Supporting Information for An Optical trap combined with three-color ...
Supporting Information for An Optical trap combined with three-color FRET
Sanghwa Lee, and Sungchul Hohng
Department of Physics and Astronomy, Department of Biophysics and Chemical Biology, and National Center for Creative Research Initiatives, Seoul National University, Seoul 151747, Korea
Component list for the setup. The following components were used to construct the hybrid instrument. Please check optical layout of the components in Figure 1a. Commercial inverted microscope: Nikon, TE2000-U Objective lens: Olympus, UPLSAPO 100x oil-immersion 532-nm fluorescence excitation laser: Spectra-Physics, Excelsior-532-50-CDRH 633-nm fluorescence excitation laser: Thorlabs, HRP050 1064-nm trapping laser: IPG photonics, YLR-5-LP 3-dimensional piezo stage: MadCityLab, LP100 Piezo-mirror scanner (PM): Physik Instrument, S-334K010 Electro-optic modulator (EOM): Conoptics, 350-50 Avalanche photo diode (APD): Perkin Elmer, SPCM-AQRH-14 Quadratic photo diode (QPD): Pacific Silicon Sensor, QP50-6SD2 CCD camera (CCD): Watec, WAT-902H2
Single-mode fiber: Thorlabs, 460HP Pinhole: Thorlabs, P100S 100-µm diameter Dichroic mirrors D1: Chroma, 900dcsp; D2: Chroma, 900dcsp; D3: Chroma, 865dcxxr; D4: Chroma, z532bcm; D5: Chroma, z532/633rpc; D6: Chroma, 640dcxr; D7: Chroma, 740dcxr Optical filters F1: Chroma, HQ580/60m-2p; F2: Chroma, HQ680/60m-2p; F3:Chroma, HQ790/80m; F4: Chroma, E950SP-2p ND filter (ND): Thorlabs, ND20B Optical lenses L1: Thorlabs, LA1805C, f = 30 mm; L2: Thorlabs, LA1608C, f = 75 mm; L3: Newport, KPX 202 AR33, f = 250 mm; L4: Newport, KPX 202 AR33, f = 250 mm; L5: Thorlabs, LA1979B, f = 200 mm; L6: focusing lens as a part of commercial Nikon microscope; L7: CVI Melles Griot, LAO-450.0-40.0, f = 450 mm; L8: CVI Melles Griot, LAO-349.9-40.0, f = 350 mm; L9: CVI Melles Griot, LAO-90.0-25.0, f = 90 mm; L10: CVI Melles Griot, LAO-90.0-25.0, f = 90 mm DNA preparation. The DNA Holliday junction was prepared as previously described.1 To construct the DNA sample containing two DNA hairpins (Figure 3a), the following DNA sequences (written from 5’ to 3’) were purchased from IDTDNA. Hairpin
strand:
CCACTGCACGCTGCTAGGTTGAGTCCTATTTTTTTTTTTAGGACT
CTT-Cy5-TTGGACCCTGTTTTTTTTTTCAGGGTCCTTGGATCGTCGCACGTCACC Stem1 strand: Cy7-CCTAGCAGCGTGCAGTGG-biotin (Stem for surface immobilization) Stem2 strand: GGGCGGCGACCTGGTGACGTGCGACGATCC-Cy3 (Stem for annealing to a λ–DNA overhang)
The strands were annealed by mixing Hairpin strand (25 µM, 6 µl), Stem1 strand (25 µM, 4 µl), Stem2 strand (25 µM, 8 µl), and slowly cooling down from 90 ºC to 4 ºC in 10 mM TrisHCl (pH 8.0) with 50 mM NaCl. λ-DNA (Promega) was used to connect the molecule of interest (Holliday junction or DNA hairpin construct) and a bead for applying force to the molecule. To construct a linear form of λ-DNA, λ-DNA (40 µl, ~10 nM) was heated to 65 ºC in the heat block for 5 min, and quickly cooled down in wet ice to prevent the intermolecular hybridization of the 12-bp complementary overhangs. And then we mixed λ-DNA solution (40 µl, ~10 nM) and the molecule of interest (1 µl, ~200 nM) at a 2:1 molar ratio with BSA (1 µl, ~20 mg/ml) and NaCl (2 µl, 1 M). The mixture was incubated at room temperature for 90 min, followed by 60 min at 4 ºC. A DNA strand labelled with digoxigenin that was complementary to a λ–DNA overhang (phosphate-AGGTCGCCGCCC-Dig, written from 5’ to 3’) was added to the mixture in 10-fold molar excess (1 µl, 4 µM) over the λ-DNA, and incubated at 4 ºC for 90 min. After the annealing process, we added 10x T4 DNA ligase buffer (5 µl) and T4 DNA ligase (400 unit, 1 µl), and mixed gently. The mixture was incubated at 16 ºC for 4 hours, and then diluted by addition of 250 µl 10 mM Tris-HCl (pH 8.0), 50 mM NaCl. Finally, the solution was stored at -20 ºC in 10 µl aliquots. References (1)
Lee, S.; Lee, J.; Hohng, S. PLoS One 2010, 5, e12270.
