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1 Supporting Information Wiley-VCH Weinheim, Germany
2 Site-Specific Control of Distances between Gold Nanoparticles using Phosphorothioate Anchors on DNA and a Short Bifunctional Molecular Fastener Jung Heon Lee, Daryl P. Wernette, Mehmet V. Yigit, Juewen Liu, Zidong Wang, and Yi Lu* Contents: 1. Further experimental section 2. Mass spectrometry results 3. Further SEM images (AuNP trimers) 4. SEM images used for histogram data processing 5. Coordinates of AuNPs on the surfaces in SEM images 6. References 1
3 1. Further experimental section Materials: BIDBE was synthesized by following the procedure reported in literature. [1]. Oligonucleotides used in the work were purchased from Integrated DNA Technologies Inc. (Coralville, IA) and were purified by polyacrylamide gel electrophoresis (PAGE). AuNPs with 13 nm diameter were synthesized by citrate reduction method [2,3]. AuNPs with 5 nm diameter were purchased from Ted Pella (Redding, CA) and were modified with phosphine as described in literature [4,5] in order to increase the stability of AuNPs by complexation with bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt. Gold thin film (30 nm) with a Cr adhesion layer (5 nm) was deposited on silicon wafer by e-beam evaporation at Å/sec deposition rate. The surface was treated with O 3 plasma and rinsed thoroughly with Millipore water (18.2 MΩ?) before use. AuNP aggregation and disassembly experiment: PS-DNA1, PS-DNA2, Non-PS-DNA1, and Non-PS-DNA2 were treated with BIDBE following the previously mentioned method. To get BF-PS-DNA1, 100 µl of 12 µm BIDBE-PS-DNA1, 1 µl of 50 mm acetate buffer (ph 5.2), and 5?µL of 1 mm TCEP were mixed and incubated at room temperature for an hour to reduce disulfide bond. The process was repeated with other strands to get BF-PS-DNA2, BF-Non-PS-DNA1, and BF-Non-PS-DNA2. After BIDBE is reduced, two BFs are formed, one attached on phosphorothioate position of DNA and another one free in solution. It is possible that free BFs in solution might react with unreacted phosphorothioate modifications. They were not intentionally removed, however, since the remaining BF can still react with unreacted phosphorothioate group and increase the AuNP binding site or react with AuNP and make them stable, and therefore benefits the assembly process. To functionalize 13 nm AuNPs with BF treated DNAs, 300 µl of 17 nm AuNP solution was added to bifunctional fastener treated DNA solutions, respectively, and incubated overnight at room temperature. 45 µl of 1 M NaCl 50 mm tris acetate buffer (ph 8.2) was added and incubated at room temperature for another night. Samples were centrifuged at 16,110g at room temperature on a bench top centrifuge for 15 minutes and the supernatant was replaced with 400 µl of 100 mm NaCl 10 mm phosphate buffer (ph 7.0). This process was repeated once again and the supernatant was finally replaced with 400 µl of 300 mm NaCl 10 mm phosphate buffer (ph 7.0). In order to check assembly of AuNPs in the presence of matching target DNA, 150 µl of AuNP functionalized with BF-PS-DNA1 and the same amount of AuNPs functionalized with BF-PS-DNA2 were mixed and 3 µl of 10 µm complementary bridging DNA was added and annealed from 60 o C to room temperature.. Table S1. Oligonucleotide sequences used for AuNP assembly/disassembly experiments Name Sequence PS-DNA1 [a] (Green) PS-DNA2 [b] (Purple) 5'-TT*T* TTA GCA TAT GAC TAT GTT ACT CGC TAT AGC-3' 5'-GTA CTT GCA ATA TGT GCA ATG GCG AGG ATT T*T*T-3' Non-PS-DNA1 5'-TTT TTA GCA TAT GAC TAT GTT ACT CGC TAT AGC-3' Non-PS-DNA2 