Supporting Information. for. Cryogenic Fluorescence Localization Microscopy of Spectrally Selected Individual FRET Pairs in a Water Matrix

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1 Supporting Information for Cryogenic Fluorescence Localization Microscopy of Spectrally Selected Individual FRET Pairs in a Water Matrix Hiroaki Tabe, Kei Sukenobe, Toru Kondo, Atsunori Sakurai, Minako Maruo, Akari Shimauchi, Mitsuharu Hirano, Shin-nosuke Uno, Mako Kamiya, Yasuteru Urano, Michio Matsushita, and Satoru Fujiyoshi Supporting Table Table S1. Photophysical properties of the FRET pairs synthesized in the present study measured in a buffer solution at 296 K. Five FRET pairs were identified by the donor and the acceptor. The photophysical parameters determined are, from left to right, quantum yield of FRET (F FRET), Förster distance (R 0) *1, and fluorescence quantum yield of the acceptor (F F_A) *2. 1 Förster distance (R 0) was determined from the fluorescence spectrum of the donor and the absorption spectrum of the acceptor assuming random orientation, which gives rise to an average of the orientation factor (k 2 ) of 2/3. The fluorescence quantum yield (F F) of the donors, i.e., not linked to the acceptor, was evaluated by using F F of AT565 in a buffer solution (= 0.90). 2 Fluorescence quantum yield (F F) of A 750 in a buffer solution, F F = 0.12, was used as a fluorescence standard for determination of F F_A. S1

2 Supporting Figures Figure S1. Fluorescence spectrum measured at 1.7 K of individual molecules of (a) AT565 and (b) Ax750 in a water matrix. AT565 and Ax750 were used as the donor and the acceptor, respectively, in the FRET pair. The excitation wavelength l ex was (a) 532 nm and (b) nm. S2

3 Figure S2. Fluorescence spectrum measured at 1.7 K of individual molecules of (a) Alexa Fluor 660, (b) Alexa 647, and (c) ATTO655 in a water matrix. The excitation wavelength l ex was (a and b) nm and (b) 635 nm. S3

4 Figure S3. Spectral-selective localization of individual FRET pairs of B581 Cy7.5 in a water matrix at 1.7 K. (a) Confocal fluorescence image of the FRET pair of B581 Cy7.5 in a water matrix at 1.7 K with excitation by broadband laser light ( nm). The polarization of the excitation light was circular. (b) The excitation spectrum of the FRET pair (spectral width of laser light ~ 0.2 nm). Spectrum b was observed at the position denoted by the cross in a. The spectrum was an average over with differently-oriented linearly-polarized lights. (c) The polarization dependence of the fluorescence excitation spectrum. (d, e) The confocal fluorescence image of the FRET pair at (d) q ex = 0 and l ex = 587 nm and (e) q ex = 60 and l ex = 600 nm. Circles having a 1/e 2 radius of the fitted 2D Gaussian function are shown by (d) green and (e) red. A circle having the 1/e 2 radius of the fitted Gaussian function. (f) The localized positions (cross) and the circles having the 1/e 2 diameter (~640 nm) of the individual FRET pairs in d and e. S4

5 Figure S4. Spectral-selective localization of individual FRET pairs of B558 scy7 in a water matrix at 1.7 K. (a) Confocal fluorescence image of the FRET pair of B558 scy7 in a water matrix at 1.7 K with excitation by broadband laser light ( nm). The polarization of the excitation light was circular. (b) The excitation spectrum of the FRET pair (spectral width of laser light ~ 0.2 nm). Spectrum b was observed at the position denoted by the cross in a. The spectrum was an average over with differently-oriented linearly-polarized lights. (c) The polarization dependence of the fluorescence excitation spectrum. (d, e) The confocal fluorescence image of the FRET pair at (d) q ex = 60 and l ex = 572 nm and (e) q ex = 90 and l ex = 560 nm. Circles having a 1/e 2 radius of the fitted 2D Gaussian function are shown by (d) red and (e) green. A circle having the 1/e 2 radius of the fitted Gaussian function. (f) The localized positions (cross) and the circles having the 1/e 2 diameter (~690 nm) of the individual FRET pairs in d and e. S5

