supplementary materials for...ahf) after the pinhole onto a second detector (τ-spad-100, picoquant)...
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www.sciencemag.org/cgi/content/full/338/6106/506/DC1
Supplementary Materials for
Fluorescence Enhancement at Docking Sites of DNA-Directed Self-Assembled Nanoantennas
G. P. Acuna,* F. M. Möller, P. Holzmeister, S. Beater, B. Lalkens, P. Tinnefeld*
*To whom correspondence should be addressed. E-mail: [email protected] (G.P.A.); [email protected] (P.T.)
Published 26 October 2012, Science 338, 506 (2012) DOI: 10.1126/science.1228638
This PDF file includes:
Materials and Methods Figs. S1 to S9 Table S1 Full Reference List
2
Materials and Methods Numerical simulations:
Numerical simulations were performed using commercial FDFD software (www.cst.com). The relative change in the excitation rate was estimated by simulating the electric field intensity in the vicinity of the nanoparticle system at the fluorophore position and averaged over the tangential and radial incident polarization. The relative change of the radiative and non radiative decay as well as the relative change in the lifetime was estimated following ref. (30), where the dye is modeled by a dipole current source with a determined orientation. The total power radiated into the far field and dissipated by the metallic objects is computed and normalized to the total power radiated into the far field in the absence of metallic objects. For these calculations, the intrinsic quantum yield of the ATTO647N dye of 0.65 was considered according to (31). All simulations were performed assuming a planar illumination with water as medium, mimicking the buffer conditions and effects arising from the glass interface were neglected. The error bars take into account the size distribution of the nanoparticles, but the deviation of particles from the spherical shape, the angular distribution of the DNA origami pillar or the possibility that not all three capturing strands are binding to a nanoparticle are not considered.
Functionalization of gold nanoparticles:
Gold nanoparticles of five different diameters (20, 40, 60, 80 and 100 nm) were purchased from BBInternational (www.bbi-gold.com). TEM measurements revealed the following size distribution, (19.4 ± 1.7 nm, 43.5 ± 4.6 nm, 61.5 ± 7 nm, 80.1 ± 7.4 nm, 105 ± 10.4 nm). Additionally some particles showed a considerable deviation from a spherical shape, see Fig. S3.
The DNA functionalization was performed as described in ref (32) with the following DNA sequence from VBC Biotech (www.vbc-biotech.com) containing a thiol modification at the 5’ end: 5’-Thiol-TTTTTTTTTTTTTTT-3’. The binding of the gold nanoparticles to the DNA origami structure was included in ref (19). The immobilized DNA origami pillars were incubated with DNA modified gold nanoparticles until the desired binding yield was achieved. This incubation was usually carried out on the microscope for several hours. Determination of intensity and fluorescence lifetime:
Fluorescence intensity and lifetime measurements were carried out on a custom-built confocal setup based on an Olympus IX-71 inverted microscope. For excitation, the light of an 80 MHz pulsed diode laser (640nm, LDH-D-C-640, Picoquant) was coupled into an oil-immersion objective (UPlanSApo 60XO / 1.35 NA, Olympus). A linear polarizer, an electrooptical modulator (EOM, LM 0202, Qioptiq) and a quarter wave plate (AQWP05M-600, Thorlabs) in the correct orientation with respect to each other allowed control over the excitation polarization (33). Depending on the rotation speed, we either measured with rotating linear polarization or average over all directions which for our measurements is equivalent to measurements with circular polarized light. Excitation and emission light were separated by a dual-band dichroic beam splitter (z532/633, AHF) and subsequently focused onto a 50 µm pinhole (Linos) before detection with an avalanche
3
photo diode (τ-SPAD-100, Picoquant) with appropriate spectral filtering (ET 700/75m, AHF and RazorEdge LP 647, Semrock). The detector signal was registered with a single photon counting PC card (SPC-830, Becker&Hickl) and further analyzed using custom-made LabView code.
For FRET measurements, an additional 532 nm laser (TECGL-30, World Star Tech) was used to excite Cy3 and the emitted light was split spectrally at 640 nm (640DCXR, AHF) after the pinhole onto a second detector (τ-SPAD-100, Picoquant) with appropriate filters (Brightline HC582/75, AHF and RazorEdge LP 532, Semrock). The two lasers were alternated with 1 ms period by use of an acousto-optical tunable filter (AOTFnc-VIS, AA optoelectronic) to separate FRET sensitized from direct Cy5 excitation.
We employed the reconvolution algorithm of the FluoFit software (Picoquant) to obtain the fluorescence lifetime from the measured decay and the instrument response function (IRF) of the setup. We used an IRF acquired at an appropriate intensity to account for count rate dependence of the detector and included the periodicity of the excitation as well as scattering in the analysis. The width of the IRF (FWHM=650 ps) limits the temporal resolution to approximately 100-200 ps. All decays could be fitted with the convolution of a monoexponential decay and the IRF.
The Holliday junction:
The Holliday junction consisted of four strands (28) called R, H, X and B with the following sequences R: 5’- ACA AAT ATC CTT GCC CCA GCA GGC GAA TTT CCC ACC GCT CGG CTC AAC TGG G -3’, H: 5’-Cy3-CCG TAG CAG CGCG AGC GGT GGG-3’, X: 5’-CCC AGT TGA GCG CTT GCT AGG G-3’ and B: 5’-Cy5-CCC TAG CAA GCC GCT GCT AGG G-3’. The first 27 base pairs of the strand R bind to the origami pillar. All four strands were incorporated together with the staples prior to the folding process.
DNA origami pillar structures:
Unmodified and modified staple strands (see Table S1) were purchased from MWG (Munich, Germany) or IBA (Göttingen, Germany) at a concentration of 100 μM and were used without further purification. DNA origamis are formed with a molar ratio of 1:30 between the viral DNA and the unmodified staple strands and 1:100 between the viral DNA and the modified staple strands. For preparation of the scaffold strands Escherichia coli strain K91 was infected with the respective M13mp18 phage (p8634) at a Multiplicity of Infection of ~1. After amplification, the phage particles were separated, purified and their ssDNA was extracted and purified similar as described before (17). The concentration was adjusted to 100 nM using a molecular weight of 330 g/mol per base and an extinction coefficient = 33 mg/ml for A260 = 1 in a NanoDrop Spectrophotometer (Peqlab, Erlangen, Germany). The DNA origami design was performed with the open-source software caDNAno (www.cadnano.org)(17). The folding buffer contained 12.5 mM MgCl2 as well as 5 mM Tris + 1 mM EDTA (pH 7.9 at 20°C). A TEM image of the DNA origami pillar is included in Fig. S8. Folding time was three days.
4
Fig. S1. Sketch of the top-view of the DNA origami pillar. The numbered circles represent DNA helices. Helices 0 to 11 form the central 12-helix bundle. The remaining helixes form the extra 6-helix bundles of the base. The helix center-to-center distance is 3 nm (16). The positions of the single dye (ATTO647N), the capturing paint strands, the Holliday Junction and the nanoparticles (NP) are also included.
5
Fig. S2. Fluorescence lifetime imaging microscopy of DNA origami pillars with 80 nm nanoparticles. Orange-red spots represent ATTO647N dyes on DNA origami pillars without nanoparticles. The shortened fluorescence lifetime (blue spots) indicates binding of nanoparticles. Commonly, intermediate binding yields were intended to use the fluorescence from DNA origami pillars without nanoparticles as internal reference (A). On the other hand, yields exceeding 70% for the dimer could be reached even for 80 nm particles (B). (C) Histogram of yields of dimers, monomers and DNA origamis without a nanoparticle for the measurement included in Fig. 3A.
6
Fig. S3. (A)-(E) TEM images of the gold nanoparticles of five different sizes. Analysis of the images revealed some deviation from perfect spherical shapes as well as a distribution of sizes (19.4 ± 1.7 nm, 43.5 ± 4.6 nm, 61.5 ± 7 nm, 80.1 ± 7.4 nm, 105 ± 10.4 nm).
7
Fig. S4. (A)-(E) Fluorescence intensity versus lifetime plot of the DNA origami pillar with binding sites for one (monomer) and two (dimer) particles of different diameters.
8
Fig. S5. Simulated quantum yield (A) and fluorescence lifetime (B) as a function of the nanoparticle diameter for the monomer and dimer system at different dye orientations, radial and tangential. The error bars take into account the size distribution of the nanoparticles. The quantum yield and fluorescence lifetime is normalized to the properties of the dye in the absence of nanoparticles. A quantum yield of 0.65 for ATTO647N was considered for both the quantum yield and fluorescence lifetime calculations.
9
Fig. S6. Time gating for signal to noise improvement at elevated background fluorescence from freely diffusing dye molecules. (A) Fluorescence decay of the ATTO647N dye located within the DNA origami pillar with two 80 nm nanoparticles (dimer). Measurements were carried out at a concentration of 0.5 µM of ATTO647N dyes in solution. The decay shows a bi-exponential behavior due to the combination of the quenched dye in the hotspot and the unquenched dyes of the background. (B) Corresponding intensity transient showing that the single ATTO647N in the hotspot is easily detected despite the high background concentration. For (C) and (D) the intensity transients were reconstructed by time gating allowing only photons arriving 1.5-6 ns and 1.5-3 ns in the fluorescence decay, respectively. The time gating further increased the signal-to-noise by removing the largest fraction of the background fluorescence.