Figure S1. FRET histograms of the two hairpins at different forces
The experiments were performed at room temperature in 10 mM Tris-HCl (pH 8.0) with 50 mM Mg2+. Experiments were repeated ten times via different molecules to make error bars in Figure 3e. All histograms were fitted to the Gaussian functions to obtain relative populations of folded/unfolded states of the two hairpins.
Figure S2. Unfolding/folding FRET time traces of the two hairpins at varying forces
The experiments were performed at room temperature in 10 mM Tris-HCl (pH 8.0) with 50 mM Mg2+. Experiments were repeated ten times via different molecules to make error bars in Figure 3f. Orange lines are the most probable FRET trajectories generated by hidden Markov modeling.
Figure S3. Mechanical unfolding of a different DNA hairpin construct
(a) DNA construct used for the experiments. (b) FRET histograms of E12 and E13 at varying forces. The experiments were performed at room temperature in 10 mM Tris-HCl (pH 8.0) buffer with 50 mM Mg2+. Folding/unfolding transitions of H1 were slow enough to be directly observed via E12. The unfolding/folding dynamics of H2, however, were too fast to be directly monitored via E13. Instead, the peak shift of E13 revealed the force-induced unfolding of H2. (C) The equilibrium constant of the unfolding reaction (Keq: the ratio of the unfolded state population to the folded state population) as a function of force (triangles for H1 and squares for H2) and their log-linear fits (black lines). In case of H1, Keq was determined from the ratio of the low FRET population and the high FRET population. In case of H2, Keq was calculated from the shift of FRET peak position.
Sanghwa Lee, and Sungchul Hohng
Department of Physics and Astronomy, Department of Biophysics and Chemical Biology, and National Center for Creative Research Initiatives, Seoul National University, Seoul 151747, Korea
Component list for the setup. The following components were used to construct the hybrid instrument. Please check optical layout of the components in Figure 1a. Commercial inverted microscope: Nikon, TE2000-U Objective lens: Olympus, UPLSAPO 100x oil-immersion 532-nm fluorescence excitation laser: Spectra-Physics, Excelsior-532-50-CDRH 633-nm fluorescence excitation laser: Thorlabs, HRP050 1064-nm trapping laser: IPG photonics, YLR-5-LP 3-dimensional piezo stage: MadCityLab, LP100 Piezo-mirror scanner (PM): Physik Instrument, S-334K010 Electro-optic modulator (EOM): Conoptics, 350-50 Avalanche photo diode (APD): Perkin Elmer, SPCM-AQRH-14 Quadratic photo diode (QPD): Pacific Silicon Sensor, QP50-6SD2 CCD camera (CCD): Watec, WAT-902H2
Single-mode fiber: Thorlabs, 460HP Pinhole: Thorlabs, P100S 100-µm diameter Dichroic mirrors D1: Chroma, 900dcsp; D2: Chroma, 900dcsp; D3: Chroma, 865dcxxr; D4: Chroma, z532bcm; D5: Chroma, z532/633rpc; D6: Chroma, 640dcxr; D7: Chroma, 740dcxr Optical filters F1: Chroma, HQ580/60m-2p; F2: Chroma, HQ680/60m-2p; F3:Chroma, HQ790/80m; F4: Chroma, E950SP-2p ND filter (ND): Thorlabs, ND20B Optical lenses L1: Thorlabs, LA1805C, f = 30 mm; L2: Thorlabs, LA1608C, f = 75 mm; L3: Newport, KPX 202 AR33, f = 250 mm; L4: Newport, KPX 202 AR33, f = 250 mm; L5: Thorlabs, LA1979B, f = 200 mm; L6: focusing lens as a part of commercial Nikon microscope; L7: CVI Melles Griot, LAO-450.0-40.0, f = 450 mm; L8: CVI Melles Griot, LAO-349.9-40.0, f = 350 mm; L9: CVI Melles Griot, LAO-90.0-25.0, f = 90 mm; L10: CVI Melles Griot, LAO-90.0-25.0, f = 90 mm DNA preparation. The DNA Holliday junction was prepared as previously described.1 To construct the DNA sample containing two DNA hairpins (Figure 3a), the following DNA sequences (written from 5’ to 3’) were purchased from IDTDNA. Hairpin