5'-GTA CTT GCA ATA TGT GCA ATG GCG AGG ATT TTT-3' CB-DNA (Red) 5'-AAT CCT CGC CAT TGC ACA TAT TGC AAG TAC GCT ATA GCG AGT AAC ATA GTC ATA TGC TAA-3' Non-CB-DNA 5'-AAT CGT ATA CTG ATA CAA TGA GCG ATA TCG CAT GAA CGT TAT ACA CGT TAC CGC TCC TAA-3' [a, b] Underlined nucleotides with * have phosphorothioate modification on the backbone. AuNP dimer formation on gold surface: BIDBE labelled phosphorothioate modified DNA (anchor DNAs) (100 nm) were mixed with CP-DNA (100 nm) in NaCl (50 mm) and acetate buffer (10 mm, ph 5) containing 400 µm TCEP and 2 mm EDTA and hybridized to form dsdna. Butane-1- thiol (Aldrich, 500 equiv) was added to the dsdna solution before immobilization on gold surface to limit the number of DNA 2
4 immobilized on and provide passivation for the surface. In order to immobilize DNA on the surface, gold thin film (~5 5 mm 2 ) was incubated into the 40 µl dsdna/butane-1-thiol solution for ~2-2.5 hours followed by thorough washing with NaCl (10mM) and acetate (10 mm, ph 5) buffer. AuNP assembly on DNA immobilized on the surface was formed by incubating gold thin film, on which dsdna is immobilized, into phosphine capped AuNP (5 nm, 400 nm) solution in NaCl (50 mm) and acetate (10 mm, ph 5) buffer with 200 µm TCEP for 12 hours at room temperature. After washing with NaCl (10mM) and acetate (10 mm, ph 5) buffer, the samples were dried with an N 2 stream. Table S2. Oligonucleotide sequences used for AuNP dimer formation Name Sequence 2PS-Anchor DNA(80) [a] 5'- GGC ATG GTC* T*A*C GTC ACC CTA GTC AGT CAT CTT GCA CTA TCT CCT TGA GAA CGT CAC GCG TCA GTA CGC TAC ATC TTA CAC CAG CAC TA*C* G*TC ACA CCT A -3' 2PS-Anchor DNA(70) [b] 5'- GGC ATG GTC* T*A*C GTC ACC CTA GTC AGT CAT CTT GCA CTA TCT CCT TGA GAA CGT CAC GCG TCA GTA CGC TAC ATC TTA C*A*C* CAG CAC TACGTC ACA CCT A -3' 2PS-Anchor DNA(60) [c] 5'- GGC ATG GTC TAC GTC ACC C*T*A* GTC AGT CAT CTT GCA CTA TCT CCT TGA GAA CGT CAC GCG TCA GTA CGC TAC ATC TTA C*A*C* CAG CAC TAC GTC ACA CCT A -3' 0PS-DNA 5'- GGC ATG GTC TAC GTC ACC CTA GTC AGT CAT CTT GCA CTA TCT CCT TGA GAA CGT CAC GCG TCA GTA CGC TAC ATC TTA CAC CAG CAC TACGTC ACA CCT A -3' CP-DNA 5'- HS-(CH 2) 6-PEG-TAG GTG TGA CGT AGT GCT GGT GTA AGA TGT AGC GTA CTG ACG CGT GAC GTT CTC AAG GAG ATA GTG CAA GAT GAC TGA CTA GGG TGA CGT AGA CCA TGC C-3' 2PS-dsDNA(80) Bifunctional fastener treated 2PS-AnchorDNA(80) and CP-DNA 2PS-dsDNA(70) Bifunctional fastener treated 2PS-AnchorDNA(70) and CP-DNA 2PS-dsDNA(60) Bifunctional fastener treated 2PS-AnchorDNA(60) and CP-DNA 0PS-dsDNA Bifunctional fastener treated 0PS-DNA and CP-DNA 2PS-dsDNA(80)- 2PS-AnchorDNA(80) with no bifunctional fastener treatment and CP-DNA nofastener [a, b, c] Underlined nucleotides with * have phosphorothioate modification on the backbone. SEM imaging: SEM images were taken with a Field Emission Scanning Electron Microscope (FE-SEM, Hitachi S-4700) at 15 kv at the working distance of ~5 cm (150 k magnification). Image processing: The distance between the AuNP dimer on the surface was measured from the coordinates of the AuNP center pixel which were collected by an in-house code using IDL (Interface Description Language). The population in the histogram was counted as two times the average number of dimers in a specific distance range per total number of AuNPs on the surface [P=2 (# of dimers in range)/(total # of AuNPs on surface)] in each of the SEM images which cover an area of nm 2. The aggregates of three or more AuNPs or clustering of two or more pairs of NP dimers in proximity were excluded since they might be formed due to non-oligonucleotide AuNP aggregation. To