10
Fig. S7. High count rates for fast dynamics. (A) Intensity transient at 1 µs binning. Off-states as short as 10 µs are clearly visualized. Therefore, the excitation intensity was increased to 7 µW and 100 µM ascorbic acid and 100 µM of methylviologen were added to the buffer to induce fast blinking of the dye molecule (34). At these high count rates, saturation effects of the setup and not by the organic dye limit photon counts. To estimate the actual rate of photons arriving at the detector in the on-state, we corrected for the dead time of the setup (B,C). Therefore, we measured the detected count rate of a micromolar concentration of ATTO647N in solution for different excitation intensities in an excitation regime where fluorescence from ATTO647N is linearly depending on excitation intensity (B). Photon count rate vs excitation intensity for a µM concentration of ATTO647N. (C) Correction factor dependence on the measured count rate. Hyperbolic fitting yields a saturation at Nmax = 6.8 ± 0.2 MHz which corresponds to a dead time of τD = 1/Nmax = 147 ± 4 ns. This yields a correction factor of f=1/(1-N τD) as displayed in (C). Accordingly, the count rate of the on-state for the transient displayed in (A) is 10.6 MHz. It should be emphasized that the blinking of the dye was purposely induced for this measurement and that all measurements discussed before were taken at lower excitation intensities to avoid saturation effects.
11
Fig. S8. Transmission electron microscopy image of the DNA origami pillar.
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1421
2835
4249
5663
7077
8491
98105
112
119
126
133
140
147
154
161
168
175
182
189
196
203
210
217
224
231
238
245
252
259
266
273
280
287
294
301
308
315
322
329
336
343
350
357
364
371
378
385
392
399
406
413
420
427
434
441
448
455
462
469
476
483
490
497
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511
518
525
532
539
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553
560
567
574
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1
TATGCCGACTCTATATCTATACCTTCAT
CGTTCGGTATTTTTAATGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATTCAAAAATATTGTCTGTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATCTCTGTTGGCCAGAATGTCCCTTTTATTACTGGTCGTGTGACTGGTGAATCTGCCAATGTAAATAATCCATTTCAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCTACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC
TTATAAACCTTCATGGAATATTTG
TGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAA
CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCC
GCCTAAATTACATGTTGGCGTTGTTAAATATGGCGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTTATACTGGTAAGAATTTGTATAACGCATATGATACTAAACAGGCTTTTTCTAGTAATTATGATTCCGGTGTTTATTCTTATTTAACGCCTTATTTATCACACGGTCGGTATTTCAAACCATTAAATTTAGGTCAGAAGATGAAATTAACTAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGCGATTGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTTAAAAAGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAATCTAAGCTATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATAGCGACGATTTACAGAAGCAAGGTTATTCACTCACATATATTGATTTATGTACTGTTTCCATTAAAAAAGGTAATTCAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCATCTTCTTTTGCTCAGGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTTGTAACTTGGTATTCAAAGCAATCAGG
CTCTTATTACTGGCTCGAAAATGCCTCT
CTACATA
CGAATCCGTTATTGTTTCTCCCGATGTAAAAGGTACTGTTACTGTATATTCATCTGACGTTAAACCTGAAAATCTACGCAATTTCTTTATTTCTGTTTTACGTGCAAATAATTTTGATATGGTAGGTTCTAACCCTTCCATTATTCAGAAGTATAATCCAAACAATCAGGATTATATTGATGAATTGCCATCATCTGATAATCAGGAATATGATGATAATTCCGCTCCTTCTGGTGGTTTCTTTGTTCCGCAAAATGATAATGTTACTCAAACTTTTAAAATTAATAACGTTCGGGCAAAGGATTTAATACGAGTTGTCGAATTGTTTGTAAAGTCTAATACTTCTAAATCCTCAAATGTATTATCTATTGACGGCTCTAATCTATTAGTTGTTAGTGCTCCTAAAGATATTTTAGATAACCTTCCTCAATTCCTTTCAACTGTTGATTTGCCAACTGACCAGATATTGATTGAGGGTTTGATATTTGAGGTTCAGCAAGGTGATGCTTTAGATTTTTCATTTGCTGCTGGCTCTCAGCGTGGCACTGTTGCAGGCGGTGTTAATACTGACCGCCTCACCTCTGTTTTATCTTCTGCTGGTGGTT
TTACCTTTTGTCGGTACTTTATATT
AACACCTTCGTGAT
ACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATTTATTTGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCC
TTGTCGTCGTCTGGACAGAATTACT
TTTTGTCTTTGGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGGTAACTTTGTTCGGCTATCTGCTTACTTTTCTTAAAAAGGGCTTCGGTAAGATAGCTATTGCTATTTCATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGATATTAGCGCTCAATTACCCTCTGACTTTGTTCAGGGTGTTCAGTTAATTCTCCCGTCTAATGCGCTTCCCTGTTTTTATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAACAAAAAATCGTTTCTTATTTGGATTGGGATAAATAATATGGCTGTTTATTTTGTAACTGGCAAATTAGGCTCTGGAAAGACGCTCGTTAGCGTTGGTAAGATTCAGGATAAAATTGTAGCTGGGTGCAAAATAGCAACTAATCTTGATTTAAGGCTT
GTTGAAATTAAACCATCTCAAGCCCAATTTACTACTCGTTCTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATGAGCAGCTTTGTTACGTTGATTTGGGTAATGAATATCCGGTTCTTGTCAAGATTACTCTTGATGAAGGTCAGCCAGCCTATGCGCCTGGTCTGTACACCGTTCATCTGTCCTCTTTCAAAGTTGGTCAGTTCGGTTCCCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACATGGAGCAGGTCGCGGATTTCGACACAATTTATCAGGCGATGATACAAATCTCCGTTGTACTTTGTTTCGCGCTTGGTATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTTGCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAAGCT
TCGCATAAGGTAATTCACAATGATTAAA
GTCTGCA
GTTTAAGAAATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTTAGTTGTTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAATCCCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTT
ACGTCCTGACTGGTATAATGAGCCAGTTCTTAAAA
