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Issue #2/2024
M. E. Stepanov, U. A. Khokhryakova, T. V. Egorova, K. A. Magaryan, A. V. Naumov
Shedding Light on DNA Origami: Practice
Shedding Light on DNA Origami: Practice
DOI: 10.22184/1993-7296.FRos.2024.18.2.166.174
Modern photonics requires technologies for the reproducible and controllable production of nanostructures, since many interesting and important optical processes occur on the subdiffractional scale characteristic for such structures. However, working with light at nanometer distances requires nanometer precision in object positioning, which is extremely difficult to achieve using standard methods. One of the new approaches that can become a response to this challenge is the use of DNA origami: structure of the polymer DNA molecule allows, on the one hand, chemically “tuning” of its geometry to obtain arbitrary shape on a nanometer scale, and on the other, addressability of DNA molecule allows nanoobjects to be placed at any position along its chain. This review considers some practical issues related to DNA origami preparation.
Modern photonics requires technologies for the reproducible and controllable production of nanostructures, since many interesting and important optical processes occur on the subdiffractional scale characteristic for such structures. However, working with light at nanometer distances requires nanometer precision in object positioning, which is extremely difficult to achieve using standard methods. One of the new approaches that can become a response to this challenge is the use of DNA origami: structure of the polymer DNA molecule allows, on the one hand, chemically “tuning” of its geometry to obtain arbitrary shape on a nanometer scale, and on the other, addressability of DNA molecule allows nanoobjects to be placed at any position along its chain. This review considers some practical issues related to DNA origami preparation.
Shedding Light
on DNA Origami: Practice
M. E. Stepanov 1, U. A. Khokhryakova 1, T. V. Egorova 1, K. A. Magaryan 1, A. V. Naumov 1, 2
Moscow Pedagogical State University (MPGU), Moscow, Russia
Lebedev Physical Institute of the Russian Academy of Sciences, Troitsk branch, Moscow, Troitsk, Russia
Modern photonics requires technologies for the reproducible and controllable production of nanostructures, since many interesting and important optical processes occur on the subdiffractional scale characteristic for such structures. However, working with light at nanometer distances requires nanometer precision in object positioning, which is extremely difficult to achieve using standard methods. One of the new approaches that can become a response to this challenge is the use of DNA origami: structure of the polymer DNA molecule allows, on the one hand, chemically “tuning” of its geometry to obtain arbitrary shape on a nanometer scale, and on the other, addressability of DNA molecule allows nanoobjects to be placed at any position along its chain. This review considers some practical issues related to DNA origami preparation.
Keywords: DNA nanotechnologies, DNA origami, numerical modeling, nanostructures.
Article received: December 12, 2023
Article accepted: January 19, 2024
Introduction
Origami is an ancient Japanese art of folding paper without cuts, giving it an arbitrary shape (the word origami literally means “folding” (ori, 折り) “paper” (gami, 紙)). Inspired by this art, the scientific community gradually came up with the idea of creating nanostructures by sequentially folding (self-assembly) a long DNA molecule (so-called scaffold) using short DNA fragments that act as staples. It turned out that the self-assembly process can be chemically “programmed” in advance due to the unique structure of the DNA molecule [1] (see the molecular basis of the DNA origami method in the first part of our review “Shedding light on DNA origami” [2]), in order to give the molecule an arbitrary shape.
However, the elegance of this idea faces certain practical difficulties. This article discusses in more detail some of the problems that arise along this path, as well as provides solutions. The first part of the article discusses the rational selection of staple molecules that guide the self-assembly of the DNA molecule, which can be done through computer modeling. The procedure of self-assembly and characterization of the resulting DNA origami nanostructures are presented in the second and third parts of the article.
There are many other interesting methods for creating nanostructures [3–23], however, DNA origami stands out among them due to the level of control and precision of the results. Using this method, molecular nanostructures based on DNA can be created with nanometer resolution allowing at the same time precise addressing of nanoobjects at preselected positions of the DNA molecule with good reproducibly. This makes DNA origami a unique tool for the controlled creation of nanocomplexes [24].