ensure unbiased evaluation of particle spacings, distances between background particles and particles in an identified predicted pair were also included in the histogram at the sacrifice of low background. The images used for population counting and coordinates of the center of all AuNPs based on pixels are provided in the last part of the Supporting Information. Since the code reads the coordination of the pixels brighter than a given threshold, it had difficulty in differentiating AuNPs from surface and collecting all AuNPs. In order to guarantee reading of all AuNPs, the center of each AuNP were collected manually and were transferred to a white background sheet ( ) to make sure that all of the AuNPs were picked and that only the center of each AuNP was considered. The coordinates of (0, 0) and (960, 720) indicate left bottom and right top corners of an image, respectively. The position of small black dots in the center of AuNPs are collected and used for distance measurements. 3
5 The distance measured are significantly above the measurement errors from the size variation of the AuNPs, the length of the fastener (~0.9 nm when fully stretched, from molecular modeling program MOE), and the resolution of the SEM equipment (~1.5 nm). 2. MALDI mass spectrometry MALDI mass spectrometry results showing the yield of the reaction between bifunctional fastener and oligonucleotide with three adjacent phosphate moieties modified to phosphorothioate are illustrated in Figure S1. 26 mer DNA with the sequence 5?-GTC CTA GAG TCA C*G*C* TAC GTC ACT CC-3? was used for analysis. Phosphorothioate groups were modified on the position of G* and two C*s. After reaction with BIDBE, oligonucleotide was treated with TCEP to reduce the disulfide Figure S1. TOF-MS scans of a (a) 26 mer oligonucleotide containing three adjacent phosphorothioate modifications and (b) the DNA with bifunctional fastener treatment bond in the BIDBE. As the oligonucleotide used has the mass of 7921 Da (see Figure S1a) and bifunctional fastener attached to phosphorothioate modification contributes to the mass of 118 Da, the DNA with only one bifunctional fastener attached should have the mass of 8039 Da and it is supposed to increase to 8158 Da and 8275 Da as the number of attached fastener increases to two and three. Figure S1b shows that the bifunctional fastener treated DNA has dominant peak of 8272 Da while there is no peak corresponding to the mass of 7921 Da and 8039 Da and only a small peak that matches the mass of 8157 Da. Considering the allowed error range ( %) of 4-8 Da, it can be concluded that most of the triplet phosphorothioate modifications which are suppose to react with one Au NP have mostly all three fasteners bound and at least have two which means that most of triplet phosphorothioate modification group on the DNA can bind to Au NPs. 4
6 3. SEM images (Au NP trimers with 40 bps between nanoparticles) (a) Figure S2. SEM images of Au NPs assembled on bifunctional fastener treated phosphorothioate modified DNA. (a) Au NPs are assembled on dsdna with 40 bps (~13.6 nm) distances between phosphorothioate modifications and (b) Au NP trimers and dimers with predicted distances are identified with yellow (Au NP trimer with 40 bps distance between NPs), red (Au NP dimer with 80 bps distance between NPs), and green (Au NP dimer with 40 bps distance between NPs). (c) Control sample with non-phosphorothioate modified DNA was shown. The scheme of Au NP-DNA assemblies on (d) 3PS-dsDNA(40, 40) and (e) 0PS-dsDNA are also shown. SEM images of the Au NP-DNA assembly with high concentration