TGGAGACAAGACAC
AAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAATGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGATCTCTCAAAAATAGCTACCCTCTCCGGCATTAATTTATCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCTTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAA
TTAGTTCGTTTTATTAACGTAGATTTTTCTTCCCA
TATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTAATGCTACTACTATTAGTAGAATTGATGCCACCTTTTCAGCTCGCGCCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTTCGCAGAATTGGGAATCAACTGTTATATGGAATGAAACTTCCAGACACCGTACTTTAGTTGCATATTTAAAACATGTTGAGCTACAGCATTATATTCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGAGCAATTAAAGGTACTCTCTAATCCTGACCTGTTGGAGTTTGCTTCCGGTCTGGTTCGCTTTGAAGCTCGAATTAAAACGCGATATTTGAAGTCTTTCGGGCTTCCTCTTAATCTTTTTGATGCAATCCGCTTTGCTTCTGACTATAATAGTCAGGGTAAAGACCTGATTT
TAACTCATTGATACTCATTTATAAACTCCTTGCAATGTATGTCGTTTCAGCTAAACGGTATCAGCAATGTTTATGTAAAGAAACAGTAAGATAATACTCAACCCGATGTTTGAGTACGGTCATCATCTGACACTACAGACTCTGGCATCGCTGTGAAGACGACGCGAAATTCAGCATTTTCACAAGCGTTATCTTTTACAAAACCGATCTCACTCTCCTTTGATGCGAATGCCAGCGTCAGACATCATATGCAGATACTCACCTGCATCCTGAACCCATTGACCTCCAACCCCGTAATAGCGATGCGTAATGATGTCGATAGTTACTAACGGGTCTTGTTCGATTAACTGCCGCAGAAACTCTTCCAGGTCACCAGTGCAGTGCTTGATAACAGGAGTCTTCCCAGGATGGCGAACAACAAGAAACTGGTTTCCGTCTTCACGGACTTCGTTGCTTTCCAGTTTAGCAATACGCTTACTCCCATCCGAGATAACACCTTCGTAATACTCACGCTGCTCGTTGAGTTTTGATTTTGCTGTTTCAAGCTCAACACGCAGTTTCCCTACTGTTAGCGCAATATCCTCGTTCTCCTGGTCGCGGCGTTTGATGTATTGCTGGTTTCTTTCCCGTTCATCCAGCAG
CGGATCTGCACAACATTGATAACGCCCAATCTTTTTGCTCAGACTC
TTCCAGCACAATCGATGGTGTTACCAATTCATGGAAAAGGTCTGCGTCAAATCCCCAGTCGTCATGCATTGCCTGCTCTGCCGCTTCACGCAGTGCCTGAGAGTTAATTTCGCTCACTTCGAACCTCTCTGTTTACTGATAAGTTCCAGATCCTCCTGGCAACTTGCACAAGTCCGACAACCCTGAACGACCAGGCGTCTTCGTTCATCTATCGGATCGCCACACTCACAACAATGAGTGGCAGATATAGCCTGGTGGTTCAGGCGGCGCATTTTTATTGCTGTGTTGCGCTGTAATTCTTCTATTTCTGATGCTGAATCAATGATGTCTGCCATCTTTCATTAATCCCTGAACTGTTGGTTAATACGCATGAGGGTGAATGCGAATAATAAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGA
TGATGAATCTTTCTACCTGTAATAATGTTGTTCCG
TTGATTTATGGTCATTC
TGCATTAGTTGAATGTGGTATTCCTAAATCTCAAC
TCGTTTTCTGAACTGTT
CTCGTAATTCCTTTTGGCGTTATGTATC
TAAAGCATTTGAGGGGGATTCAAT
ATGATAGTGTTGCTCTTACTATGC
GAATATTTATGACGATTCCGCAGTATTG
TTTTATCGTCGTCTGGTAAACGAGGGTT
GACGCTATCCAGTCTAAACATTTTA
CTATTACCCCCTCTGGCAAAACTTCTTTTGCAAAAGCCTCTCGCTATTTTGGT
AATGGGGATTTGTCTTCATTAGAC
TTGTGAGCGGATAACAATTTCACACAGG
TCAGTCATTTCTCGCACATTGCAG
AAACAGCTATGACCATGATTACGAATTC
GTACTCGTGATAATAATTTTGCACGGTA
GAGCTCGGTACCCGGGGATCCATTC
AGAGAGGGCAAGTATCGTTTCCACC
TCCTGTGACTCGGAAGTGCATTTATCAT
TGGGTTTAATCATCTATATGTTTTGTAC
CTCCATAAAACAAAACCCGCCGTA
GCGAGTTCAGATAAAATAAATCCCCGCGAGTGCGAGGATTGTTATGTAATAT
CTGCATTAGCTGAACATGTTGTTTA
CAAAACCTCCCGCAAGTCGGGAGGTTCG
TCTATTGTTGATAAACAGGCGCGTT
CTAAAACGCCTCGCGTTCTTAGAATACC
GGATATTATTTTTCTTGTTCAGGACTTA
GGATAAGCCTTCTATATCTGATTT
TCTACATGCTCGTAAATTAGGATG
GCTTGCTATTGGGCGCGGTAATGATTCC
CCGATTATTGATTGGTT
TACGATGAAAATAAAAACGGCTTGCTTGTTCTCGA
TGAGTGCGGTACTTGGTTTAATACCCGTTCTTGGAATGATAAGGAAAGACAG
TAGATATAGAGTCG
GAAGGTA
GAACGAT
AAATACC
CCATTAA
AACATCG
GCCCTAA
ACTGATA
TGCGCGA
TCTTTAA
CTATTAG
TGAATGG
ATATTTT
ACAGACA
ACGTGGC
TAAGAAT
GAAAGCG
CTGACCT
AACCCTT
GAGATAG
GCCAACA
CATTCTG
AAAGGGA
AGTAATA
CACGACC
CCAGTCA
AGATTCA
CATTGGC
TTATTTA
AAATGGA
TCGTCTG
CGCTCAA
ATTTTGA
TACCTAC
ATGGAAA
AACGCTC
CAGGAAA
ATTGCAA
GCCAGCC
TATTACC
AGAACAA
AATATCC
TGCTGGT
TCGGCCT
CAAACTA
AAGAACT
TGAGTAG
ACTTGCC
TAACATC
TTAGTAA
TCTTTGA
CAATACT
GTTGTAG
ATTAACC
CACGCAA
TGTCCAT
AAGAGTC
CGAGTAA
AGGCCAC
ATCAGTG
TTTTATA
GAAGTGT
ATCCTGA
CGCCAGA
AACGGTA
AGACAGG
GGATTTT
ATTAAAG
GAGGCCG
TAAACAG
CGGGAGC
ATCAGAG
CGTTAGA
CTTTCCT
TAACGTG
GCACGTA
TTGACGA
GGTTGCT
GTACTAT
AGGGCGC
CCGCTAC
TAATGCG
CCGCGCT
ACACCCG
AACCACC
TGCGCGT
GTCACGC
GGCAAGTGTAGCG
CAAATATTCCATGAAGGTTTATAATT
CCACACAACATACG
AGCCGGA
AGCATAAAGTGTAA
AGCCTGG
GGTGCCTAATGAGT
GAGCTAA
CTCACATTAATTGC
GTTGCGC
TCACTGCCCGCTTT
CCAGTCG
GGAAACCTGTCGTG
CCAGCTG
CATTAATGAATCGG
CCAACGC
GCGGGGAGAGGCGG
TTTGCGT
ATTGGGCGCCAGGG
TGGTTTT
TCTTTTCACCAGTG
AGACGGG
CAACAGCTGATTGC
CCTTCAC
CGCCTGGCCCTGAG
AGAGTTG
CAGCAAGCGGTCCA
CGCTGGT
TTGCCCCAGCAGGC
GAAAATC
CTGTTTGATGGTGG
TTCCGAA
ATCGGCAAAATCCC
TTATAAA
TCAAAAGAATAGCC
CGAGATA
GGGTTGAGTGTTGT
TCCAGTT
TGGAACAAGAGTCC
ACTATTA
AAGAACGTGGACTC
CAACGTC
AAAGGGCGAAAAAC
CGTCTAT
CAGGGCGATGGCCC
ACTACGT
GAACCATCACCCAA
ATCAAGT
TTTTTGGGGTCGAG
GTGCCGT
AAAGCACTAAATCG
GAACCCT
AAAGGGAGCCCCCG
ATTTAGA
GCTTGACGGGGAAA
GCCGGCG
AACGTGGCGAGAAA
GGAAGGG
AAGAAAGCGAAAGGAGCGGG
TAACAACGCCAACATGTAA
CCATATT
AGAATCG
TTAATTG
CTCAACAGTAGGGC
GCCAACG
CTTACCAGTATAAA
ACAAATT
ATCATATGCGTTAT
GTTTAGT
ACTAGAAAAAGCCT
CATAATT
ATAAACACCGGAAT
AATAAGA
TAAATAAGGCGTTA
CGTGTGA
TTTGAAATACCGAC
TTAATGG
CTTCTGACCTAAAT
ATTTCAT
ATATATTTTAGTTA
TTTTCAA
AACGCGAGAAAACT
GACAAAG
AAATCCAATCGCAA
CTGATGC
ACTATATGTAAATG
TTATATA
CGGCTTAGGTTGGG
TAACCTC
AGAGACTACCTTTT
AGGTCTG
TTTATCAAAATCAT
TAGTGAA
CTGAGAAGAGTCAA
TAAGACG
GCGATAGCTTAGAT
AAACATA
CTTAGAATCCTTGA
ATTTTCC
GTCGCTATTAATTA
GTAAATC
ATAACCTTGCTTCT
TGAGTGA
TAAATCAATATATG
CAGTACA
TTTTTTAATGGAAA
AATTACC
AACAATTTCATTTG
TACATTT
AAAACAAAATTAAT
CATCAAG
GATGATGAAACAAA
AAAAGAA
TCAATTACCTGAGC
ATTCATT
CGCAGAGGCGAATT
AAAATCG
TTGCTTTGAATACCAAGTTAC
AGAGGCATTTTCGAGCCAGTAATAAG
AGTATGTAGAACCACCAGCAG
AAGATAA
AACAGAG
GTGAGGC
GGTCAGT
ATTAACA
CCGCCTG
CAACAGT
GCCACGC
TGAGAGC
CAGCAGC
AAATGAA
AAATCTA
AAGCATC
ACCTTGC
TGAACCT
CAAATAT
CAAACCC
TCAATCA
ATATCTG
GTCAGTT
GGCAAAT
CAACAGT
TGAAAGG
AATTGAG
GAAGGTT
ATCTAAA
ATATCTT
TAGGAGC
ACTAACA
ACTAATA