1. DNA origami modeling
The principle behind constructing DNA origami is to provide a long circular single-stranded DNA molecule (scaffold) with desired geometry by fastening its fragments with molecular staples (oligonucleotides, short DNA fragments) complementarily connecting required sections of the scaffold [2]. The problem lies in the fact that even a simple DNA origami design can include hundreds of molecular staples of individual composition. Errors in their choice can lead to a sharp deterioration in the quality of DNA origami self-assembly, making their rational selection an extremely important task. The answer to this challenge has been the development of specialized software that simplifies and automates the process of selecting a specific set of staples that can facilitate the folding of the chosen scaffold into a specific geometric shape (DNA origami).
Given the recent emergence of DNA nanotechnology practice for using CAD tools (Computer Aided Design) for its design is still not yet fully established, so the actual design process may be extremely non-intuitive. In the work [1], it was proposed to divide all DNA origami design environments into three generations according to their functional capabilities.
CaDNAno is a first-generation DNA-design program that allows the development of custom DNA nanostructures for a wide range of applications. The program has a user-friendly interface that enables users to design 2D DNA models (Fig. 1a). Due to the lack of a 3D interface, there is difficulty in designing 3D structures, however, CaDNAno supports model export to various file formats for more complex modeling and future use. The second-generation programs are considered as more user-friendly and less demanding in terms of the technical assembly. For example, TALOS (Three-dimensional Algorithmically-generated Library of DNA Origami Shapes) automatically selects staples to obtain 3D DNA origami of one of 20 different geometric frames (Fig. 1b).
The third-generation design software further expands the available functionality. For instance, it can provide the user with ability of DNA structure fine-tuning, visualization of just-built model parameters and internal interactions. Example of 3rd generation program is Adenita Samson (2021).
It should be noted that the approaches to predictable self-assembly of DNA origami nanostructures problem have great potential for optimization and are constantly being improved [25].
2. Assembly AND purification
of DNA-origami
During the assembly of DNA origami (Fig. 2), high purity of components and precision in the selection procedure conditions are required. After the optimal staple (oligonucleotides) design is achieved through modeling, their synthesis is outsourced to specialized companies (in Russia they are “Syntol”, “Evrogen”, “DNA-synthesis”, etc.).
The scaffold selection for DNA origami is determined by the size and complexity of the desired DNA origami structure. Most common choice is a single-stranded circular DNA molecule of the M13 bacteriophage genome with a length of 7249 nucleotides. Obviously, the size of a DNA origami structure is limited by the scaffold length used for assembly. This issue can be circumvented by using the longer single-stranded DNA molecules as the scaffolds, or by applying additional staples that are partially complement of other staples or the scaffold, allowing for the formation of additional elements extending beyond the primary DNA origami structure and increasing its size. The stability of base pairing in the DNA molecule significantly depends on the cation presence in the solution [2]. In most protocols for assembling DNA origami, the reaction occurs at pH = 8 in the tris-acetate buffer (TAE) with a concentration of Mg2+ ions from 5 to 20 mM. The DNA scaffold and staples are added to the buffer solution with the required concentration of Mg2+ ions in a ratio of 1:10 or 1:20 (to reduce the number of nonspecific aggregates generated) and exposed to the procedure of a specific annealing. During the annealing procedure, the mixture is first heated briefly almost to the boiling temperature, and then slowly cooled, resulting in the spontaneous self-assembly of the DNA origami structure. The cooling process duration depends on the complexity of DNA origami structure and ranges from several hours for small 2D structures to a week for multi-layered 3D structures. Creating complex hierarchical DNA origami structures (super-origami) requires assembly in multiple stages. The next step (purification and concentration of the resulting DNA origami structure) is critical for the optical/biomedical applications. The quality and purity of the assembled DNA origami construction is evaluated using the agarose gel electrophoresis, as the gel migration rates of “correct” and “by-product” assembly products differs. Purification of DNA origami is usually done using agarose gel electrophoresis followed by extraction of the “correct” product from a cut gel piece, ultrafiltration, polyethylene glycol (PEG) precipitation, ultracentrifugation or gel filtration (size exclusion chromatography). The choice of purification method depends on the ultimate use of the DNA origami structure.
