Limits...
The structure of DNA by direct imaging.

Marini M, Falqui A, Moretti M, Limongi T, Allione M, Genovese A, Lopatin S, Tirinato L, Das G, Torre B, Giugni A, Gentile F, Candeloro P, Di Fabrizio E - Sci Adv (2015)

Bottom Line: In the image, all relevant lengths of A-form DNA are measurable.A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment.Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.

View Article: PubMed Central - PubMed

Affiliation: SMILEs Lab, Physical Science and Engineering (PSE) and Biological and Environmental Science and Engineering (BESE) Divisions, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.

ABSTRACT
The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.

No MeSH data available.


Related in: MedlinePlus

A-DNA simulations.(A) Atomic model of A-DNA and corresponding HRTEM image simulations calculated using three defocus (Δf) values. (B) The A-DNA filament was subdivided into four plane slices of ¼t = 6.0 Å parallel to the helix axis. The black arrow indicates the electron beam propagation direction perpendicular to the planes. The corresponding electron exit wave functions at ¼t, ½t, ¾t, and t show phase variations directly correlated to specimen potential and atom position. Amplitude changes are negligible due to weak phase object approximation. In both (A) and (B), the lattice fringes form angles of 18° with respect to the helix axis, and the periodicity of minor and major grooves is in accordance with the double-helix atomic model.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4643809&req=5

Figure 3: A-DNA simulations.(A) Atomic model of A-DNA and corresponding HRTEM image simulations calculated using three defocus (Δf) values. (B) The A-DNA filament was subdivided into four plane slices of ¼t = 6.0 Å parallel to the helix axis. The black arrow indicates the electron beam propagation direction perpendicular to the planes. The corresponding electron exit wave functions at ¼t, ½t, ¾t, and t show phase variations directly correlated to specimen potential and atom position. Amplitude changes are negligible due to weak phase object approximation. In both (A) and (B), the lattice fringes form angles of 18° with respect to the helix axis, and the periodicity of minor and major grooves is in accordance with the double-helix atomic model.

Mentions: To complete our analysis, in Fig. 3A, we report a numerical HRTEM simulation by the exit wave reconstruction method of a single dsDNA image formation, where the microscope parameters used (electron energy, beam current, spherical aberration coefficient, incidence semiangle, and focus) are taken equal to those corresponding to the actual experimental conditions of our experiments. This simulation is realistic because our sample is background-free. Furthermore, in Fig. 3B, we report the front wave phase distortion (exit wave function) due to the phase change of the incidence plane wave under TEM conditions. This image helps in understanding the physical reason why a single DNA molecule gives enough phase change for a 1.5 Å resolution and a good image contrast. The phase shift modulation in a TEM is as follows:ϕ(x,y)=πtλU0Φ(x,y)where ϕ(x, y) is the phase-contrast modulation of an object of thickness t (t = 21 Å for DNA) with atomic potential energy Φ(x, y), and the electron wavelength λ, in our case, is equal to 4.2 pm at U0 accelerating energy of 80 keV. From the image simulation, we can see that after the TEM plane wave passes through 21 Å of DNA, the incidence wave function becomes distorted as ϕ(x, y) because of the atomic potential energy Φ(x, y) of the DNA molecule [ϕ(x, y), changes in the range of an appreciable fraction, 2π]. It is worth to say that this is under the hypothesis that spurious scattering from the substrate and from possible salt inclusions is absent. Both of these conditions are fulfilled by our preparation technique.


The structure of DNA by direct imaging.

Marini M, Falqui A, Moretti M, Limongi T, Allione M, Genovese A, Lopatin S, Tirinato L, Das G, Torre B, Giugni A, Gentile F, Candeloro P, Di Fabrizio E - Sci Adv (2015)

A-DNA simulations.(A) Atomic model of A-DNA and corresponding HRTEM image simulations calculated using three defocus (Δf) values. (B) The A-DNA filament was subdivided into four plane slices of ¼t = 6.0 Å parallel to the helix axis. The black arrow indicates the electron beam propagation direction perpendicular to the planes. The corresponding electron exit wave functions at ¼t, ½t, ¾t, and t show phase variations directly correlated to specimen potential and atom position. Amplitude changes are negligible due to weak phase object approximation. In both (A) and (B), the lattice fringes form angles of 18° with respect to the helix axis, and the periodicity of minor and major grooves is in accordance with the double-helix atomic model.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643809&req=5

Figure 3: A-DNA simulations.(A) Atomic model of A-DNA and corresponding HRTEM image simulations calculated using three defocus (Δf) values. (B) The A-DNA filament was subdivided into four plane slices of ¼t = 6.0 Å parallel to the helix axis. The black arrow indicates the electron beam propagation direction perpendicular to the planes. The corresponding electron exit wave functions at ¼t, ½t, ¾t, and t show phase variations directly correlated to specimen potential and atom position. Amplitude changes are negligible due to weak phase object approximation. In both (A) and (B), the lattice fringes form angles of 18° with respect to the helix axis, and the periodicity of minor and major grooves is in accordance with the double-helix atomic model.
Mentions: To complete our analysis, in Fig. 3A, we report a numerical HRTEM simulation by the exit wave reconstruction method of a single dsDNA image formation, where the microscope parameters used (electron energy, beam current, spherical aberration coefficient, incidence semiangle, and focus) are taken equal to those corresponding to the actual experimental conditions of our experiments. This simulation is realistic because our sample is background-free. Furthermore, in Fig. 3B, we report the front wave phase distortion (exit wave function) due to the phase change of the incidence plane wave under TEM conditions. This image helps in understanding the physical reason why a single DNA molecule gives enough phase change for a 1.5 Å resolution and a good image contrast. The phase shift modulation in a TEM is as follows:ϕ(x,y)=πtλU0Φ(x,y)where ϕ(x, y) is the phase-contrast modulation of an object of thickness t (t = 21 Å for DNA) with atomic potential energy Φ(x, y), and the electron wavelength λ, in our case, is equal to 4.2 pm at U0 accelerating energy of 80 keV. From the image simulation, we can see that after the TEM plane wave passes through 21 Å of DNA, the incidence wave function becomes distorted as ϕ(x, y) because of the atomic potential energy Φ(x, y) of the DNA molecule [ϕ(x, y), changes in the range of an appreciable fraction, 2π]. It is worth to say that this is under the hypothesis that spurious scattering from the substrate and from possible salt inclusions is absent. Both of these conditions are fulfilled by our preparation technique.

Bottom Line: In the image, all relevant lengths of A-form DNA are measurable.A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment.Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.

View Article: PubMed Central - PubMed

Affiliation: SMILEs Lab, Physical Science and Engineering (PSE) and Biological and Environmental Science and Engineering (BESE) Divisions, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.

ABSTRACT
The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.

No MeSH data available.


Related in: MedlinePlus