CTATCGGTAGXTCCTATATGGTCAACTGCTCXTACTCAGCGACAGATTTGTGXAAAACTCTGCCGTAAGAGGAXCAACTAGCGG CACTGGTCAGXTCCTATATGGTCAACTGCTCXCTACCGATAGCCGCTAGTTGXGCCTGGCCTTGGTCCATTTGXGGTTTGCTGA CCACACTTGCXTCCTATATGGTCAACTGCTCXCTGACCAGTGTCAGCAAACCXTAACAGGATTAGCAGAGCGAXGTGTGCGTGC CCACACTTGCXTCCTATATGGTCAACTGCTCXCTACCGATAGCCGCTAGTTGXGTAGTAATACCAGATGGAGTXGTGTGCGTGC TACCGGATCGXTCCTATATGGTCAACTGCTCXCTGACCAGTGTCAGCAAACCXGTAGTAATACCAGATGGAGTXCCGTAATTGC CCACACTTGCXTCCTATATGGTCAACTGCTCXCGATCCGGTAGCAATTACGGXGTAGTAATACCAGATGGAGTXGTGTGCGTGC S2

3 Figure S1. Denaturing PAGE (12%, TBE buffer) analysis of clip strands. Figure S2. Denaturing PAGE (15%, TBE) analysis of other strands: (left) stained by GelRed; (right) overlay of three-channel fluorescence scan showing orthogonal excitation and emission between dyes and lack of emission from quencher. S3

4 Assembly of DNA Cubes For gel studies, DNA nanostructures were assembled using 5 M stock solutions in 1x TAMg buffer of the particular strands needed in each case, added in stoichiometric quantities. The strands were mixed together and then annealed slowly from 90 to 4 C over four hours in an Eppendorf Mastercycler pro Thermal Cycler. Before loading on gels, 1 L of glycerine solution per 6 L of sample was mixed in to ensure that the DNA sank to the bottom of the appropriate lane. The prisms discussed in this manuscript are given names according to their shape (TP, C, or PP), and the number of A sequences they display. They were assembled using the following combinations: Table S2. Structure and strand composition of DNA prisms. Prism Diagram Clips used C1 1-TT, 2-AB, 3-TT, 4-TT C2 1-TT, 2-AB, 3-TT, 4-AB C2 1-TT, 2-AA, 3-TT, 4-TT C3 C4 1-AB, 2-AB, 3-AB, 4-TT 1-AB, 2-AB, 3-AB, 4-AB C4 1-TT, 2-AA, 3-TT, 4-AA C6 C8 1-AA, 2-AA, 3-AA, 4-TT 1-AA, 2-AA, 3-AA, 4-AA C4* 1-AB, 2-AC, 3-AD, 4-AE TP PP 1-AB, 2-AB, TP3-AB 1-AB, 2-AB, 3-AB, PP4-AB, PP5-AB S4

5 Figure S3. Native PAGE (6%, TAMg buffer) showing step-wise assembly of DNA prisms. Mixtures are simply mixed at room temperature, except when designated with a, meaning that they were subjected to a 4 hour anneal from 95 to 4 C. Left: TP, centre: C4, right: PP. All three gels are run under identical conditions, and the first two bands (1-AB, and 1-AB + 2-AB) are kept constant to enable accurate comparison. Figure S4. Native PAGE (5%, TAMg buffer) showing DNA cubes with different single stranded regions. Minor variations in mobility are to be expected. S5