3. DNA origami characterization methods
The study methods for the synthesized DNA origami structures can be divided into two groups: ensemble methods and methods for studying at the level of single molecules. Ensemble methods (gel electrophoresis, fluorescence spectroscopy, circular dichroism spectroscopy) allow for creating a general picture, averaged over a large number of assembled structures: whether the planned DNA origami structure was assembled, how many side products were concomitant.
Second (electron and atomic force microscopy) are techniques suitable for single molecule analysis [26, 27]. In combination with millisecond time resolution, the atomic force microscopes can register fast dynamic processes in the single DNA origami structures, and the use of modified probe tips allows studying the elasticity of DNA origami [28]. High-resolution electron microscopy makes it capable of registering individual particles attached to opposite sides of a single DNA nucleotide, with a distance of only 0.34 nm between them [29]. Such high resolution allows studying topological changes in the single structures, but it leads to rapid destruction of the DNA structure due to the need for sample preparation in a vacuum. To overcome this difficulty, more expensive methods of cryogenic electron microscopy are used [30].
Each of the given methods has its advantages and limitations. Combining different methods can provide the best way to achieve a complete understanding of DNA origami structural properties.
Conclusion
DNA origami method allows for controllably shaping a DNA molecule at a natural scale for this molecule (nanometer-scale), just like the Japanese art of origami allows shaping a piece of paper at a corresponding scale. The difference lies in the fact that for manipulating molecules, special chemical and physical conditions are organized in which the DNA origami assembles itself. The process requires careful preparation, but its results (including those in the field of photonics) can justify all efforts, as they provide an opportunity not only to control the shape of DNA molecules but also to place nanoobjects with nanometer precision at any position along this newly acquired shape. Furthermore, the DNA origami method is relatively young and its capabilities are far from being exhausted.
Acknowledgments
The work was carried out with financial support from the Ministry of Education of the Russian Federation within the framework of the state assignment of the Moscow Pedagogical State University “Physics of nanostructured materials and highly sensitive sensors: synthesis, fundamental research and applications in photonics, life sciences, quantum and nanotechnologies”.
AUTHORS
Stepanov Maxim Evgenievich – junior researcher, Moscow Pedagogical State University (MPGU), Shpolskiy Department of Theoretical Physics, junior researcher at the Laboratory of Physics of Advanced Materials and Nanostructures, Moscow, Russia.
RSCI ID: 334465
Scopus ID: 57195265809
ResearcherID: AAB‑6181-2022
ORCID: 0000-0002-0332-1235
Khokhryakova Uliana Aleksandrovna – Bachelor in fundamental physics of Moscow Pedagogical State University, research assistant at the Youth Laboratory of Biophotonics and Nanoengineering, e-mail: ua_khokhryakova@mpgu.su.
Egorova Tatiana Vladimirovna – Ph.D. in biological sciences, head of the Youth Laboratory of Biophotonics and Nanoengineering at Moscow Pedagogical State University, Moscow, Russia.
Scopus ID: 56868341400
ORCID: 0000-0002-7554-5246
ResearcherID: P‑9982-2017
Magaryan Konstantin Arutyunovich – Ph.D. in physical and mathematical sciences, Moscow Pedagogical State University (MPGU), Shpolskiy Department of Theoretical Physics, senior researcher at the Laboratory of Physics of Advanced Materials and Nanostructures.