strand:
CCACTGCACGCTGCTAGGTTGAGTCCTATTTTTTTTTTTAGGACT
CTT-Cy5-TTGGACCCTGTTTTTTTTTTCAGGGTCCTTGGATCGTCGCACGTCACC Stem1 strand: Cy7-CCTAGCAGCGTGCAGTGG-biotin (Stem for surface immobilization) Stem2 strand: GGGCGGCGACCTGGTGACGTGCGACGATCC-Cy3 (Stem for annealing to a λ–DNA overhang)
The strands were annealed by mixing Hairpin strand (25 µM, 6 µl), Stem1 strand (25 µM, 4 µl), Stem2 strand (25 µM, 8 µl), and slowly cooling down from 90 ºC to 4 ºC in 10 mM TrisHCl (pH 8.0) with 50 mM NaCl. λ-DNA (Promega) was used to connect the molecule of interest (Holliday junction or DNA hairpin construct) and a bead for applying force to the molecule. To construct a linear form of λ-DNA, λ-DNA (40 µl, ~10 nM) was heated to 65 ºC in the heat block for 5 min, and quickly cooled down in wet ice to prevent the intermolecular hybridization of the 12-bp complementary overhangs. And then we mixed λ-DNA solution (40 µl, ~10 nM) and the molecule of interest (1 µl, ~200 nM) at a 2:1 molar ratio with BSA (1 µl, ~20 mg/ml) and NaCl (2 µl, 1 M). The mixture was incubated at room temperature for 90 min, followed by 60 min at 4 ºC. A DNA strand labelled with digoxigenin that was complementary to a λ–DNA overhang (phosphate-AGGTCGCCGCCC-Dig, written from 5’ to 3’) was added to the mixture in 10-fold molar excess (1 µl, 4 µM) over the λ-DNA, and incubated at 4 ºC for 90 min. After the annealing process, we added 10x T4 DNA ligase buffer (5 µl) and T4 DNA ligase (400 unit, 1 µl), and mixed gently. The mixture was incubated at 16 ºC for 4 hours, and then diluted by addition of 250 µl 10 mM Tris-HCl (pH 8.0), 50 mM NaCl. Finally, the solution was stored at -20 ºC in 10 µl aliquots. References (1)
Lee, S.; Lee, J.; Hohng, S. PLoS One 2010, 5, e12270.
Figure S1. FRET histograms of the two hairpins at different forces
The experiments were performed at room temperature in 10 mM Tris-HCl (pH 8.0) with 50 mM Mg2+. Experiments were repeated ten times via different molecules to make error bars in Figure 3e. All histograms were fitted to the Gaussian functions to obtain relative populations of folded/unfolded states of the two hairpins.
Figure S2. Unfolding/folding FRET time traces of the two hairpins at varying forces
The experiments were performed at room temperature in 10 mM Tris-HCl (pH 8.0) with 50 mM Mg2+. Experiments were repeated ten times via different molecules to make error bars in Figure 3f. Orange lines are the most probable FRET trajectories generated by hidden Markov modeling.
Figure S3. Mechanical unfolding of a different DNA hairpin construct
(a) DNA construct used for the experiments. (b) FRET histograms of E12 and E13 at varying forces. The experiments were performed at room temperature in 10 mM Tris-HCl (pH 8.0) buffer with 50 mM Mg2+. Folding/unfolding transitions of H1 were slow enough to be directly observed via E12. The unfolding/folding dynamics of H2, however, were too fast to be directly monitored via E13. Instead, the peak shift of E13 revealed the force-induced unfolding of H2. (C) The equilibrium constant of the unfolding reaction (Keq: the ratio of the unfolded state population to the folded state population) as a function of force (triangles for H1 and squares for H2) and their log-linear fits (black lines). In case of H1, Keq was determined from the ratio of the low FRET population and the high FRET population. In case of H2, Keq was calculated from the shift of FRET peak position.