6 Dynamic Light Scattering Samples for DLS were prepared at 0.5 µm with respect to total DNA strand concentration. 15µL aliquots were then analysed on a DynaPro (model MS) molecular-sizing instrument using a laser wavelength of 824 nm. Transmission Electron Microscopy Samples (2 μl at 0.5 µm w.r.t. total DNA) were deposited on carbon film coated copper EM grids for one minute, followed by blotting off the excess liquid with the edge of a filter paper, and washing three times with 20 µl of water, before drying under vacuum. The samples were imaged using a Tecnai 12 microscope (FEI electron optics) equipped with a Lab6 filament at 120kV. Images were acquired using a Gatan 792 Bioscan 1k x 1k Wide Angle Multiscan CCD Camera (Gatan Inc.). Contrast was adjusted automatically - note that in the presence of any high-contrast foreign matter, this results in the micelles being almost invisible. Images were analysed using ImageJ, which required manually setting threshold levels and placing limits on the size and circularity of features to ensure correct particle picking. The area values obtained were converted into radii (for comparison with DLS), making the assumption that the features are circular, which can be readily validated by eye. High resolution images were acquired using a FEI Tecnai G2 F20 operating at 120 kv equipped with a Gatan Ultrascan k x 4k CCD Camera System Model 895, after staining the grids for 1 minute with uranyl formate. Atomic Force Microscopy Samples (5 μl at 0.5 µm w.r.t. total DNA) were deposited for on freshly cleaved mica for ca. 5 seconds, followed by wicking off excess liquid with the edge of a filter paper, and washing with 5 x 50 µl water, blowing-off of excess liquid under a stream of air, and drying under vacuum for 20 minutes. Measurements were acquired using a Multimode 8 scanning probe microscope and Nanoscope V controller (Bruker, Santa Barbara, CA), running NanoScope 8 in PeakForce tapping with silicon nitride probes (Bruker type ScanAsyst) of nominal spring constant of 0.4 N/m, resonant frequency of 70 khz and tip radius < 5 nm. Images were processed using NanoScope Analysis Fluorescence Measurements Fluorescence scans of gels were taken using a BioRad ChemiDoc MP, with BioRad Image Lab 5.0 software, and the settings below. Table S3. Excitation and emission settings for gel imaging of fluorophores. Experiment A488 Cy3 Cy5 A488 Cy3 Cy3 Cy5 A488 Cy5 Illumination Blue epi Green epi Red epi Blue epi Green epi Blue epi Measured λ filter 530/28 nm 605/50 nm 695/55 nm 605/50 nm 695/55 nm 695/55 nm Fluorescence spectra were recorded on a BioTek Synergy HT microplate reader using 60 ul of sample with a fixed DNA cube concentration of µm, and dyes or polymers in stoichiometric ratios as appropriate. A488 was excited at 488 nm, Cy3 at 545 nm, and Cy5 at 646 nm. Emission was measured from 500 (A488), 555 (Cy3), or 655 (Cy5) nm up to 800 nm. Each well was scanned with every excitation wavelength in triplicate. S6

7 Figure S5. Further AFM images and analysis of structures arising from C4/HE 7. Height = 2.2 ± 0.4 nm, diameter = 25.5 ± 4.6 nm (184 features measured). Note preponderance of triangular and linear (aspect ratio = 2) structures suggesting dimeric and tetrahedral (or possibly trigonal planar) structures. S7

8 Figure S6. AFM images of structures arising from C4/HE 8. Height = 2.4 ± 0.3 nm, diameter = 27.9 ± 5.2 nm (184 features measured). Note presence of quadrilateral structures in addition to triangular and linear structures, suggesting projections of octahedral products. S8

9 Figure S7. Native PAGE (5%, TAMg buffer) of products arising from C4 and HE x at varying ratios. Figure S8. Native PAGE (5%, TAMg buffer) analysis of effect of Mg 2+ concentraion upon assembly of C4/HE 7 self-assembly. S9

10 Figure S9. The double-stranded regions of the cube (20 base pairs = 6.8 nm) are expected to be aligned with the radius of the micelle (17.4 nm), meaning that the polymers occupy a sphere of radius 10.6 nm. The surface area of such a sphere upon which the cubes must sit is 4πr 2 = 1412 nm 2. The area of the face of cube is less certain, since there are flexible single-stranded regions. Assuming that this effect is not significant, side length would be nm to accommodate the width of the duplexes as well as their length, giving an area of 81.0 nm 2. This would fit just over 17 times onto the surface of the polymer sphere. A compression of the cube side area is possible, which would increase this number, however since squares do not pack well onto the surface of a sphere, and there ratio of the total radius taken up by cubes would be higher if duplex width is accounted for, the value is more likely to be lower. S10

11 Figure S10. PAGE analysis of ExoVII degradation assay. Left: Native (5%, TAMg buffer), right: denaturing (12%, TBE buffer). S11