RSCI ID: 723988
ResearcherID: A‑4208-2014
ORCID: 0000-0003-4754-4657
Naumov Andrey Vitalievich – corresponding member of the Russian Academy of Sciences, doctor of physical and mathematical sciences, head of the Troitsk branch of the Lebedev Physical Institute, head of the department of Moscow Pedagogical State University (MPGU), corresponding member of the Russian Academy of Sciences, associate professor, Moscow, Russia.
RSCI ID: 35867
Scopus ID: 7201349036
ResearcherID: E‑8905-2010
ORCID: 0000-0001-7938-9802.
CONFLICT OF INTERESTS
The authors declare that they have no conflict of interests. All authors took part in preparation of the article and supplemented the manuscript in terms of their scope of work.
CONTRIBUTION
OF THE COMPOSITE AUTHORS
The article has been prepared based on the work of all composite authors.
on DNA Origami: Practice
M. E. Stepanov 1, U. A. Khokhryakova 1, T. V. Egorova 1, K. A. Magaryan 1, A. V. Naumov 1, 2
Moscow Pedagogical State University (MPGU), Moscow, Russia
Lebedev Physical Institute of the Russian Academy of Sciences, Troitsk branch, Moscow, Troitsk, Russia
Modern photonics requires technologies for the reproducible and controllable production of nanostructures, since many interesting and important optical processes occur on the subdiffractional scale characteristic for such structures. However, working with light at nanometer distances requires nanometer precision in object positioning, which is extremely difficult to achieve using standard methods. One of the new approaches that can become a response to this challenge is the use of DNA origami: structure of the polymer DNA molecule allows, on the one hand, chemically “tuning” of its geometry to obtain arbitrary shape on a nanometer scale, and on the other, addressability of DNA molecule allows nanoobjects to be placed at any position along its chain. This review considers some practical issues related to DNA origami preparation.
Keywords: DNA nanotechnologies, DNA origami, numerical modeling, nanostructures.
Article received: December 12, 2023
Article accepted: January 19, 2024
Introduction
Origami is an ancient Japanese art of folding paper without cuts, giving it an arbitrary shape (the word origami literally means “folding” (ori, 折り) “paper” (gami, 紙)). Inspired by this art, the scientific community gradually came up with the idea of creating nanostructures by sequentially folding (self-assembly) a long DNA molecule (so-called scaffold) using short DNA fragments that act as staples. It turned out that the self-assembly process can be chemically “programmed” in advance due to the unique structure of the DNA molecule [1] (see the molecular basis of the DNA origami method in the first part of our review “Shedding light on DNA origami” [2]), in order to give the molecule an arbitrary shape.
However, the elegance of this idea faces certain practical difficulties. This article discusses in more detail some of the problems that arise along this path, as well as provides solutions. The first part of the article discusses the rational selection of staple molecules that guide the self-assembly of the DNA molecule, which can be done through computer modeling. The procedure of self-assembly and characterization of the resulting DNA origami nanostructures are presented in the second and third parts of the article.
There are many other interesting methods for creating nanostructures [3–23], however, DNA origami stands out among them due to the level of control and precision of the results. Using this method, molecular nanostructures based on DNA can be created with nanometer resolution allowing at the same time precise addressing of nanoobjects at preselected positions of the DNA molecule with good reproducibly. This makes DNA origami a unique tool for the controlled creation of nanocomplexes [24].
1. DNA origami modeling
The principle behind constructing DNA origami is to provide a long circular single-stranded DNA molecule (scaffold) with desired geometry by fastening its fragments with molecular staples (oligonucleotides, short DNA fragments) complementarily connecting required sections of the scaffold [2]. The problem lies in the fact that even a simple DNA origami design can include hundreds of molecular staples of individual composition. Errors in their choice can lead to a sharp deterioration in the quality of DNA origami self-assembly, making their rational selection an extremely important task. The answer to this challenge has been the development of specialized software that simplifies and automates the process of selecting a specific set of staples that can facilitate the folding of the chosen scaffold into a specific geometric shape (DNA origami).