of DNA immobilized on the surface are depicted in Figure S2. The surface with dsdna containing three triplet phosphorothioate modifications with 40 base pair gap between them [3PS-dsDNA(40, 40)] had a large amount of Au NPs bound on the surface (see Figure S2a). Au NP trimers with the matching distance (40 bps~13.6 nm between Au NPs) could be found on the surface (circled yellow, see Figure S2b) but their number was limited. However, Au NPs dimers with 80 bps distance (~27.2 nm, circled red) was found to be dominant while there are also a large portion of dimers with 40 bps distance (~13.6 nm, circled green). This shows that the reaction yield is high but short distances between phosphorothioate modifications (~13.6 nm) might have increased steric hindrance for the reaction between Au NP and bifunctional fasteners to form Au NPs trimers. The control surface immobilized with dsdna without phosphorothioate modification [0PS-dsDNA] had sparse Au NPs due only to nonspecific binding of Au NPs to Au surface (see Figure S2c). The sequences of DNA used are listed in Table S3. 50 nm Au thin film with 5 nm Cr adhesion layer deposited on Si wafer was used to immobilize DNA. The surface was deposited with thermal deposition method and the deposition rate was ~3-5 Å/sec. 5 nm Au NPs were used to assemble on DNA. Final solution of dsdna immobilized on the surface is 200 nm and no butane thiol was used to passivate the surface to get high concentration of Au NP/DNA assembly. 150 nm of Au NP solution in 50 mm NaCl 10 mm acetate (ph 5) was used for Au NP and fastener reaction and the reaction was made for 20 hours. Other reaction conditions are identical to the processes noted in the Experimental Section. 5
7 Table S3. Oligonucleotides used to form Au NP trimers Name Sequence 3PS-AnchorDNA(40, 40) 5'- GGC ATG GTC* T*A*C GTC ACC CTA GTC AGT CAT CTT GCA CTA TCT CCT TGA (Green) G*A*A* CGT CAC GCG TCA GTA CGC TAC ATC TTA CAC CAG CAC TA*C* G*TC ACA CCT A -3' 0PS-DNA (Green) C-DNA (Red) 5'- GGC ATG GTC TAC GTC ACC CTA GTC AGT CAT CTT GCA CTA TCT CCT TGA GAA CGT CAC GCG TCA GTA CGC TAC ATC TTA CAC CAG CAC TACGTC ACA CCT A -3' 5'- HS-(CH 2) 6-TAG GTG TGA CGT AGT GCT GGT GTA AGA TGT AGC GTA CTG ACG CGT GAC GTT CTC AAG GAG ATA GTG CAA GAT GAC TGA CTA GGG TGA CGT AGA CCA TGC C-3' Bifunctional fastener treated 3PS-AnchorDNA(40,40) and C-DNA Bifunctional fastener treated 0PS-DNA and CP-DNA 3PS-dsDNA(40, 40) 0PS-dsDNA Underlined nucleotides with * are phosphorothioate modified 6
8 4. SEM images used for histogram data processing 2PS-dsDNA(80): 2PS-dsDNA(80)_1 2PS-dsDNA(80)_2 2PS-dsDNA(80)_3 7
9 2PS-dsDNA(70): 2PS-dsDNA(70)_1 2PS-dsDNA(70)_2 2PS-dsDNA(70)_3 8
10 2PS-dsDNA(60): 2PS-dsDNA(60)_1 2PS-dsDNA(60)_2 2PS-dsDNA(60)_3 9
11 0PS-dsDNA: 0PS-dsDNA_1 0PS-dsDNA_2 0PS-dsDNA_3 10
12 2PS-dsDNA(80)-nofastener: 2PS-dsDNA(80)-nofastener_1 2PS-dsDNA(80)-nofastener_2 2PS-dsDNA(80)-nofastener_3 11
13 5. Coordinates of AuNPs on the surfaces in SEM images 2PS-dsDNA(80)_1 12
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18 6. References [1] R. F. Luduena, M. C. Roach, P. P. Trcka, S. Weintraub, Anal. Biochem. 1981, 117, 76. [2] J. J. Storhoff, R. Elghanian, R. C. Mucic, C. A. Mirkin, R. L. Letsinger, J. Am. Chem. Soc. 1998, 120, [3] J. Liu, Y. Lu, Nat. Protoc. 2006, 1, 246 [4] C. J. Loweth, W. B. Caldwell, X. Peng, A. P. Alivisatos, P. G. Schultz, Angew. Chem. 1999, 111, 1925; Angew. Chem. Int. Ed. 1999, 38, [5] Z. Deng, Y. Tian, S.-H. Lee, A. E. Ribbe, C. Mao, Angew. Chem. 2005, 117, 3648; Angew. Chem. Int. Ed. 2005, 44,
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