6 Figure S5. Spectral-selective localization of individual FRET pairs of B558 scy7 in a water matrix at 1.7 K. (a) Confocal fluorescence image of the FRET pair of B558 scy7 in a water matrix at 1.7 K with excitation by broadband laser light ( nm). The polarization of the excitation light was circular. (b) The excitation spectrum of the FRET pair (spectral width of laser light ~ 0.2 nm). Spectrum b was observed at the position denoted by the cross in a. The spectrum was an average over with differently-oriented linearly-polarized lights. (c) The polarization dependence of the fluorescence excitation spectrum. (d, e) The confocal fluorescence image of the FRET pair at (d) q ex = 30 and l ex = 574 nm and (e) q ex = 120 and l ex = 565 nm. Circles having a 1/e 2 radius of the fitted 2D Gaussian function are shown by (d) red and (e) green. A circle having the 1/e 2 radius of the fitted Gaussian function. (f) The localized positions (cross) and the circles having the 1/e 2 diameter (~640 nm) of the individual FRET pairs in d and e. S6

7 Supporting Materials and general information General chemicals were purchased from commercial suppliers (Wako Pure Chemical, Tokyo Chemical Industries, Aldrich Chemical Company, Dojindo, Lumiprobe and Thermo Fisher Scientific) and were used without further purification. 1 H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCEIII 400 (400 MHz) with chemical shifts (δ) reported in ppm relative to the solvent residual signals of CD 3OD (3.31 ppm), and coupling constants reported in Hz. High-resolution mass spectra (HRMS) were measured on a Bruker microtofii with electron spray ionization (ESI). HPLC purification and analyses were performed on a reversephase column (GL Sciences (Tokyo, Japan), Inertsil ODS-3 10 mm 250 mm and Inertsil ODS-3 20 mm 250 mm for purification and Inertsil ODS mm 250 mm for analyses) using an HPLC system composed of a pump (Jasco PU-2080 or PU-2087) and a detector (Jasco MD-2010 or MD-2018). AT565 amine AT565 NHS ester AT565 amine To a solution of AT565 NHS ester (100 ml of a 14 mm DMSO solution, 1.4 µmol, 1.0 eq.), N-Boc-transcyclohexyl-1,4-diamine 21 (25.4 µl of a 100 mm DMSO solution, 2.5 µmol, 1.8 eq.) and DIEA (2.0 µl) were added. The reaction mixture was stirred at r.t. for 1 h under dark conditions. H 2O (10 µl) was added. After stirring for 30 min, TFA (250 µl) was added, and the reaction mixture was stirred at r.t. for 5 h. The crude product was purified by semipreparative HPLC using eluent A (H 2O with 0.1% TFA and 1% CH 3CN) and eluent B (CH 3CN with 1% H 2O) (A/B = 90/10 to 0/100 for 40 min) to give AT565 amine. 1 H NMR (400 MHz, MeOD): 8.62 (d, J = 7.8 Hz, 1 H), 8.39 (d, J = 8.2 Hz, 1 H), 8.17 (dd, J = 8.2, 1.8 Hz, 1 H), 7.76 (d, J = 1.6 Hz, 1 H), 6.93 (s, 2 H), 6.72 (s, 2 H), 3.91 (m, 1 H), 3.67 (q, J = 7.4 Hz, 4 H), 3.58 (t, J = 5.7 Hz, 4 H), 3.11 (m, 1 H), 2.69 (t, J = 6.0 Hz, 4 H), 2.11 (m, 4 H), 1.93 (m, 4 H), 1.53 (m, 4 H), 1.33 (t, J = 7.1 Hz, 6 H); HRMS (m/z): [M+H] + calcd. for C 37H 43N 4O 4, ; found, S7