GATTAGA
GCCGTCA
ATAGATA
ATACATT
TGAGGAT
TTAGAAG
TATTAGA
CTTTACA
AACAATT
CGACAAC
TCGTATT
AAATCCT
TTGCCCG
AACGTTA
TTAATTT
TAAAAGT
TTGAGTA
ACATTAT
CATTTTG
CGGAACA
AAGAAAC
CACCAGA
AGGAGCG
GAATTAT
CATCATA
TTCCTGA
TTATCAG
ATGATGG
CAATTCA
TCAATAT
AATCCTG
ATTGTTT
GGATTAT
ACTTCTG
AATAATG
GAAGGGT
TAGAACC
TACCATA
TCAAAAT
TATTTGC
ACGTAAA
ACAGAAA
TAAAGAA
ATTGCGT
AGATTTT
CAGGTTT
AACGTCA
GATGAAT
ATACAGT
AACAGTA
CCTTTTA
CATCGGG
AGAAACA
ATAACGG
TATAAAGTACCGACA
CACGAAGGTGTTAA
TGAGTAT
AGTGCCT
CGGGGTC
GTTTTAA
GTAATAA
TGTACTG
ACAGGAG
TGATGAT
TGGCTTT
TCATACA
GTAAGCG
CGTTCCA
AATTTAC
GTCTCTG
AAGCGCA
GAATGGA
AAAGCCA
CCTCATT
AATAAAT
ACAAACA
GATATTC
TGGCCTT
AGACGAT
GCAGGTC
GGTTGAG
GACAGGA
CAGCATT
CCGCCGC
ACCAGAG
AACCACC
CCACCAG
AGAGCCG
CACCCTC
GAGCCAC
ACCCTCA
AACCGCC
CCCTCAG
GCCGCCA
CCTCAGA
CCGCCTC
ACCGGAA
AGCCACC
GAACCAG
ATCACCG
AATCAAA
TTTTCAT
TGCCATC
TAGCGTT
CCCTTAT
CATAGCC
TTTCGGT
TCGGCAT
GTTTTCA
GTAGCGC
TCAGACT
TTTAGCG
GTTTGCC
GAATCAA
AGCGACA
AATCAGT
GCACCGT
GATAGCA
AACCATC
CCAATGA
AACGTCA
GGCCGGA
TTAGCAA
ATTACCA
TAGCACC
TCACCAG
AGCAAAA
TAGAGCC
TGGGAAT
AGCCATT
CGACTTG
CCGTCAC
ATTATCA
AAGGTGA
TTCATTA
GAAATTA
ATTGACG
GGTAAAT
GAGGGAA
ATTGAGG
ACATTCAACCG
AGTAATTCTGTCCAGACGACGACAAAAGCC
TTAAATC
AAGATTAGTTGCTA
TTTTGCA
CCCAGCTACAATTT
TATCCTG
AATCTTACCAACGC
TAACGAG
CGTCTTTCCAGAGC
CTAATTT
GCCAGTTACAAAAT
AAACAGC
CATATTATTTATCC
CAATCCA
AATAAGAAACGATT
TTTTGTT
TAACGTCAAAAATG
AAAATAG
CAGCCTTTACAGAG
AGAATAA
CATAAAAACAGGGA
AGCGCAT
TAGACGGGAGAATT
AACTGAA
CACCCTGAACAAAG
TCAGAGG
GTAATTGAGCGCTA
ATATCAG
AGAGATAACCCACA
AGAATTG
AGTTAAGCCCAATA
ATAAGAG
CAAGAAACAATGAA
ATAGCAA
TAGCTATCTTACCG
AAGCCCT
TTTTAAGAAAAGTA
AGCAGAT
AGCCGAACAAAGTT
ACCAGAA
GGAAACCGAGGAAA
CGCAATA
ATAACGGAATACCC
AAAAGAA
CTGGCATGATTAAG
ACTCCTT
ATTACGCAGTATGT
TAGCAAA
CGTAGAAAATACAT
ACATAAA
GGTGGCAACATATA
AAAGAAA
CGCAAAGACACCAC
GGAATAA
GTTTATTTTGTCAC
AATCAAT
AGAAAATTCATATG
GTTTACC
AGCGCCAAAGAC
TTTAATT
GAGATGG
TGGGCTT
AGTAAAT
ACACCAGAACGAGT
TGCCCTGACGAGAA
TAAGGCT
GCTCATTCAGTGAA
CAAAGCT
CCAAATCAACGTAA
TCATTAC
CAAGAACCGGATAT
ATCTTGA
CTTCATCAAGAGTA
GGCTGAC
CAGGCGCATAGGCT
TACAGAC
ACAGATGAACGGTG
AAAGAGG
ACTGACCAACTTTG
GAACCGA
GGTCAATCATAAGG
CGCAGAC
TAGCCGGAACGAGG
TGTTACT
CGCGACCTGCTCCA
CGAAATC
CTGATAAATTGTGT
TCATCGC
AACGGAGATTTGTA
AAAGTAC
ACCAAGCGCGAAAC
CGATTAT
TCTTTGACCCCCAG
ACACTCA
AAGAATACACTAAA
GAGGCAA
AACCTAAAACGAAA
AGGCACC
TAATGCCACTACGA
AAATACG
CCATTAAACGGGTA
GAAGTTT
GACTTTTTCATGAG
GACTAAA
ACAGAGGCTTTGAG
AACGGCT
GGAACGAGGGTAGC
CAGCATC
TCAGCAGCGAAAGA
GTCACCC
GCTTTTGCGGGATC
AAAGGCC
GCTTGCAGGGAGTT
CGCTGAG
CCGATATATTCGGT
CGCATAA
ACAACCATCGCCCA
AATGACA
ATACCGATAGTTGCGCCGAC
TTTAATCATTGT
GAATTAC
CTTATGC
GATGCAGACAACAG
TGCCCGT
ATAAACA
GTTAATG
CCCCCTG
CCTATTT
CGGAACC
TATTATT
CTGAAAC
ATGAAAG
TATTAAG
AGGCTGA
GACTCCT
CAAGAGA
AGGATTA
GGATTAG
CGGGGTT
TTGCTCA
GTACCAG
GCGGATA
AGTGCCG
TCGAGAG
GGTTGAT
ATAAGTA
TAGCCCG
GAATAGG
TGTATCA
CCGTACT
CAGGAGG
TTTAGTA
CCGCCAC
CCTCAGA
ACCGCCA
CCCTCAG
AACCGCC
ACCCTCA
GAGCCAC
CACCCTC
ATTTTCA
GGGATAG
CAAGCCC
AATAGGA
ACCCATG
TACCGTA
ACACTGA
GTTTCGT
CACCAGT
ACAAACT
ACAACGC
CTGTAGC
ATTCCAC
AGACAGC
CCTCATA
GTTAGCG
TAACGAT
CTAAAGT
TTTGTCG
TCTTTCC
AGACGTT
AGTAAAT
GAATTTT
CTGTATG
GGATTTT
GCTAAAC
AACTTTC
AACAGTT
TCAGCGG
AGTGAGA
ATAGAAA
GGAACAA
CTAAAGG
AATTGCG
AATAATA
ATTTTTT
CACGTTG
AAAATCT
CCAAAAA
AAAGGCT
CCAAAAG
GAGCCTT
TAATTGT
ATCGGTT
TATCAGC
TTGCTTT
CGAGGTGAATTTC
ATTATACCAGTCA
CTGGCTC
TTAAGAA
GTCTTGTCTCCATT
TCCAGCCAGCTTGT
ATCGCAC
CAGGAAG
TCGGCCT
GACAGTA
GGACGAC
TTTGAGG
CTGCCAG
CGTGCAT
TCGTAAC
GGGCGCA
TGTAGAT
ACGTTGG
ATAGGTC
TAATGGG
TTGACCG
CGGCGGA
GAACAAA
TCCGTGG
CGGATTC
AACCCGT
GAGTAAC
TGTGAGC
CATTAAA
TCATCAA
CCAGCTT
CCTGTAG
TGGCCTT
TCGCGTC
AAATAAT
CCATCAA
AGGAACG
AACCAAT
ATTTTTT
TCAGCTC
TGTTAAA
AAATTTT
TCGCATT
TTAAAAT
TATTTTG
ACGTTAA
ATTGTAA
TATTTAA
AAGCAAA
ATTGTAT
CAGGAAG
CCAAAAA
AAAAGCC
TAATCAG
CGGTTGA
TGTACCC
AATCATA
GCATGTC
AAAACTA
TAATCGT
TGAACGG
GAATCGA
AACAAGA
TGGAGCA
GAGAGTC
ATTGCCT
TCAGGTC
AAGGCTA
ATCTACA
TTGAGAG
GCTATTT
GAGGGTA
TGCCGGA
AAATTAA
AGCTGAT
CCGTTCT
TATTCAA
AATATGA
CACCATC
GTCAAAT
GGAGACA
AAAGGCC
GGGTGAG
TTCAAAA
GTAAAGA
TGTGTAG
TGAGTAA
CAATGCC
TGGGAAGAAAAATCTACGT
TAATAAAACGAACT
AAAAATCAGGTCTT
TACCCTGACTATTA
TAGTCAG
AAGCAAAGCGGATT
GCATCAA
AAAGATTAAGAGGA
AGCCCGA
AAGACTTCAAATAT
CGCGTTT
TAATTCGAGCTTCA
AAGCGAA
CCAGACCGGAAGCA
AACTCCA
ACAGGTCAGGATTA
GAGAGTA
CCTTTAATTGCTCC
TTTTGAT
AAGAGGTCATTTTT
GCGGATG
GCTTAGAGCTTAAT
TGCTGAA
TATAATGCTGTAGC
TCAACAT
GTTTTAAATATGCA
ACTAAAG
TACGGTGTCTGGAA
GTTTCAT
TCCATATAACAGTT
GATTCCC
AATTCTGCGAACGA
GTAGATT
TAGTTTGACCATTA
GATACAT
TTCGCAAATGGTCA
ATAACCT
GTTTAGCTATATTT
TCATTTG
GGGCGCGAGCTGAA
AAGGTGG
CATCAATTCTACTA
ATAGTAG
TAGCATTAACATCC
AATAAAT
CATACAGGCAAGGC
AAAGAAT
TAGCAAAATTAAGC
AATAAAG
CCTCAGAGCATAAA
GCTAAAT
CGGTTGTACCAAAA
ACATTAT
GACCCTGTAATACT
TTTGCGG
GAGAAGCCTTTATT
TCAACGC
AAGGATAAAAATTTTTAGAA
GAGTATCAAT
TATAAAT
AGGAGTT
CATTGCA
CGACATA
CCGTTTAGCTGAAA
AAACATTGCTGATA
TTTACAT
TATCTTACTGTTTC
TGAGTAT
CTCAAACATCGGGT
GACCGTA
TAGTGTCAGATGAT
GAGTCTG
TCACAGCGATGCCA
GTCGTCT
ATGCTGAATTTCGC
TGTGAAA
TAAAAGATAACGCT
GGTTTTG
AGGAGAGTGAGATC
GCATCAA
TGACGCTGGCATTC
TGATGTC
TGAGTATCTGCATA
ATGCAGG
TCAATGGGTTCAGG
TTGGAGG
TCGCTATTACGGGG
TTACGCA
AACTATCGACATCA
CGTTAGT
AATCGAACAAGACC
GGCAGTT
GGAAGAGTTTCTGC
GTGACCT
CAAGCACTGCACTG
CTGTTAT
TCCTGGGAAGACTC
TTCGCCA
CCAGTTTCTTGTTG
ACGGAAA
CGAAGTCCGTGAAG
AAAGCAA
TATTGCTAAACTGG
GTAAGCG
TATCTCGGATGGGA
AAGGTGT
GCGTGAGTATTACG
ACGAGCA
AAAATCAAAACTCA
AAACAGC
GCGTGTTGAGCTTG
GGAAACT
TGCGCTAACAGTAG
AGGATAT