12 C4/H 12 micelle +30 base strand: R H = 19.7 nm (13.4% PD) C4/H 12 micelle +60 base strand: R H = 21.0 nm (13.8% PD) C4/H 12 micelle +100 base strand: R H = 24.0 nm (28.6% PD) Figure S11. DLS correlation functions and size distributions for micelles of prisms with extension strands after incubation of the preannealed micelle with 1.1 equivalents of the extension strand for 30 minutes at 37 C. (0.5 µm total DNA, TAMg buffer, 25 C). For comparison: the C4/H 12 micelle has R h of 17.4 nm. S12

13 Normalised particle count C4/H 12 micelle + 30 base strands: R = 16.6 ± 2.8 nm (493 particle counted) C4/H 12 micelle + 60 base strands: R = 18.3 ± 2.0 nm (241 particles counted) C4/H 12 micelle base strands: R = 20.3 ± 3.8 nm (98 particles counted) Radius (nm) Figure S12. TEM images and size analysis of micelles hybridised to extension strands. S13

14 TP/HE 12 micelle: R H = 16.6 nm (12.4% PD) C4/HE 12 micelle: R H = 17.4 nm (8.5% PD) PP/HE 12 micelle: R H = 18.6 nm (21.0% PD) Figure S13. DLS correlation functions and size distributions for micelles of prisms (0.5 µm total DNA, TAMg buffer, 25 C). For comparison: DNA cubes have R h of about 6 nm, 2, 3 while micelles of HE 12-DNA are sized at 11 nm 1 S14

15 Particle count More Radius (nm) Figure S 14. TEM images of TP/HE 12 micelle, with size analysis. Radius = 12.0 ± 1.7 nm (742 particles measured). S15

16 Particle count More Radius (nm) Figure S15. Further TEM images of C4/HE 12 micelle, with size analysis. Radius = 13.2 ± 1.9 (174 particles measured). S16

17 Particle count More Radius (nm) Figure S16. TEM images of PP/HE 12 micelle, with size analysis. Radius = 12.8 ± 1.7 (483 particles measured) S17

18 Figure S17. AFM images of TP/HE 12 micelles. Measuring 157 particles: height = 7.45 ± 0.56 nm; diameter = ± 2.00 nm. S18

19 Figure S18. Further AFM images of C4/HE 12 micelles. Measuring 97 particles: height = 6.06 ± 2.18 nm; diameter = ± 8.78 nm. S19

20 Figure S19. AFM images of PP/HE 12 micelles. Measuring 90 particles: height = 8.00 ± 1.04 nm; diameter = ± 1.17 nm. S20

21 Figure S20. Native PAGE (5%, TAMg buffer) of displacment of DNA prisms from micellar superstructures. Displacment was achieved by incubating the prism/micelles with 1.2 equivalents of A20 which is complementary to the full length of the side of the prism. The difference in mobility between the displacement product band and the prism is expected, being due to the presence of the A20. S21

22 Figure S21. Further TEM images of crosslinked C4/HE 12 micelles. In this experiment, since the linking strands (XL1 and XL2) are complementary to the A sequence, HE 12-DNA with the complement the B-sequence was used. The initial micelle created in this way is otherwise identical to that made using the HE 12-DNA with the A sequence. S22

23 Figure S22. Additional AFM images of concentration-mediated superstructures of C4/HE 12 micelles, and height cross-section analysis. The average height over 194 features (7.7 ± 1.9 nm) is similar to that of individual micelles (6 nm). The separation between peaks (ca. 30 nm) is slightly less than the measured diameter of free micelles (42 nm), which is expected given the greater influence of tip convolution on individual features and potential compression of the micelles as they pack. S23