8 AT565 Ax750 Ax750 Ax750 Ax750 NHS ester AT565-Ax750 AT565 amine To a solution of AT565 amine (70 µl of a 10 mm DMSO solution, 0.7 µmol, 1.0 eq.), Alexa 750 NHS ester (70 µl of a 10 mm DMSO solution, 0.7 µmol, 1.8 eq.) and DIEA (0.5 µl) were added. The reaction mixture was stirred at r.t. for 7 h under dark conditions. The crude product was purified by semipreparative HPLC using eluent A (H 2O with 0.1% TFA and 1% CH 3CN) and eluent B (CH 3CN with 1% H 2O) (A/B = 90/10 to 0/100 for 40 min) to give ATTO565-Alexa 750. HRMS (m/z): [M-H] - calcd. for C 75H 85N 6Na 2O 417 S 4, ; found, nm 750nm 1. Caron, K., Lachapelle V., Keillor J. W. Dramatic increase of quench efficiency in "spacerless" dimaleimide fluorogens. Organic & Biomolecular Chemistry 9, (2011). S8

9 B581 scy7 scy7 amine B581-sCy7 B581 NHS ester To a solution of B581 NHS ester (200 µl of a 10 mm DMSO solution, 2.0 µmol, 1.0 eq.), cyanine 7 amine (200 µl of a 10 mm DMSO solution, 2.0 µmol, 1.0 eq.) and N,N-diisopropylethylamine (DIEA) (3.8 µl, 20 µmol, 10 eq.) were added. The reaction mixture was stirred at r.t. for 14 h under dark conditions. The crude product was purified by semipreparative HPLC using eluent A (H 2O with 0.1% TFA and 1% CH 3CN) and eluent B (CH 3CN with 1% H 2O) (A/B = 90/10 to 0/100 for 40 min) and by semipreparative HPLC using eluent C (H 2O with 100 mm TEAA and 1% CH 3CN) and eluent D (CH 3CN with 1% H 2O) (C/D = 90/10 to 0/100 for 40 min) to give B581-sCy7. HRMS (m/z): [M] - calcd. for C 65H 74BF 2N 6O 8S 2, ; found, nm 750nm S9

10 B581 Cy7.5 Cy7.5 amine B581 NHS ester B581-Cy7.5 To a solution of B581 NHS ester (120 µl of a 10 mm DMSO solution, 1.2 µmol, 1.0 eq.), Cy7.5 amine (120 µl of a 10 mm DMSO solution, 1.2 µmol, 1.0 eq.) and N,N-diisopropylethylamine (DIEA) (2.3 µl, 13 µmol, 11 eq.) were added. The reaction mixture was stirred at r.t. for 14 h under dark conditions. The crude product was purified by semipreparative HPLC using eluent A (H 2O with 0.1% TFA and 1% CH 3CN) and eluent B (CH 3CN with 1% H 2O) (A/B = 90/10 to 0/100 for 40 min) to give BODIPY581/591-cyanine 7.5. HRMS (m/z): [M] + calcd. for C 73H 80BF 2N 6O 3, ; found, ; [M] - calcd. for C 73H 78BF 2N 6O 3, ; found, (detected as an OH-adduct under the measurement conditions). 580nm 750nm S10

11 B558 scy7 scy7 amine B558-sCy7 B558 NHS ester To a solution of B558 NHS ester (200 µl of a 10 mm DMSO solution, 2.0 µmol, 1.0 eq.), sulfocyanine7 amine (200 µl of a 10 mm DMSO solution, 2.0 µmol, 1.0 eq.) and N,N-diisopropylethylamine (DIEA) (3.8 µl, 20 µmol, 10 eq.) were added. The reaction mixture was stirred at r.t. for 14 h under dark conditions. The crude product was purified by semipreparative HPLC using eluent A (H 2O with 0.1% TFA and 1% CH 3CN) and eluent B (CH 3CN with 1% H 2O) (A/B = 90/10 to 0/100 for 40 min) to give BODIPY558/568-sulfocyanine7. HRMS (m/z): [M] + calcd. for C 59H 70BF 2N 6O 8S 3, ; found, ; [M] - calcd. for C 59H 68BF 2N 6O 8S 3, ; found, nm 750nm S11

12 AT647N Ax base-long single-stranded DNA (ssdna) labeled with AT647N and Ax750 was purchased from Integrated DNA Technology. The 5 -end of the oligonucleotide was modified with an amine group (5 -amino modifier C6), and Ax750 NHS ester was labeled with the amine group. The 3 -end of the oligonucleotide was modified with a hexynyl group, and AT647N-azide was labeled with the hexynyl group (3 -hexynyl). The molecular structure of the modified ssdna is shown below. S12