CGACCAGGAGAACG
AACGCCG
CCAGCAATACATCA
AAAGAAA
GATGAACGGG
GAGTCTGAGCAAAAAG
ATTGGGC
GTTATCA
ATGTTGT
GCAGATC
CGTCCGG
CACCGCT
TCTGGTG
CCGGAAA
CCAGGCA
AAGCGCC
ATTCGCC
ATTCAGG
CTGCGCA
ACTGTTG
GGAAGGG
CGATCGG
TGCGGGC
CTCTTCG
CTATTAC
GCCAGCT
GGCGAAA
GGGGGAT
GTGCTGC
AAGGCGA
TTAAGTT
GGGTAAC
GCCAGGG
TTTTCCC
AGTCACG
ACGTTGT
AAAACGA
CGGCCAG
TGCCAAG
CTTTATT
ATTCGCA
TTCACCC
TCATGCG
TATTAAC
CAACAGT
TCAGGGA
TTAATGA
AAGATGG
CAGACAT
CATTGAT
TCAGCAT
CAGAAAT
AGAAGAA
TTACAGC
GCAACAC
AGCAATA
AAAATGC
GCCGCCT
GAACCAC
CAGGCTA
TATCTGC
CACTCAT
TGTTGTG
AGTGTGG
CGATCCG
ATAGATG
AACGAAG
ACGCCTG
GTCGTTC
AGGGTTG
TCGGACT
TGTGCAA
GTTGCCA
GGAGGAT
CTGGAAC
TTATCAG
TAAACAG
AGAGGTT
CGAAGTG
AGCGAAA
TTAACTC
TCAGGCA
CTGCGTG
AAGCGGC
AGAGCAG
GCAATGC
ATGACGA
CTGGGGA
TTTGACG
CAGACCT
TTTCCAT
GAATTGG
TAACACC
ATCGATTGTG
CAGGTAGAAAGAT
ATTATTA
GAACAAC
ACCATAAATCAACG
GTTGAGATTTAGGAATACCACATTCA
ACTAATGCAAACAGTTCAGAA
TACATAACGCCAAAAGGAAT
ATCCCCCTCAAATGCTTTAGA
GCATAGTAAGAGCAACACTATCATCA
ATACTGCGGAATCGTCATAAA
CCAGACGACG
TCGTTTA
GATAGCGTCAACCC
ATGTTTAGACTG
ACCAAAATAGCGAGAG
GCTTTTG
CAAAAGA
AGTTTTG
CCAGAGG
GGGTA
CAAATC
ATGAAGA
AAGTCTA
CGCTCAC
TGTTATC
TGAAAT
CTGCAATGTGCGAGAAATGACTGAGA
ATTCGTAATCATGGTCATAGC
ATCCCCGGGTACCGAGCTCTACCGTGCAAAATTATTATCA
GGTGGAAACGATACTTGCCCTCTCTATGATAAATGCACTTCCGAGTC
ACAAAACATATAGATGATTA
CGGGTTTTGTTTTATGGAGGT
ATATTACATAACAATCCTC
GCACTCG
CGGGGAT
TTATTTT
ATCTGAA
AACAACATGTTCAGCTA
TTTTGTA
CGGGAGG
CGACTTG
AACCTCC
AACGCGCCTGTTTATCAACAATAGAGGTATTCTAAGA
ACGCGAGG
AGTCCTGAACAAGAAAAA
GATATAGAAGGCTTATCCTA
CATCCTAATTTACGAGCATGTAGAGG
AATCATT
ACCGCGC
CCAATA
TTCATCGTAAACCAATCAAT
TTTTATT
AACAAGCAAGCCGT
CTGTCTTTCCTT
ATCATTC
CAAGAAC
GGGTATT
AAACCAA
GTACCG
Fig.
S9.
Pillar
de
sig
n s
che
ma
tic.
12
Start End Sequence Modified in structure Modification 6[279] 10[266] CGCGACCTGCTCCAACCGCCACCATCAATTCACCCATGCAGG unmodified 2[146] 6[133] ATAAACACCGGAATAAGCATCGTCTCTGCAAGAGAATCTTGA unmodified 6[363] 10[350] TCTTTGACCCCCAGACACTGATATTTAATTACAGCGGCAGTTAATCGAA unmodified 5[35] 6[28] TTAAATCCACGAAGGTGTTAACTTATGCTGGGCTT unmodified 10[370] 2[357] GGAAGAGTTTCTGCGCAACACTAACATCTTAATTTTAGTGAA unmodified 2[125] 6[112] ACTAGAAAAAGCCTCAGCAGCGTAAGCGTATTAAGTCATTAC unmodified 9[567] 5[580] ACATTATCACCATCAAAATCTCCGTCACCGCAAAGACACCAC unmodified 6[174] 10[161] CAGGCGCATAGGCTTTGCTCATTGACCGGGCGAAAGTCGTCT unmodified 9[126] 5[139] CGCGTTTGGGCGCAGACTCCTAATTTACGCCAGTTACAAAAT unmodified 5[518] 1[531] TAGCAAATAGCACCTACCATAATCAGAGTTTTTGGGGTCGAG unmodified 2[104] 6[91] ATCATATGCGTTATCAACAGTTGATGATTATTATTCAAAGCT unmodified 1[280] 9[293] AGAGTTGATTGCAATATTAACAACCAATGTTTTAAATATGCA unmodified 1[238] 9[251] AGACGGGCGCTCAACGGCCAGCCTGTAGGCTTAGAGCTTAAT unmodified 10[244] 2[231] TGACGCTGGCATTCAAAACGATCGTCTGATATCTTTTTTCAA unmodified 10[517] 2[504] GCGTGAGTATTACGTTATCAGTAAACAGGAAGGGTAATTACC unmodified 2[314] 6[301] CGGCTTAGGTTGGGTATTAGAGCCGCCACACCCTCTCATCGC unmodified 1[364] 9[377] TTATAAATTAGTAAAGCAATAATTGTATTAGTTTGACCATTA unmodified 1[343] 9[356] TTCCGAATGAGTAGAGAAGAAATTGTAAAATTCTGCGAACGA unmodified 9[294] 5[307] ACTAAAGTCAGCTCGAGCCACCCCTCAGGTAATTGAGCGCTA unmodified 9[357] 5[370] GTAGATTAAGCAAAGTTTCGTTTTTCATCAAGAAACAATGAA unmodified 5[329] 1[342] ACCCACAAGAATTGAGCCACCTCGTATTCAAACTACTGTTTGATGGTGG Fig. 4A ATACATCTA 9[189] 5[202] GAGAGTACGGATTCTCGAGAGTGGCCTTTAACGTCAAAAATG unmodified 6[384] 10[371] AAGAATACACTAAAACAAACTCAGGAAGAAAATGCGTGACCT unmodified 27[35] 7[48] ACCGCGCTTTTATTGGGTATTAGTAAATGATGCAGACAACAG unmodified 5[476] 1[489] AAAAGAAAACCATCATTGTTTAGACAGGCAGGGCGATGGCCC unmodified 10[328] 2[315] AACTATCGACATCACAGACATTGCTGGTAACAATTTAACCTC Dimers A15 5[455] 1[468] CGCAATAAATCAGTCAATTCAATCCTGAAAAGGGCGAAAAAC unmodified 2[41] 19[55] AGAATCGTTATTTTCGCTCACATTCGTAATCATGGTCATAGC unmodified 10[307] 2[294] TCGCTATTACGGGGTCAGGGATATTACCTTAGAAGTTATATA unmodified 7[49] 25[49] TGCCCGTACACCAGAACGAGTAAACCAACGACTTGACGCGAGG unmodified 1[490] 9[503] ACTACGTATTAAAGGGAGGATAAGGCTACATACAGGCAAGGC unmodified 5[161] 1[174] CAATCCACCTCATTCAAACCCGCCAACACATTAATGAATCGG unmodified 5[287] 1[300] TCAGAGGAACCGCCTGAGGATGCCAGCCCAGCAAGCGGTCCA unmodified 11[56] 17[53] CACCGCTCCGTTTAGCTGAAAGGGTA unmodified 9[147] 5[160] AAGCGAAATAGGTCGGATTAGGAATGGACATATTATTTATCC unmodified 1[301] 9[314] CGCTGGTAGAACAATTAATGATGTTAAATACGGTGTCTGGAA Monomers, Dimers A15 2[83] 6[70] CTTACCAGTATAAAGGTCAGTGTAATAACCCCCTGTAAGGCT unmodified 9[462] 5[475] ATAGTAGTGGAGCAGAATTTTGCACCGTATAACGGAATACCC unmodified 5[56] 1[69] TTTTGCACGGGGTCAACAGAGAACATCGAGCATAAAGTGTAA unmodified
13
9[441] 5[454] AAGGTGGTGAACGGTCTTTCCGAATCAAGGAAACCGAGGAAA unmodified 5[413] 1[426] AGCAGATGTAGCGCAGGAGCGAAGAGTCTGGAACAAGAGTCC unmodified 2[167] 6[154] TAAATAAGGCGTTACAAATATAAAGCCACGGGGTTGGCTGAC unmodified 1[217] 9[230] TGGTTTTTTATTTAAGTCACGCATTAAAAAGAGGTCATTTTT unmodified 10[580] 2[567] TGCGCTAACAGTAGAGAGCAGGTACTATAGATTTTAAAAGAA unmodified 9[231] 5[244] GCGGATGCCAGCTTCCGTACTCCGCCGCCATAAAAACAGGGA unmodified 18[27] 22[8] ATGAAGAGCACTCGACAAAACATATAGATGATTA unmodified 18[54] 11[55] TGAAATAGCCGGACCATTAACGTCCGG unmodified 10[433] 2[420] CCAGTTTCTTGTTGAGTGTGGCGAGTAAGAATTATATTTTCC unmodified 10[76] 2[63] AAACATTGCTGATATCTGGTGGCCCTAAGTGAGGCGCCAACG unmodified 2[503] 6[490] TTTTTTAATGGAAAACTTCTGAACGTCAAACTTTCAACGGCT unmodified 2[335] 6[322] TTTTCGACAACACCGGAACAAGCCCAAAGTAC Fig. 4A ATACATCTA 9[483] 5[496] AATAAATTCAGGTCGCTAAACCCAATGACTGGCATGATTAAG unmodified 6[153] 10[140] CTTCATCAAGAGTAAGGATTAACGTTGGCTCTTCGGAGTCTG