RNA-templated DNA origami structures

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1 Electronic Supplementary Information RNA-templated DNA origami structures Masayuki Endo,* a,c Seigi Yamamoto, b Koichi Tatsumi, b Tomoko Emura, b Kumi Hidaka, b and Hiroshi Sugiyama* a,b,c a Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshidaushinomiyacho, Sakyo-ku, Kyoto , Japan. b Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawaoiwakecho, Sakyo-ku, Kyoto , Japan. c CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo , Japan. endo@kuchem.kyoto-u.ac.jp; hs@kuchem.kyoto-u.ac.jp. Materials. Reagents and solvents for the synthesis were purchased from standard suppliers and used without further purification. PCR amplification was carried out using GoTaq Green Master Mix (Promega). Single-stranded M13mp18 DNA and streptavidin magnetic beads were purchased from New England Biolabs (Ipswich, MA). The staple strands and primers were purchased from Operon Biotechnology (Tokyo, Japan). Modified oligonucleotides were purchased from Nippon Bio Service (Saitama, Japan). The gel-filtration column and the Sephacryl S-300 were purchased from Bio-rad Laboratories, Inc. (Hercules, CA) and GE Healthcare (Buckinghamshire, UK), respectively. 1. Preparation of template dsdna for RNA synthesis. The PCR amplification of the template dsdna was carried out from a plasmid-containing EGFP coding region using Go Taq (Promega) with two primers by following the manufacture s protocol, and the product was purified using a PCR purification kit (Qiagen). S1

2 2. Preparation of RNA template. A T7 promoter-containing template dsdna was used for the transcription to prepare RNA template. RNA synthesis was performed using 10 nm template dsdna, 40 mm Tris-HCl (ph 8.0), 5 mm DTT, 8 mm MgCl 2, 2 mm spermidine, 0.5 mm NTP, and 0.25μM T7 RNA polymerase (Takara) at 42 o C for 1.5 h. In the case of the preparation of modified RNA templates, UTP was replace to 5-aminoally UTP and 5-biotinylated UTP. The transcribed RNA was purified using RNeasy Mini kit (Qiagen). The product was confirmed by gel electrophoresis. 3. Preparation of RNA-templated DNA origami. The RNA-templated DNA origami was designed by a cadnano software 1 using the geometry of 11.00, 10.67, and bp/turn helical pitch. The sequences of the staple strands are listed in Table S1. For the tile formation, sample solution (40 L) containing 0.02 M RNA template, 1 M staple strands (20-50 eq), 20 mm Tris-HCl (ph 7.6), 1 mm EDTA, and 10 mm MgCl 2 was annealed from 65 C to 15 C at a rate of 1.0 C/min. For the tube formation, sample solution (40 L) containing 0.02 M RNA template, 0.4 M staple strands (20 eq), 20 mm Tris-HCl (ph 7.6), 1 mm EDTA, and 10 mm MgCl 2 was annealed from 65 C to 15 C at a rate of 0.1 C/min. These structures were purified using a gel filtration column (Sephacryl 300, GE Helthcare). 4. Purification by streptavidin magnetic beads. For the streptavidin-magnetic beads purification, the biotin-ss-dna was added by replacing the corresponding unmodified staple DNA, the mixtures were assembled using the same condition. A mixture of RNA-templated DNA origami (20 μl) was incubated with pre-washed streptavidin magnetic beads (New England Biolabs) at rt for 40 min. The sample in a microtube was placed in the magnetic separation stand and washed three times with the buffer. Then the target product attached on the streptavidin magnetic beads was recovered by cleavage of disulfide bond with 50 mm DTT, 20 mm Tris buffer (ph 7.6), and 100 mm MgCl 2 (32 μl) at rt for 1 h. The collected samples were analyzed by native PAGE. 5. Preparation of DNA-templated DNA origami. S2

3 For the preparation of the DNA-templated DNA origami, template single-stranded DNA was obtained using a reported method. 2 PCR amplified double-stranded DNA with biotin-attached reverse primer was denatured by NaOH, and then the bottom strand was removed by streptavidin magnetic beads (New England Biolabs). The solution was neutralized by ammonium acetate. Finally the sample was purified by PCR purification kit (Qiagen). 6. Preparation of RNA-templated DNA origami with modified UTP. For the preparation of modified RNA templates, in vitro transcription was performed with ATP, CTP, GTP, and 5-aminoallyl UTP or 5-biotinylated UTP. The purified modified RNA templates and staple strands were assembled as described above. 7. High-speed AFM imaging of the RNAP movement and transcription: AFM images were obtained using an AFM system (Nano Live Vision, RIBM, Tsukuba, Japan) with a silicon nitride cantilever (Olympus BL-AC10EGS). Samples (2 L) were adsorbed onto a aqueous 3-aminopropyltriethoxysilane (APTES, 0.1%) passivated mica plate for 5 min at room temperature and then washed three times using the same buffer solution. Scanning was performed in the same buffer solution using a tapping mode. Fig. S1. AFM images of RNA/DNA hybrid tiles after anealing. 7HB-tile with bp/turn pitch (left) and 7HB-tile with bp/turn pitch. S3