24 Figure S23. Fluorescence scans of native PAGE (5%, TAMg buffer) analysis of dyes appended to free C4 DNA cubes. Dye strands (1.0 eqv) were incubated with pre-formed cubes for 30 minutes at 37 C. Lane 0 = no dyes, 1 = Alexa488, 2 = Cy3, 3 = Cy5, 4 = EQ, 5 = Alexa488 + Cy3, 6 = Cy3 + Cy5, 7 = Alexa488 + Cy5, 8 = Alexa488 + EQ, 9 = Cy3 + EQ, 10 = Cy5 + EQ, 11 = Alexa488 + Cy3 + Cy, 12 = Alexa488 + Cy3 + Cy5 + EQ. Left: 2-dye FRET. Red channel = excitation of Alexa488, observing emission of Cy3, blue channel = excitation of Cy3, observing emission of Cy5. Centre: 3-dye FRET, exciting Alexa488 and observing Cy5 emission. Right: Visualising DNA using GelRed. Figure S24. Fluorescence scans of native AGE (2.5%, TAMg buffer) analysis of dyes appended to C4/HE 12 micelles. Dye strands (1.0 eqv) were incubated with pre-formed micelles for 30 minutes at 37 C. Lane 0 = no dyes, 1 = Alexa488, 2 = Cy3, 3 = Cy5, 4 = EQ, 5 = Alexa488 + Cy3, 6 = Cy3 + Cy5, 7 = Alexa488 + Cy5, 8 = Alexa488 + EQ, 9 = Cy3 + EQ, 10 = Cy5 + EQ, 11 = Alexa488 + Cy3 + Cy, 12 = Alexa488 + Cy3 + Cy5 + EQ. (a) 2-dye FRET. Red channel = excitation of Alexa488, observing emission of Cy3, blue channel = excitation of Cy3, observing emission of Cy5. (b) 3-dye FRET, exciting Alexa488 and observing Cy5 emission. (c) Visualising DNA using GelRed. (d) Individual dye emission scan (green = Alexa488, red = Cy3, blue = Cy5) of whole double-decker AGE gel. In images (a) to (c) and in Fig. 4c the lower level (lanes 8 12) has been positioned next to the top level (lanes 0 7). No scaling or contrast enhancement was applied in this manipulation. S24

25 C-A488 M-A488 C-Cy3 M-Cy3 C-Cy5 M-Cy Figure S25. Direct fluorescence spectra of dyes on C4 (designated C) and C4/HE 12 micelles (M). Alexa 488 λ ex = 488 nm, Cy3 λ ex =545 nm, Cy5 λ ex =645 nm C-A488-Cy3 M-A488-Cy3 C-Cy3-Cy5 M-Cy3-Cy5 C-A488-Cy5 M-A488-Cy Figure S26. Two-dye FRET fluorescence spectra of dyes on C4 (designated C) and C4/HE 12 micelles (M). Alexa 488-Cy3 λ ex = 488 nm, Cy3-Cy5 λ ex = 545 nm, Alexa488-Cy5 λ ex = 488 nm. S25

26 C-A488-EQ M-A488-EQ C-Cy3-EQ M-Cy3-EQ C-Cy5-EQ M-Cy5-EQ Figure S27. Difference fluorescence spectra quenched dyes on C4 (designated C) and C4/HE 12 micelles (M), minus the appropriate unquenched spectra. Alexa 488 λ ex = 488 nm, Cy3 λ ex =545 nm, Cy5 λ ex =645 nm C-A488-Cy3-Cy5 C-A488-Cy3-Cy5-EQ M-A488-Cy3-Cy5 M-A488-Cy3-Cy5-EQ C quench difference M quench difference Figure S28. Three-dye FRET fluorescence spectra of dyes on C4 (designated C) and C4/HE 12 micelles (M), in the presence and absence of quencher. λ ex = 488 nm. Quench difference lines are the subtraction of the quenched system from the unquenched version, illustrating which dyes are preferentially quenched. References 1. Edwardson, T.G.W., Carneiro, K.M.M., Serpell, C.J. & Sleiman, H.F. An Efficient and Modular Route to Sequence-Defined Polymers Appended to DNA. Angew. Chem. Int. Ed. 53, (2014). 2. McLaughlin, C.K. et al. Three-Dimensional Organization of Block Copolymers on DNA-Minimal Scaffolds. J. Am. Chem. Soc. 134, (2012). 3. Edwardson, T.G.W., Carneiro, K.M.M., McLaughlin, C.K., Serpell, C.J. & Sleiman, H.F. Site-specific positioning of dendritic alkyl chains on DNA cages enables their geometry-dependent self-assembly. Nature Chem. 5, (2013). S26