Given the recent emergence of DNA nanotechnology practice for using CAD tools (Computer Aided Design) for its design is still not yet fully established, so the actual design process may be extremely non-intuitive. In the work [1], it was proposed to divide all DNA origami design environments into three generations according to their functional capabilities.
CaDNAno is a first-generation DNA-design program that allows the development of custom DNA nanostructures for a wide range of applications. The program has a user-friendly interface that enables users to design 2D DNA models (Fig. 1a). Due to the lack of a 3D interface, there is difficulty in designing 3D structures, however, CaDNAno supports model export to various file formats for more complex modeling and future use. The second-generation programs are considered as more user-friendly and less demanding in terms of the technical assembly. For example, TALOS (Three-dimensional Algorithmically-generated Library of DNA Origami Shapes) automatically selects staples to obtain 3D DNA origami of one of 20 different geometric frames (Fig. 1b).
The third-generation design software further expands the available functionality. For instance, it can provide the user with ability of DNA structure fine-tuning, visualization of just-built model parameters and internal interactions. Example of 3rd generation program is Adenita Samson (2021).
It should be noted that the approaches to predictable self-assembly of DNA origami nanostructures problem have great potential for optimization and are constantly being improved [25].
2. Assembly AND purification
of DNA-origami
During the assembly of DNA origami (Fig. 2), high purity of components and precision in the selection procedure conditions are required. After the optimal staple (oligonucleotides) design is achieved through modeling, their synthesis is outsourced to specialized companies (in Russia they are “Syntol”, “Evrogen”, “DNA-synthesis”, etc.).
The scaffold selection for DNA origami is determined by the size and complexity of the desired DNA origami structure. Most common choice is a single-stranded circular DNA molecule of the M13 bacteriophage genome with a length of 7249 nucleotides. Obviously, the size of a DNA origami structure is limited by the scaffold length used for assembly. This issue can be circumvented by using the longer single-stranded DNA molecules as the scaffolds, or by applying additional staples that are partially complement of other staples or the scaffold, allowing for the formation of additional elements extending beyond the primary DNA origami structure and increasing its size. The stability of base pairing in the DNA molecule significantly depends on the cation presence in the solution [2]. In most protocols for assembling DNA origami, the reaction occurs at pH = 8 in the tris-acetate buffer (TAE) with a concentration of Mg2+ ions from 5 to 20 mM. The DNA scaffold and staples are added to the buffer solution with the required concentration of Mg2+ ions in a ratio of 1:10 or 1:20 (to reduce the number of nonspecific aggregates generated) and exposed to the procedure of a specific annealing. During the annealing procedure, the mixture is first heated briefly almost to the boiling temperature, and then slowly cooled, resulting in the spontaneous self-assembly of the DNA origami structure. The cooling process duration depends on the complexity of DNA origami structure and ranges from several hours for small 2D structures to a week for multi-layered 3D structures. Creating complex hierarchical DNA origami structures (super-origami) requires assembly in multiple stages. The next step (purification and concentration of the resulting DNA origami structure) is critical for the optical/biomedical applications. The quality and purity of the assembled DNA origami construction is evaluated using the agarose gel electrophoresis, as the gel migration rates of “correct” and “by-product” assembly products differs. Purification of DNA origami is usually done using agarose gel electrophoresis followed by extraction of the “correct” product from a cut gel piece, ultrafiltration, polyethylene glycol (PEG) precipitation, ultracentrifugation or gel filtration (size exclusion chromatography). The choice of purification method depends on the ultimate use of the DNA origami structure.
3. DNA origami characterization methods
The study methods for the synthesized DNA origami structures can be divided into two groups: ensemble methods and methods for studying at the level of single molecules. Ensemble methods (gel electrophoresis, fluorescence spectroscopy, circular dichroism spectroscopy) allow for creating a general picture, averaged over a large number of assembled structures: whether the planned DNA origami structure was assembled, how many side products were concomitant.