4 Fig. S2. Purification of RNA/DNA hybrid tiles using streptavidin-magnetic beads. Native polyacryl gel electrophoresis (4%) of RNA/DNA hybrid tiles with bp/turn and bp/turn. Fig. S3. Purification of RNA/DNA hybrid tube using streptavidin-magnetic beads. Native polyacryl gel electrophoresis (4%) of RNA/DNA hybrid tube. S4

Modified UTP ( M) UTP ( M) Lane 1 0 500 Lane 2 125 375 Lane 3 250 250 Lane 4 375 125 Lane 5 500 0 Fig. S4.

Gel electrophoresis (1% agarose) analysis of modified RNA transcripts.

5 Modified UTP ( M) UTP ( M) Lane Lane Lane Lane Lane Fig. S4. Preparation of modified RNA transcripts with 5-aminoallyl UTP (A) and 5-biotinylated UTP (B). Gel electrophoresis (1% agarose) analysis of modified RNA transcripts. The concentrations of modified UTP and UTP were changed as listed in the table. Fig. S5. AFM images of modified RNA/DNA hybrid tiles with bp/turn pitch. RNA templates were prepared with 5-aminoally UTP (left) or 5-biotinylated UTP (right). S5

RNA/DNA hybrid tube (right middle), and 5- biotinylated-uridine-containing RNA/DNA hybrid tube

6 Fig. S6. Sectional analysis of tubular structures. DNA/DNA tube (left), RNA/DNA hybrid tube (left middle), 5-aminoallyl-uridine-containing RNA/DNA hybrid tube (right middle), and 5- biotinylated-uridine-containing RNA/DNA hybrid tube (right). Fig. S7. Native PAGE (4%) analysis of 5-aminoally-uracil and 5-biotinylated uracil modified RNA/DNA hybrid tiles with bp/turn. S6

Fig. S8. AFM images of modified RNA/DNA hybrid tiles with 11.00 bp/turn pitch. For imaging, mica was not passivated with APTES.

7 Fig. S8. AFM images of modified RNA/DNA hybrid tiles with bp/turn pitch. For imaging, mica was not passivated with APTES. In these cases, monomers may be washed out, and the stacked tiles were left on the mica surface. References 1. S. M. Douglas, A. H. Marblestone, S. Teerapittayanon, A. Vazquez, G. M. Church, W. M. Shih, Nucleic Acids Res. 2009, 37, E. Pound, J. R. Ashton, H. A. Becerril, A. T. Woolley, Nano Lett. 2009, 9, S7

