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An unexpected conclusion about the abnormal conformational states of DNA molecules on the surface of charged monomolecular films and their fundamental differences from the strictly periodic conformation of the double helix in the volume of the solution was made in a work carried out jointly by scientists from the Russian Federation and the United States, five of whom are graduates of MIPT. The obtained results open up prospects for research in a new field of highly perturbed surface conformations of DNA with complex and very rich physics and nonlinear mechanics in response to the action of previously unsuspected huge lateral forces on a molecular scale.

The conformation of a polymer is a stable configuration of the spatial arrangement of the atoms of its chain. Of the several conformations of the DNA inheritance molecule, the most well-known is the so — called B-form-a right-twisted double helix with a step of 3.4 nm and a diameter of 2 nm, in which the planes of the nitrogenous bases (A,T,G,C) are perpendicular to the helix axis and are at a stacking distance (~0.34 nm). It is also known about DNA that it is one of the most rigid linear-chain polymers.

Due to the high bending stiffness, the mechanical properties of DNA molecules for weak and moderate bending moments are well interpreted within the framework of a simple mechanical worm-like chain model (WLC model). This model considers a short segment of DNA as a homogeneous bending rod obeying Hooke's law, that is, with a linear relationship between the applied external bending moment and the bending caused by it.

The validity of the WLC model was confirmed for a huge array of experimental data obtained in the volume of the solution in vivo and in vitro,and for them it has long become canonical. On the other hand, over the past two decades, the nonlinear mechanics of DNA has been developed, corresponding to the region of very large external forces. For this purpose, micromanipulations with single DNA molecules are used using laser tweezers (tweezers), for the invention of which and their application to study the mechanical properties of biomolecules, Arthur Ashkin was awarded the Nobel Prize in 2018.

The large bending stiffness of DNA molecules provides a large lateral size of two-dimensional conformations during adsorption to the surface, which is necessary for microscopic observations using atomic force microscopy (AFM). In the AFM method, the sample surface is scanned by sharp probes with a radius of curvature at the end of ~10 nm, and the minimum size of the visualized details (spatial resolution) is several nanometers.

For observation using AFM, DNA is adsorbed from a solution onto atomically smooth mica or graphite surfaces covered with films ~1 nm thick formed by positively charged modifier substances (DNA has a negative charge in solution). Until now, it was assumed by default that adsorption on such surfaces leads only to some compression of the DNA double helix by vertically directed attractive forces, and the conformation of the B-form remains intact.

This consideration ignores the tangential (directed along the surface) electrostatic forces due to the uneven distribution of the surface charge in positively charged modifier films (Fig. 1a). It comes from idealized representations of a uniformly charged surface, when the lateral lateral forces on both sides of the DNA strand are balanced (Fig. 1b).

"We have shown that it is these lateral forces, overlooked from consideration, that have the strongest influence on the structure of DNA at the nanoscale. They appear due to the fact that positively charged modifier molecules themselves have a linear size exceeding the diameter of DNA, and the charge is concentrated at the ends of the molecules. At the molecular scale, these forces are enormous and make the state of DNA internally highly overstressed, which leads to numerous radical conformational rearrangements. Such an extreme structural reorganization turns out to be completely unexpected and counterintuitive within the framework of the concepts of DNA as a rigid polymer chain. As a result, the DNA conformation becomes oversaturated with structural anomalies, which fundamentally distinguishes it from all previously studied conformations, " says Valery Prokhorov, one of the co-authors of the work who proposed the idea of interpretation.

Until now, microscopy of DNA molecules on the surface has been reduced to fairly trivial measurements of the size of their two-dimensional conformations, from which the persistent length of DNA was determined and compared with those obtained by other methods — the only parameter of the WLC model that quantitatively characterizes the flexibility of the polymer chain (the persistent length of a linear-chain polymer corresponds to the length of a segment of the polymer chain, which is approximately straight under conditions of constant change in the shape of the chain due to thermal motion; the persistent length of rigid-chain DNA is ~50 nm and is ~1 nm for flexible-chain polymers such as polyethylene). Nanoscale measurements were considered of little interest.

