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Amplified stretch of bottlebrush-coated DNA in nanofluidic channels.

Zhang C, Hernandez-Garcia A, Jiang K, Gong Z, Guttula D, Ng SY, Malar PP, van Kan JA, Dai L, Doyle PS, Vries Rd, van der Maarel JR - Nucleic Acids Res. (2013)

Bottom Line: The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy.Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA.The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, National University of Singapore, 2 Science Drive 3, 117542 Singapore, Laboratory of Physical Chemistry and Colloid Science, Wageningen University, 6703 HB Wageningen, The Netherlands, Food and Biobased Research, Wageningen University, 6700 AA Wageningen, The Netherlands, BioSystems and Micromechanics (BioSym) IRG, Singapore MIT Alliance for Research and Technology (SMART), 117576 Singapore and Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

ABSTRACT
The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.

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(A–D) Tapping mode atomic force microscopy images of linearized pUC18 DNA complexed with increasing amounts of polypeptide. The N/P ratios are 0, 0.1, 1.0 and 2.0, in panels A, B, C and D, respectively. (E, F) As in panels (A–D), but for DNA with N/P = 2.0. The scale bar denotes 1 μm. Panels B–D are a montage.
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gkt783-F6: (A–D) Tapping mode atomic force microscopy images of linearized pUC18 DNA complexed with increasing amounts of polypeptide. The N/P ratios are 0, 0.1, 1.0 and 2.0, in panels A, B, C and D, respectively. (E, F) As in panels (A–D), but for DNA with N/P = 2.0. The scale bar denotes 1 μm. Panels B–D are a montage.

Mentions: The persistence lengths were obtained from analysis of atomic force microscopy images of bottlebrush-coated DNA on silica. For this purpose, linearized pUC18 plasmid (2686 bp) was used at a concentration of 0.2 mg/l. The C4 blocks of the diblock polypeptide weakly adsorb to silica such that no additions to the buffer are necessary to promote adhesion. The C4-block mediated adsorption of bottlebrush-coated DNA is relatively weak because it is found that the molecules are more easily removed by flushing with water as compared with DNA molecules that have been adsorbed in the presence of either MgCl2 or high molecular weight polylysine. Accordingly, the bottlebrush is not kinetically trapped in a 3D conformation (30). For the reference case of bare DNA, molecules were bound to a mica surface by the use of a buffer comprising 10 mM MgCl2. Excess polypeptide and MgCl2 were removed from the interface by immersion of the specimens in ultrapure water for 30 min. During this development time, the molecules equilibrate on the surface in a 2D conformation. Subsequently, all specimens were N2-dried. A series of typical images for increasing C4K12 to DNA ratios are shown in Figure 6A–D. The relatively small plasmids are imaged in their entirety with a field of view of 3 × 3 μm2. We have also imaged polypeptide-coated DNA with N/P = 2.0. However, as shown in panels E and F of Figure 6, only segments of these relatively long molecules can be visualized. Nevertheless, their appearance in terms of thickness and rigidity is similar to the one of the shorter plasmid molecules.Figure 6.


Amplified stretch of bottlebrush-coated DNA in nanofluidic channels.

Zhang C, Hernandez-Garcia A, Jiang K, Gong Z, Guttula D, Ng SY, Malar PP, van Kan JA, Dai L, Doyle PS, Vries Rd, van der Maarel JR - Nucleic Acids Res. (2013)

(A–D) Tapping mode atomic force microscopy images of linearized pUC18 DNA complexed with increasing amounts of polypeptide. The N/P ratios are 0, 0.1, 1.0 and 2.0, in panels A, B, C and D, respectively. (E, F) As in panels (A–D), but for DNA with N/P = 2.0. The scale bar denotes 1 μm. Panels B–D are a montage.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814371&req=5

gkt783-F6: (A–D) Tapping mode atomic force microscopy images of linearized pUC18 DNA complexed with increasing amounts of polypeptide. The N/P ratios are 0, 0.1, 1.0 and 2.0, in panels A, B, C and D, respectively. (E, F) As in panels (A–D), but for DNA with N/P = 2.0. The scale bar denotes 1 μm. Panels B–D are a montage.
Mentions: The persistence lengths were obtained from analysis of atomic force microscopy images of bottlebrush-coated DNA on silica. For this purpose, linearized pUC18 plasmid (2686 bp) was used at a concentration of 0.2 mg/l. The C4 blocks of the diblock polypeptide weakly adsorb to silica such that no additions to the buffer are necessary to promote adhesion. The C4-block mediated adsorption of bottlebrush-coated DNA is relatively weak because it is found that the molecules are more easily removed by flushing with water as compared with DNA molecules that have been adsorbed in the presence of either MgCl2 or high molecular weight polylysine. Accordingly, the bottlebrush is not kinetically trapped in a 3D conformation (30). For the reference case of bare DNA, molecules were bound to a mica surface by the use of a buffer comprising 10 mM MgCl2. Excess polypeptide and MgCl2 were removed from the interface by immersion of the specimens in ultrapure water for 30 min. During this development time, the molecules equilibrate on the surface in a 2D conformation. Subsequently, all specimens were N2-dried. A series of typical images for increasing C4K12 to DNA ratios are shown in Figure 6A–D. The relatively small plasmids are imaged in their entirety with a field of view of 3 × 3 μm2. We have also imaged polypeptide-coated DNA with N/P = 2.0. However, as shown in panels E and F of Figure 6, only segments of these relatively long molecules can be visualized. Nevertheless, their appearance in terms of thickness and rigidity is similar to the one of the shorter plasmid molecules.Figure 6.

Bottom Line: The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy.Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA.The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, National University of Singapore, 2 Science Drive 3, 117542 Singapore, Laboratory of Physical Chemistry and Colloid Science, Wageningen University, 6703 HB Wageningen, The Netherlands, Food and Biobased Research, Wageningen University, 6700 AA Wageningen, The Netherlands, BioSystems and Micromechanics (BioSym) IRG, Singapore MIT Alliance for Research and Technology (SMART), 117576 Singapore and Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

ABSTRACT
The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.

Show MeSH
Related in: MedlinePlus