unmodified 9[609] 5[622] TCAACGCGTAAAGAATCGGTTGGTAAATAGAAAATTCATATG unmodified 20[51] 20[12] ATCCCCGGGTACCGAGCTCTACCGTGCAAAATTATTATCA unmodified 6[510] 10[497] GGAACGAGGGTAGCAACAGTTATCTACACTGGAACAAGGTGT unmodified 2[608] 6[595] CGCAGAGGCGAATTATACAGTGAAATTAGAGCCTTCGCATAA unmodified 12[48] 16[28] ACCATAAATCAACGAGTTTTGGATAGCGTCAACCC unmodified 9[210] 5[223] TTTTGATTGTGAGCTAGCCCGGGTTGAGCAGCCTTTACAGAG unmodified 10[475] 2[462] TATTGCTAAACTGGAGGGTTGCGCCAGATCAATATTGAGTGA unmodified 14[55] 15[55] ATCCCCCTCAAATGCTTTAGAATACTGCGGAATCGTCATAAA unmodified 10[412] 2[399] TCCTGGGAAGACTCTATCTGCCACGCAAAAGAAACAAACATA unmodified 1[385] 9[398] CGAGATAGTTGTAGGAACCACAAAAGCCTTCGCAAATGGTCA unmodified 5[581] 1[594] GGAATAAAAGGTGAAACGTCACCGCTACGCTTGACGGGGAAA unmodified 2[482] 6[469] TAAATCAATATATGAATCCTGGATAGCACTGTATGGACTAAA unmodified 9[168] 5[181] AACTCCACGGCGGAGTACCAGAATAAATAATAAGAAACGATT unmodified 3[28] 2[42] AGTATGTAGAACCACCAGCAGTTAATTG unmodified 2[62] 4[49] CTCAACAGTAGGGCAAGATAAAGTGCCT unmodified 1[70] 9[83] AGCCTGGACTGATACCGGAAACAGGAAGAAGCAAAGCGGATT unmodified 1[595] 9[608] GCCGGCGCCGCGCTTTTGACGGGGTGAGGAGAAGCCTTTATT unmodified 9[273] 5[286] TCAACATAGGAACGCCCTCAGGAGCCACCACCCTGAACAAAG unmodified 6[468] 10[455] GACTTTTTCATGAGAGTAAATAACAAGAGTCGTTCAAAGCAA unmodified 1[511] 9[524] ATCAAGTCGGGAGCTAAACAGGCTATTTTAGCAAAATTAAGC unmodified 8[27] 4[13] CTGGCTCGAATTACTATAAAGTACCGACA unmodified 5[497] 1[510] ACTCCTTGGCCGGAAATAATGGAGGCCGGAACCATCACCCAA unmodified 6[195] 10[182] ACAGATGAACGGTGAGTGCCGTCCGTGGAAGGCGATGTGAAA unmodified 1[322] 9[335] GAAAATCTCGGCCTCATTGATTTAAAATTCCATATAACAGTT Monomers, Dimers A15 10[223] 2[210] AGGAGAGTGAGATCTTTTCCCCATTGGCAATTGAGATTTCAT unmodified 9[378] 5[391] GATACATCCAAAAAACAACGCCCCTTATTAGCTATCTTACCG unmodified 2[188] 6[175] TTTGAAATACCGACATATCTGACAAACAGCGGATATACAGAC unmodified
14
6[69] 9[62] TGCCCTGACGAGAAATAAACATCCAGCCAGCTTGTTACCCTGACTATTA unmodified 9[504] 5[517] AAAGAATTTGAGAGTCAGCGGTTAGCAAATTACGCAGTATGT unmodified 9[63] 5[76] TAGTCAGATCGCACGTTAATGGTTTTAACCCAGCTACAATTT unmodified 10[454] 2[441] CGAAGTCCGTGAAGAACGAAGTTTTATATTATCAGGTAAATC unmodified 2[356] 6[343] TTTATCAAAATCATTTGCCCGATCACCGTACCGTACGATTAT unmodified 5[371] 1[384] ATAGCAATAGCGTTTTGAGTATCTTTGATCAAAAGAATAGCC unmodified 10[34] 13[48] AGGAGTTCAAAAGAGAACAACACTAATGCAAACAGTTCAGAA unmodified 2[230] 6[217] ATATATTTTAGTTAGAAGGTTGACAGGAGAATAGGGAACCGA unmodified 6[342] 10[329] ACCAAGCGCGAAACAATAGGATATTTTGTCAGCATCGTTAGT Monomers, Dimers A15 2[587] 6[574] TCAATTACCTGAGCCAGGTTTATTATCACCAAAAACGCTGAG unmodified 10[496] 2[483] TATCTCGGATGGGAGTTGCCAGGATTTTGGATTATCAGTACA unmodified 1[553] 9[566] GAACCCTTTGACGACTGCGTGTATTCAACGGTTGTACCAAAA unmodified 5[350] 1[363] CCCAATAATAAGAGAATCAAAAACGTTAACTTGCCATCGGCAAAATCCC unmodified 1[406] 9[419] TCCAGTTTGTCCATCACTCATTGTACCCGTTTAGCTATATTT unmodified 2[566] 6[553] GATGATGAAACAAATAAAGAAAGCCATTATTTTTTAAAGGCC unmodified 6[216] 10[203] ACTGACCAACTTTGATAAGTAGAGTAACGCCAGGGGGTTTTG unmodified 10[559] 2[546] GCGTGTTGAGCTTGTCAGGCAGCACGTAACAGAAACATCAAG unmodified 1[133] 9[146] CCAGTCGGAAAGCGTGCGGGCTGTAGATTAATTCGAGCTTCA unmodified 10[622] 2[609] CCAGCAATACATCATTTCCATAACCACCCCTTTTAAAAATCG unmodified 16[53] 0[42] ATGTTTAGACTGCCAGAGGCGACATAGCAGATCAAATACC unmodified 2[524] 6[511] AACAATTTCATTTGTAGAACCATTACCAAGTGAGACAGCATC unmodified 9[588] 5[601] TTTGCGGAAAGGCCCCAAAAGTTCATTAGTTTATTTTGTCAC unmodified 2[209] 6[196] CTTCTGACCTAAATCAACAGTAGACGATGGTTGATAAAGAGG unmodified 5[602] 1[615] AATCAATATTGACGAACAGTAACACCCGAACGTGGCGAGAAA unmodified 9[35] 10[35] AAAAATCAGGTCTTGTCTTGTCTCCATTATGTTGTCATTGCA unmodified 5[140] 1[153] AAACAGCAAGCGCAACCTTGCCTGACCTGGAAACCTGTCGTG unmodified 5[245] 1[258] AGCGCATAACCACCACTAACAATTTTGACAACAGCTGATTGC unmodified 10[139] 2[126] TAGTGTCAGATGATCGATCGGTAAGAATAAATCTACATAATT unmodified 2[251] 6[238] AACGCGAGAAAACTTAGGAGCACCAGAGCAGGAGGCGCAGAC unmodified 1[259] 9[272] CCTTCACATGGAAAATTCGCAAAATAATTATAATGCTGTAGC unmodified 1[91] 9[104] GAGCTAACTATTAGATTCGCCGGACGACAAAGATTAAGAGGA unmodified 5[119] 1[132] CTAATTTCGTTCCAAAATGAAACGTGGCTCACTGCCCGCTTT unmodified 6[321] 10[308] AACGGAGATTTGTAATTTTCAAAATTTTAAGATGGTTACGCA Fig. 4A ATACATCTA 1[532] 9[545] GTGCCGTCTTTCCTAGCGAAAAAATTAACCTCAGAGCATAAA unmodified 28[55] 27[47] AACAAGCAAGCCGTCCAATA unmodified 1[448] 9[461] CAACGTCGAAGTGTACGCCTGGAATCGACATCAATTCTACTA unmodified 22[48] 2[28] CGGGTTTTGTTTTATGGAGGTCGGGGATCCATATT unmodified 6[237] 10[224] GGTCAATCATAAGGTGTATCATCATCAAACGTTGTGCATCAA unmodified 24[34] 28[15] TTTTGTACAAGAACTTCATCGTAAACCAATCAAT unmodified 6[90] 10[77] GCTCATTCAGTGAACCTATTTTCGGCCTCCAGGCATTTACAT unmodified 6[489] 10[476] ACAGAGGCTTTGAGGGATTTTATTGCCTTGTGCAAGTAAGCG unmodified
15