Phenomenologically, two types of two-dimensional surface conformations were identified (Fig. 1 c, d). On the surface of weakly charged mica (Fig. 1d), DNA has smooth contours, which corresponds to the expectations for a rigid-chain polymer. In the more compact, so-called "projection" conformations observed on the surface of the modifier films, there are additionally numerous strong local bends at the scale of ~10 nm (Fig. 1c), unexpected for hard-stranded DNA.

We noticed that these bends have an abnormally large curvature, indicating that these areas are kinks. The conformation of the kink (break), in which the continuity of the regular helical structure of DNA is broken due to a local break in the stacking of neighboring bases, was introduced by the author of the double helix model by Crick in 1974.

The unexpected experimental conclusion about the presence of a large number of kinks was supported by general physical analysis and specific calculations within the framework of the model schematically shown in Figure 1e. In the proposed model, DNA electrostatically interacts with a one-dimensional periodically charged lamellar sublayer of a modifier substance film formed at the boundary with graphite. The model predicts the appearance of a supercritical (kink) bending moment in DNA (τ>30pNnm) at small angles between it and the lamellae (Fig. 1e, right part).

The proposed scheme was expanded to include other structural anomalies (other than kinks), such as melting eyes, which were also observed on AFM images and had no reasonable explanation until now. In this case, an unexpected overlap is found between the mechanics of DNA on the surface, which is the source of large lateral electrostatic forces, and nonlinear mechanics, in which forces are applied to DNA artificially in micromanipulations with laser tweezers. The obtained results open up research in a new field of highly perturbed surface conformations of DNA with complex and very rich physics and nonlinear mechanics in response to the action of previously unsuspected huge lateral forces on a molecular scale.

The development of this field will require great efforts of theorists, molecular modeling and new AFM measurements of extra-resolution sufficient to visualize individual strands of the helical structure of DNA. The qualitative differences in the scale of kink bends, which require nonlinear non-WLC mechanics to describe, from DNA bends described by the standard WLC model are shown in Figure 1f.

"Our work is also interesting in another context," says Dmitry Klinov, head of the Laboratory of Medical Nanotechnologies at FKHM FMBA, associate professor of the Department of Molecular and Translational Medicine at MIPT, one of the co-authors, who for the first time drew attention to the unexpected features of AFM images of DNA on the surface of charged films. - Abnormal bends were observed on DNA images obtained with a fairly good resolution from the very beginning of using AFM for its visualization. However, they did not attract the attention of researchers and were not commented on in any way"

Thanks to the high-resolution AFM probes we developed earlier, we were able to observe DNA with a minimum recorded width close to 3 nm, which allowed us to increase the accuracy of determining the curvature of the bending sections of DNA and, most importantly, to understand that it exceeds the critical one corresponding to the threshold for the formation of kinks. Now it turns out that a number of fundamentally important and complex physical phenomena were viewed due to the uncritical use of the stereotype of a rigid DNA polymer chain and an unacceptable idealization of the properties of a surface that was considered uniformly charged." The work was carried out with the support of the Russian Foundation for Basic Research and the Ministry of Education and Science of the Russian Federation.

The article was published in the journal Nano-Micro Letters
PICTURE: Diagram of bending conformational anomalies of DNA on the surface. (a) The appearance of DNA bending at the nanoscale due to the imbalance of the attractive forces (different length of the red arrows) between the DNA and the nearest surface charges. The characteristic distance between the charges s is greater than the diameter of the DNA (s > dDNA). (b) Compensation of lateral lateral forces on a uniformly charged surface (s << dDNA). (c) AFM images of abnormally bent DNA on the surface of a positively charged graphite modifier film. (d) DNA with smooth contours on the mica surface. (e) A scheme of the appearance of supercritical bending of DNA at the nanoscale due to the action of lateral forces caused by interaction with a one-dimensional periodic charge distribution in the lamellar sublayer of a two-layer modifier film. (f) Qualitative difference between weakly and moderately curved DNA conformations in the volume of the solution and wound on the nucleosome, and kink DNA conformations on lamellar surfaces with a one-dimensional periodic charge distribution © Valery Prokhorov

Source: naked-science.ru, sci-dig.ru