8 DNA strands for RNA-templated DNA tile with a bp/turn pitch 1A 1B 1C 2A 2B 2C 2D 3A 3B 3C 4A 4B 4C 4D 5A 5B 5C 6A 6B 6C 6D AAGCACTGGTAGGTCAGGGTGGTCCCTCGCCCTTGCTCAC TGCCCCAGGCCGTCCTCCTTGAAGAGCGGCTG AGGGCGGAGCTCAGGTAGTGGTTGCCAGCTTG TGGGCACCACCCCGGTGAACAGCTACGAGGGT GGGCCAGGTTCATGTGGTCGGGGTTCGATGCC CTTCAGCTCTGTTGTAGTTGTACTTCGGGCAG CAGCACGGCGCTTCTCGTTGGGGTCTTTGCTC CGTGCTGCGCACGGGCAGCTTGCCGACCAGGA CGTTGTGGCGATGCGGTTCACCAGGAAGAAGT TGTGATCGGGCCGTCGCCGATGGGGATATAGA GCCGTTTACGTCGCCGTCCAGCTCGGTGGTGC AGATGAACCGGGCATGGCGGACTTGGTGTCGC CCTCGAACTCTGCTTGTCGGCCATGGTGTTCT GCTGGTAGCGGTCACGAACTCCAGCAGGACCA GTAGCCTTTTCAGGGTCAGCTTGCAACTTGTG GCCGTTCTTTCACCTCGGCGCGGGTCCTGGAC CCCGGCGGTGGTCGGCGAGCTGCAACCTTGAT CTCGCCGGACACGCTGCGTAGGTG GCATCGCCGAAGAAGATGGTGCGCTCTTGTAG TTGCCGTCGCGGATCTTGAAGTTCCGCTGCCG TCCTCGATCAGCTCGTCCATGCCGAGAGTGAT DNA strands for RNA-templated DNA tile with a bp/turn pitch 1A 1B 1C 2A 2B 2C 2D 3A 3B 3C 4A 4B 4C 4D 5A 5B 5C 6A 6B 6C 6D AGCACTGCCGTAGGTCAGGGTGGTCTCGCCCTTGCTCACC GCCCCAGGTGCCGTCCTCCTTGAAGCGGCTGA GGGCGGACTGCTCAGGTAGTGGTTCAGCTTGT TGGGCACCACCCCGGTGAACAGCTCCACGAGGG TGGGCCAGGTTCATGTGGTCGGGGTAGTCGATGC CCTTCAGCTCTGTTGTAGTTGTACTCGTCGGGCA GCAGCACGGCGCTTCTCGTTGGGGTCTTTGCTCA CGTGCTGCGCACGGGCAGCTTGCCGACCAGGA CGTTGTGGCGATGCGGTTCACCAGGAAGAAGT TGTGATCGGGCCGTCGCCGATGGGGATATAGA GGCCGTTTACGTCGCCGTCCAGCTCGGTGGTGC AGATGAACTTCGGGCATGGCGGACTTGGTGTCGC CCTCGAACTTTCTGCTTGTCGGCCATGGTGTTCT GCTGGTAGTGCGGTCACGAACTCCAGCAGGACCA CGTAGCCTTCAGGGTCAGCTTGCCGAACTTGT TGCCGTTCTCACCTCGGCGCGGGTCTCCTGGA TCCCGGCGGGTCGGCGAGCTGCACCACCTTGA CCCTCGCCGGACACGCTGTAGGTGG CATCGCCCTTTGAAGAAGATGGTGCGCTTGTAGT TGCCGTCGTTGGCGGATCTTGAAGTTGCTGCCGT CCTCGATGTTACAGCTCGTCCATGCCGAGAGTGA S8

9 DNA strands for RNA-templated DNA tube with a bp/turn pitch 1 TCATGTGGTCGGGGCCCTTGCTCGTCCA 2 GAAGTCGTGTCGGCCCTTGATGCCGTTC 3 TGAACAGCTCCTCGTAGCGGCACTTGAA 4 TGCCGAGAAGTTCACATGATATAGACGT 5 ATGGCGGTGAAGCACTGCACGACCCCGGCCGGCGGCGGTCAC 6 CGTAGCCTTCGGGCTGTGGCTTGTGGCGGATCTTGAGTGATC 7 TCGATGTGTTGTAGTTGTACTTCCTGGATCAGGGTGGTCACG 8 GCACGCTGCCGTCCGAACTCCCCAGGATGGGCACCCCGTAGG 9 AGCTCGAAGCAGGACCATGTGGCGAGCTGTGCCCCAGGATGT 10 TTACGTCGCCGTCCAGGGTGGAGAAGATGGTGCGCCCAGCTT 11 TCCTTGAGCCAGGGCACGGGCTGGCCGTTTCTCGTTGGGGTC 12 TGTAGTTGCCGTCGTGCCGTCGCTGGTAGTGGTCGATCGCGC 13 GTGTTCTCTCCTTGAAGTCGACGGGTCTCGGTGGTGCAGATG 14 CGTCGCCGATGGGGTTTGCTCACACGCTGAACTTGAGCTTGC 15 TCGCCGGAGGGCGGACTGGGTACGGGGCCAGCTCG 16 CTCGCCCAACTTCAACTTCACCTCGGCGTGCCCTT 17 ATGCGGTCCCTCGAGGGTCAGCTTGCCG 18 TGTCGGGCAGCAGCGCTCAGGCATCGCC Biotin-SS-attached DNA strands for streptavidin magnetic beads purification RNA-templated DNA tile (R32-6A-SS-biotin) 5 -Biotin-SS-TTTT-CTCGCCGGACACGCTGCGTAGGTG-3 RNA-templated DNA tile (R33-6A-SS-biotin) 5 -Biotin-SS-TTTT-CCCTCGCCGGACACGCTGTAGGTGG-3 RNA-templated DNA tube (tube-18-ss-biotin) 5 -Biotin-SS-TTTT-TGTCGGGCAGCAGCGCTCAGGCATCGCC-3 S9

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XactEdit Cas9 Nuclease with NLS User Manual

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