5[203] 1[216] AAAATAGGCAGGTCTGAAAGGAGATTCAATTGGGCGCCAGGG unmodified 10[160] 2[147] TCACAGCGATGCCACTATTACAACCCTTTGAACCTAATAAGA unmodified 5[434] 1[447] ACCAGAAGTTTGCCTTCCTGAATCAGTGAAGAACGTGGACTC unmodified 9[399] 5[412] ATAACCTCGGTTGAAGACAGCTCGGCATTTTTAAGAAAAGTA unmodified 5[266] 1[279] AACTGAACACCCTCGCCGTCAAACGCTCCGCCTGGCCCTGAG unmodified 5[560] 1[573] AAAGAAACGACTTGATTGCGTGGTTGCTAAAGGGAGCCCCCG unmodified 5[182] 1[195] TTTTGTTGATATTCGTCAGTTAGTAATAGCGGGGAGAGGCGG unmodified 1[427] 9[440] ACTATTAAGGCCACCGATCCGAAAACTAGGGCGCGAGCTGAA unmodified 6[594] 10[581] CCGATATATTCGGTAAAGGCTGGAGACAATGACGAAGGATAT unmodified 1[469] 9[482] CGTCTATAACGGTATCGGACTGAGAGTCTAGCATTAACATCC unmodified 5[539] 1[552] ACATAAATAGAGCCACGTAAATAACGTGAAAGCACTAAATCG unmodified 1[574] 9[587] ATTTAGAAGGGCGCGCAATGCGTCAAATGACCCTGTAATACT unmodified 6[447] 10[434] CCATTAAACGGGTATTTGTCGTAATCGTATAGATGACGGAAA unmodified 10[391] 2[378] CAAGCACTGCACTGGCCGCCTCAATACTACATTATTAAGACG unmodified 10[202] 2[189] TAAAAGATAACGCTTTAAGTTCACGACCGGCAAATTTAATGG unmodified 2[272] 6[259] AAATCCAATCGCAAGATTAGAAGAGCCGCCTCAGATGTTACT unmodified 6[426] 10[413] TAATGCCACTACGAGTTAGCGAATCATATGTTGTGTTCGCCA unmodified 9[546] 5[559] GCTAAATCCGTTCTAATAATATGGGAATGGTGGCAACATATA unmodified 2[545] 6[532] AAAACAAAATTAATTATTTGCAGCAAAACTAAAGGGTCACCC unmodified 1[196] 9[209] TTTGCGTCCAGTCAGGGTAACAACCCGTCCTTTAATTGCTCC unmodified 6[405] 10[392] AACCTAAAACGAAAATTCCACTAATCAGCAGGCTACTGTTAT unmodified 2[398] 6[385] GCGATAGCTTAGATCATTTTGCATAGCCCTGTAGCGAGGCAA unmodified 10[181] 2[168] ATGCTGAATTTCGCGGGGGATCATTCTGTCAATCACGTGTGA unmodified 1[154] 9[167] CCAGCTGGAGATAGGCCAGCTTAATGGGCCAGACCGGAAGCA unmodified 9[315] 5[328] GTTTCATTCGCATTGGGATAGCCGCCTCAGAGAT Dimer, Fig. 4 A, E A15 9[315] 5[328] GTTTCATTCGCATTGGGATAGCCGCCTCAGAGAT Dimer, Fig 1A A15, ATTO647N 10[538] 2[525] AAAATCAAAACTCACGAAGTGCGTTAGATCAAAATTACATTT unmodified 1[112] 9[125] GTTGCGCACAGACAACTGTTGCGTGCATAAGACTTCAAATAT unmodified 10[349] 2[336] CAAGACCCAGAAATAAGAACTAAATCCTAGGTCTGAGAGACTACC Dimer A15 2[440] 6[427] GTCGCTATTAATTACATCATATTTAGCGCTAAAGTAAATACG
unmodified
9[336] 5[349] TATAACAGTTGATTCCCACGTTAAACCCATGGAACCAGAGTTAAG Fig. 4A ATACATCTA 6[111] 10[98] CCAAATCAACGTAACTGAAACTTTGAGGATTCAGGTGAGTAT unmodified 10[286] 2[273] TCAATGGGTTCAGGTCATGCGCAGGAAAATAGATACTGATGC unmodified 9[420] 5[433] TCATTTGGCATGTCTAACGATTCAGACTAGCCGAACAAAGTT unmodified 9[525] 5[538] AATAAAGTGCCGGAGGAACAATCACCAGCGTAGAAAATACAT unmodified 6[258] 10[245] TAGCCGGAACGAGGTTTAGTATGGCCTTTGCCAAGTGATGTC unmodified 9[105] 5[118] AGCCCGACTGCCAGATGAAAGTCATACACGTCTTTCCAGAGC unmodified 1[175] 9[188] CCAACGCAAAGGGAGTGCTGCGAACAAAACAGGTCAGGATTA unmodified 16[27] 0[14] TCGTTTAGCTTTTGTATAAATATTGGGCTAGATATAGAGTCG unmodified 10[601] 2[588] CGACCAGGAGAACGCTGGGGATAATGCGGATGAATATTCATT unmodified 6[573] 10[560] GCTTGCAGGGAGTTCACGTTGAATATGAAAGCGGCGGAAACT unmodified
16
2[293] 6[280] ACTATATGTAAATGATACATTACCCTCAAACCGCCCGAAATC unmodified 2[377] 6[364] CTGAGAAGAGTCAATAAAAGTTGCCATCCACCAGTACACTCA unmodified 10[118] 2[105] CTCAAACATCGGGTCTGCGCAATATTTTTGAGAGCGTTTAGT unmodified 26[47] 27[34] GATATAGAAGGCTTATCCTAAATCATT unmodified 4[48] 29[54] TGAGTATAAGATTAGTTGCTAAACCTCCGTACCG unmodified 5[98] 1[111] TAACGAGTGGCTTTGCCACGCTGAATGGCTCACATTAATTGC unmodified 10[265] 2[252] TGAGTATCTGCATACTTTATTTACCTACACTAATAGACAAAG unmodified 2[419] 6[406] CTTAGAATCCTTGACACCAGAGTTTTCACCTCATAAGGCACC unmodified 6[300] 10[287] CTGATAAATTGTGTACCCTCAATTTTTTCAACAGTTTGGAGG unmodified 0[41] 23[48] GAACGATCCACACAACATACGTGTTATCATCTGAA unmodified 10[97] 2[84] TATCTTACTGTTTCAAGCGCCTCTTTAACCGCCTGACAAATT unmodified 9[84] 5[97] GCATCAAGACAGTACGGAACCACAGGAGAATCTTACCAACGC unmodified 5[308] 1[321] ACAAATATCCTTGCCCCAGCAGGCGAA Fig. 4A ATACATCTA
5[308] 1[321] ACAAATATCCTTGCCCCAGCAGGCGAA Fig. 4E TTTCCCACCGCTCG GCTCAACTGGG
5[77] 1[90] TATCCTGTGTACTGATTAACATGCGCGAGGTGCCTAATGAGT unmodified 5[224] 1[237] AGAATAACAGCATTATCTAAAAAATGGATCTTTTCACCAGTG unmodified 5[392] 1[405] AAGCCCTTTTCGGTCGGAACAATTAACCGGGTTGAGTGTTGT unmodified 9[252] 5[265] TGCTGAATCGCGTCCCGCCACCCACCAGTAGACGGGAGAATT unmodified 6[531] 10[518] TCAGCAGCGAAAGAATAGAAAGAGGGTAAGAGGTTACGAGCA unmodified 6[132] 10[119] CAAGAACCGGATATAGGCTGATCGTAACGGAAGGGGACCGTA unmodified 6[552] 10[539] GCTTTTGCGGGATCAATTGCGAGCTGATTTAACTCAAACAGC unmodified 2[461] 6[448] ATAACCTTGCTTCTATGATGGAGCGACAAGACGTTGAAGTTT unmodified 9[2] 12[8] TGGGAAGAAAAATCTACGTCAGGTAGAAAGAT 5'-Biotin 25[5] 24[35] AACGCGCCTGTTTATCAACAATAGAGGTATTCTAAGACGGGAGG 5'-Biotin 17[5] 16[11] ACCAAAATAGCGAGAGCCAGACGACG 5'-Biotin 11[5] 10[11] GAGTCTGAGCAAAAAGGAGTATCAAT 5'-Biotin 7[9] 8[8] TTTAATCATTGTATTATACCAGTCA 5'-Biotin 5[5] 5[34] AGTAATTCTGTCCAGACGACGACAAAAGCC 5'-Biotin 3[2] 2[9] AGAGGCATTTTCGAGCCAGTAATAAGTAACAACGCCAACATGTAA 5'-Biotin 1[9] 8[28] CAAATATTCCATGAAGGTTTATAATTGAAGGTAGTTATCATTAAGAA 5'-Biotin 13[2] 9[34] GTTGAGATTTAGGAATACCACATTCAATTATTATAATAAAACGAACT 5'-Biotin 23[2] 18[15] ATATTACATAACAATCCTCCAAATC 5'-Biotin 29[9] 6[14] CTGTCTTTCCTTTTTAATT 5'-Biotin 19[9] 18[28] CTGCAATGTGCGAGAAATGACTGAGAAAGTCTA 5'-Biotin 15[9] 14[15] GCATAGTAAGAGCAACACTATCATCATACATAACGCCAAAAGGAAT 5'-Biotin 21[5] 21[51] GGTGGAAACGATACTTGCCCTCTCTATGATAAATGCACTTCCGAGTC 5'-Biotin 27[2] 26[10] CATCCTAATTTACGAGCATGTAGAGGAGTCCTGAACAAGAAAAA 5'-Biotin 6[27] 24[11] GAGATGGATCATTCAACAACATGTTCAGCTA unmodified 6[615] 10[602] ACAACCATCGCCCATAATTGTTTCAAAACAGACCTAACGCCG unmodified
17
Table S1. List of unmodified and modified oligonucleotides from the 5’ to the 3’ used for the different DNA pillar structures sketched in Fig. 1A, Fig. 4A and E. The nomenclature follows the numbering used in Fig. S9.
References and Notes 1. C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, T. Ha, Advances in single-molecule
fluorescence methods for molecular biology. Annu. Rev. Biochem. 77, 51 (2008). doi:10.1146/annurev.biochem.77.070606.101543 Medline
2. P. Tinnefeld, M. Sauer, Branching out of single-molecule fluorescence spectroscopy: Challenges for chemistry and influence on biology. Angew. Chem. Int. Ed. 44, 2642 (2005). doi:10.1002/anie.200300647