Second (electron and atomic force microscopy) are techniques suitable for single molecule analysis [26, 27]. In combination with millisecond time resolution, the atomic force microscopes can register fast dynamic processes in the single DNA origami structures, and the use of modified probe tips allows studying the elasticity of DNA origami [28]. High-resolution electron microscopy makes it capable of registering individual particles attached to opposite sides of a single DNA nucleotide, with a distance of only 0.34 nm between them [29]. Such high resolution allows studying topological changes in the single structures, but it leads to rapid destruction of the DNA structure due to the need for sample preparation in a vacuum. To overcome this difficulty, more expensive methods of cryogenic electron microscopy are used [30].
Each of the given methods has its advantages and limitations. Combining different methods can provide the best way to achieve a complete understanding of DNA origami structural properties.
Conclusion
DNA origami method allows for controllably shaping a DNA molecule at a natural scale for this molecule (nanometer-scale), just like the Japanese art of origami allows shaping a piece of paper at a corresponding scale. The difference lies in the fact that for manipulating molecules, special chemical and physical conditions are organized in which the DNA origami assembles itself. The process requires careful preparation, but its results (including those in the field of photonics) can justify all efforts, as they provide an opportunity not only to control the shape of DNA molecules but also to place nanoobjects with nanometer precision at any position along this newly acquired shape. Furthermore, the DNA origami method is relatively young and its capabilities are far from being exhausted.
Acknowledgments
The work was carried out with financial support from the Ministry of Education of the Russian Federation within the framework of the state assignment of the Moscow Pedagogical State University “Physics of nanostructured materials and highly sensitive sensors: synthesis, fundamental research and applications in photonics, life sciences, quantum and nanotechnologies”.
AUTHORS
Stepanov Maxim Evgenievich – junior researcher, Moscow Pedagogical State University (MPGU), Shpolskiy Department of Theoretical Physics, junior researcher at the Laboratory of Physics of Advanced Materials and Nanostructures, Moscow, Russia.
RSCI ID: 334465
Scopus ID: 57195265809
ResearcherID: AAB‑6181-2022
ORCID: 0000-0002-0332-1235
Khokhryakova Uliana Aleksandrovna – Bachelor in fundamental physics of Moscow Pedagogical State University, research assistant at the Youth Laboratory of Biophotonics and Nanoengineering, e-mail: ua_khokhryakova@mpgu.su.
Egorova Tatiana Vladimirovna – Ph.D. in biological sciences, head of the Youth Laboratory of Biophotonics and Nanoengineering at Moscow Pedagogical State University, Moscow, Russia.
Scopus ID: 56868341400
ORCID: 0000-0002-7554-5246
ResearcherID: P‑9982-2017
Magaryan Konstantin Arutyunovich – Ph.D. in physical and mathematical sciences, Moscow Pedagogical State University (MPGU), Shpolskiy Department of Theoretical Physics, senior researcher at the Laboratory of Physics of Advanced Materials and Nanostructures.
RSCI ID: 723988
ResearcherID: A‑4208-2014
ORCID: 0000-0003-4754-4657
Naumov Andrey Vitalievich – corresponding member of the Russian Academy of Sciences, doctor of physical and mathematical sciences, head of the Troitsk branch of the Lebedev Physical Institute, head of the department of Moscow Pedagogical State University (MPGU), corresponding member of the Russian Academy of Sciences, associate professor, Moscow, Russia.
RSCI ID: 35867
Scopus ID: 7201349036
ResearcherID: E‑8905-2010
ORCID: 0000-0001-7938-9802.
CONFLICT OF INTERESTS
The authors declare that they have no conflict of interests. All authors took part in preparation of the article and supplemented the manuscript in terms of their scope of work.
CONTRIBUTION
OF THE COMPOSITE AUTHORS
The article has been prepared based on the work of all composite authors.
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