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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 direct image and metrology.(A) HRTEM phase-contrast image of a single A-DNA helix bound to a 100 Å DNA bundle obtained by stacking two images acquired with 50 e/s Å2 at 80 keV. (B) Dotted line sharpens the DNA location. Major and minor grooves and the helix pitch of 26.5 Å are highlighted. (C) The principal lengths (the backbones, the base pairs (BPs), the diameter, and the rise per base pair) are indicated and reported in Table 1. The length difference between the purine and pyrimidine bases is also shown: a.u., arbitrary unit. (D) The tilt of the base pairs with respect to the helix axis is reported and measures 19°.
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Figure 1: A-DNA direct image and metrology.(A) HRTEM phase-contrast image of a single A-DNA helix bound to a 100 Å DNA bundle obtained by stacking two images acquired with 50 e/s Å2 at 80 keV. (B) Dotted line sharpens the DNA location. Major and minor grooves and the helix pitch of 26.5 Å are highlighted. (C) The principal lengths (the backbones, the base pairs (BPs), the diameter, and the rise per base pair) are indicated and reported in Table 1. The length difference between the purine and pyrimidine bases is also shown: a.u., arbitrary unit. (D) The tilt of the base pairs with respect to the helix axis is reported and measures 19°.

Mentions: The TEM image is reported in Fig. 1, with the metrology throughout its four panels. In particular, we show the direct image of a single dsDNA, highlighting the characteristic lengths and other relevant features such as the diameter, major and minor groove visualization, their relative distance, inter–base pair distance, base pair length, and angle measured between the base pair and the helix axis (5–9). The A-DNA micrograph was taken in a CS-corrected HRTEM operating at 80 keV electron beam energy, with a magnification of about 1 million and a resolution of 1.5 Å, calculated according to the equations reported in (10).


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 direct image and metrology.(A) HRTEM phase-contrast image of a single A-DNA helix bound to a 100 Å DNA bundle obtained by stacking two images acquired with 50 e/s Å2 at 80 keV. (B) Dotted line sharpens the DNA location. Major and minor grooves and the helix pitch of 26.5 Å are highlighted. (C) The principal lengths (the backbones, the base pairs (BPs), the diameter, and the rise per base pair) are indicated and reported in Table 1. The length difference between the purine and pyrimidine bases is also shown: a.u., arbitrary unit. (D) The tilt of the base pairs with respect to the helix axis is reported and measures 19°.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A-DNA direct image and metrology.(A) HRTEM phase-contrast image of a single A-DNA helix bound to a 100 Å DNA bundle obtained by stacking two images acquired with 50 e/s Å2 at 80 keV. (B) Dotted line sharpens the DNA location. Major and minor grooves and the helix pitch of 26.5 Å are highlighted. (C) The principal lengths (the backbones, the base pairs (BPs), the diameter, and the rise per base pair) are indicated and reported in Table 1. The length difference between the purine and pyrimidine bases is also shown: a.u., arbitrary unit. (D) The tilt of the base pairs with respect to the helix axis is reported and measures 19°.
Mentions: The TEM image is reported in Fig. 1, with the metrology throughout its four panels. In particular, we show the direct image of a single dsDNA, highlighting the characteristic lengths and other relevant features such as the diameter, major and minor groove visualization, their relative distance, inter–base pair distance, base pair length, and angle measured between the base pair and the helix axis (5–9). The A-DNA micrograph was taken in a CS-corrected HRTEM operating at 80 keV electron beam energy, with a magnification of about 1 million and a resolution of 1.5 Å, calculated according to the equations reported in (10).

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