3. J. Eid et al., Real-time DNA sequencing from single polymerase molecules. Science 323, 133 (2009). doi:10.1126/science.1162986 Medline
4. B. Huang, H. Babcock, X. Zhuang, Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143, 1047 (2010). doi:10.1016/j.cell.2010.12.002 Medline
5. M. J. Levene et al., Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299, 682 (2003). doi:10.1126/science.1079700 Medline
6. S. Uemura et al., Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012 (2010). doi:10.1038/nature08925 Medline
7. L. Novotny, N. van Hulst, Antennas for light. Nat. Photonics 5, 83 (2011). doi:10.1038/nphoton.2010.237
8. J. A. Schuller et al., Plasmonics for extreme light concentration and manipulation. Nat. Mater. 9, 193 (2010). doi:10.1038/nmat2630 Medline
9. T. H. Taminiau, F. D. Stefani, F. B. Segerink, N. F. van Hulst, Optical antennas direct single-molecule emission. Nat. Photonics 2, 234 (2008). doi:10.1038/nphoton.2008.32
10. A. G. Curto et al., Unidirectional emission of a quantum dot coupled to a nanoantenna. Science 329, 930 (2010). doi:10.1126/science.1191922 Medline
11. A. Kinkhabwala et al., Large single-molecule fluorescence enhancements produced by a bowtie nanoantenna. Nat. Photonics 3, 654 (2009). doi:10.1038/nphoton.2009.187
12. M. Ringler et al., Shaping emission spectra of fluorescent molecules with single plasmonic nanoresonators. Phys. Rev. Lett. 100, 203002 (2008) and references therein. doi:10.1103/PhysRevLett.100.203002 Medline
13. H. Lin et al., Mapping of surface-enhanced fluorescence on metal nanoparticles using super-resolution photoactivation localization microscopy. ChemPhysChem 13, 973 (2012) and references therein. doi:10.1002/cphc.201100743 Medline
14. H. Cang et al., Probing the electromagnetic field of a 15-nanometre hotspot by single molecule imaging. Nature 469, 385 (2011) and references therein. doi:10.1038/nature09698 Medline
15. M. P. Busson, B. Rolly, B. Stout, N. Bonod, S. Bidault, Accelerated single photon emission from dye molecule-driven nanoantennas assembled on DNA. Nat. Commun. 3, 962 (2012) and references therein. doi:10.1038/ncomms1964 Medline
16. P. W. Rothemund, Folding DNA to create nanoscale shapes and patterns. Nature 440, 297 (2006). doi:10.1038/nature04586 Medline
17. S. M. Douglas et al., Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414 (2009). doi:10.1038/nature08016 Medline
18. See the supplementary materials on Science Online.
19. G. P. Acuna et al., Distance dependence of single-fluorophore quenching by gold nanoparticles studied on DNA origami. ACS Nano 6, 3189 (2012). doi:10.1021/nn2050483 Medline
20. E. A. Coronado, E. R. Encina, F. D. Stefani, Optical properties of metallic nanoparticles: Manipulating light, heat and forces at the nanoscale. Nanoscale 3, 4042 (2011). doi:10.1039/c1nr10788g Medline
21. P. Anger, P. Bharadwaj, L. Novotny, Enhancement and quenching of single-molecule fluorescence. Phys. Rev. Lett. 96, 113002 (2006). doi:10.1103/PhysRevLett.96.113002 Medline
22. A. Bek et al., Fluorescence enhancement in hot spots of AFM-designed gold nanoparticle sandwiches. Nano Lett. 8, 485 (2008). doi:10.1021/nl072602n Medline
23. S. Kühn, U. Håkanson, L. Rogobete, V. Sandoghdar, Enhancement of single-molecule fluorescence using a gold nanoparticle as an optical nanoantenna. Phys. Rev. Lett. 97, 017402 (2006). doi:10.1103/PhysRevLett.97.017402 Medline
24. J. Vogelsang et al., A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew. Chem. Int. Ed. 47, 5465 (2008). doi:10.1002/anie.200801518
25. N. Di Fiori, A. Meller, The effect of dye-dye interactions on the spatial resolution of single-molecule FRET measurements in nucleic acids. Biophys. J. 98, 2265 (2010). doi:10.1016/j.bpj.2010.02.008 Medline
26. R. Jungmann et al., Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett. 10, 4756 (2010). doi:10.1021/nl103427w Medline
27. S. A. McKinney, A.-C. Déclais, D. M. J. Lilley, T. Ha, Structural dynamics of individual Holliday junctions. Nat. Struct. Biol. 10, 93 (2003). doi:10.1038/nsb883 Medline
28. A. Gietl, P. Holzmeister, D. Grohmann, P. Tinnefeld, DNA origami as biocompatible surface to match single-molecule and ensemble experiments. Nucleic Acids Res. 40, e110 (2012). doi:10.1093/nar/gks326 Medline
29. H. S. Chung, K. McHale, J. M. Louis, W. A. Eaton, Single-molecule fluorescence experiments determine protein folding transition path times. Science 335, 981 (2012). doi:10.1126/science.1215768 Medline
30. T. H. Taminau, F. D. Stefani, N. F. Van Hulst, Single emitters coupled to plasmonic nano-antennas: Angular emission and collection efficiency. New J. Phys. 10, 105005 (2008).
31. P. Bharadwaj, L. Novotny, Spectral dependence of single molecule fluorescence enhancement. Opt. Express 15, 14266 (2007). doi:10.1364/OE.15.014266 Medline
32. C. A. Mirkin, R. L. Letsinger, R. C. Mucic, J. J. Storhoff, A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382, 607 (1996). doi:10.1038/382607a0 Medline
33. K. D. Weston, L. S. Goldner, Orientation imaging and reorientation dynamics of single dye molecules. J. Phys. Chem. B 105, 3453 (2001). doi:10.1021/jp001373p
34. J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, P. Tinnefeld, Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy. Proc. Natl. Acad. Sci. U.S.A. 106, 8107 (2009). doi:10.1073/pnas.0811875106 Medline