All DNA samples were mixed in TBE buffer (Medicago, 10 x TBE tablets) diluted in milli-Q water to the desired ionic strength. The oxygen scavenger β-mercaptoethanol (Sigma-Aldrich) was added to the buffer (3% v/v) to suppress photonicking of the DNA. YOYO-1 was purchased from Invitrogen and DNA from phage lambda (λ-DNA, 48.5 kb) was purchased from New England Biolabs.
The nanofluidic chips were fabricated in fused silica according to methods described elsewhere (6 (link)), with a cross-section of ∼100 × 150 nm2 and a length of ∼500 µm. All experiments were conducted in a nanofluidic chip consisting of pairs of microchannels that are spanned by nanochannels. The DNA sample solution was loaded into one of the microchannels of the chip and transferred to the nanochannel array by pressure. By applying pressure over two connected microchannels, the DNA was subsequently injected into the nanochannels. The DNA was imaged using an epi-fluorescence microscope (Zeiss AxioObserver.Z1) equipped with a Photometrics Evolve EMCCD camera and a 100× oil immersion TIRF objective (NA = 1.46) from Zeiss. Stacks of 200 images were recorded for each molecule, using the AxioVision software, with an exposure time of 100 ms per image at maximum speed, corresponding to approximately seven frames per second. Data analysis was performed with the freeware ImageJ (www.imagej.com ) and a custom-written MatLab based software. For each molecule, kymographs (timetraces) were first extracted. Subsequently, each line in the kymograph—corresponding to one frame in the original time series—was fitted with a convolution of a box function and an error function (6 (link)). This gives the position and extension of the molecule in each frame that is used to align the kymograph as well as obtaining average intensities and fluctuations.
The fluorescence intensity was normalized to a distinct upper limit in fluorescence intensity identified from measurements. This upper limit is assumed to correspond to the fluorescence intensity of fully intercalated DNA, ∼1 YOYO molecule every 4 bp (21 (link),22 (link)). Extrapolation of the extension of native DNA was obtained by a linear fit of the first eight binned values for each ionic strength. The straight lines inFigure 3 B were calculated from the extrapolated extension of the native DNA, again assuming a maximal intercalation of one YOYO every 4 bp and adding the resulting increase in contour length of 0.51 nm per YOYO molecule (22 (link)), taking the relative extension of the native DNA compared with the full contour length into account.
The nanofluidic chips were fabricated in fused silica according to methods described elsewhere (6 (link)), with a cross-section of ∼100 × 150 nm2 and a length of ∼500 µm. All experiments were conducted in a nanofluidic chip consisting of pairs of microchannels that are spanned by nanochannels. The DNA sample solution was loaded into one of the microchannels of the chip and transferred to the nanochannel array by pressure. By applying pressure over two connected microchannels, the DNA was subsequently injected into the nanochannels. The DNA was imaged using an epi-fluorescence microscope (Zeiss AxioObserver.Z1) equipped with a Photometrics Evolve EMCCD camera and a 100× oil immersion TIRF objective (NA = 1.46) from Zeiss. Stacks of 200 images were recorded for each molecule, using the AxioVision software, with an exposure time of 100 ms per image at maximum speed, corresponding to approximately seven frames per second. Data analysis was performed with the freeware ImageJ (
The fluorescence intensity was normalized to a distinct upper limit in fluorescence intensity identified from measurements. This upper limit is assumed to correspond to the fluorescence intensity of fully intercalated DNA, ∼1 YOYO molecule every 4 bp (21 (link),22 (link)). Extrapolation of the extension of native DNA was obtained by a linear fit of the first eight binned values for each ionic strength. The straight lines in
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