RNA Origami: Engineering Self Assembling Structures at the Nanoscale

Chapter 1: RNA origami

The nanoscale folding of RNA is known as RNA origami. This folding process enables RNA to transform into specific structures that can be used to organize molecules. Both Aarhus University and the California Institute of Technology contributed to the development of this innovative approach, which was developed by researchers. Enzymes are responsible for the synthesis of RNA origami, which involves folding RNA into certain forms. Folding of the RNA takes place in living cells under conditions that are natural to the environment. RNA origami is a representation of a DNA gene, which, when present in cells, can be transcribed into RNA by RNA polymerase.  There are a great number of computer algorithms that can assist with the folding of RNA; however, none of them can accurately predict the folding of RNA based on a single sequence.

Molecular components that are capable of self-assembling into stable structures are the focus of nucleic acid nanotechnology. These artificial nucleic acids are designed to build molecular components that can be used for a variety of applications, including targeted medication delivery and programmable biomaterials. Through the application of DNA motifs, DNA nanotechnology is able to construct target structures and groupings. Nanorobotics, algorithmic arrays, and sensor applications are just some of the many applications that have made use of this technology.  There is a wide range of potential applications that could be developed using DNA nanotechnology in the future.

Designers have been able to establish RNA nanotechnology as a burgeoning discipline as a result of the success of DNA nanotechnology. The method of RNA nanotechnology combines the straightforward construction and manipulation that is typical of DNA with the added flexibility in structure and diversity in function that is comparable to that of proteins.  The diversity of RNA in terms of both structure and function, as well as its advantageous in vivo properties and bottom-up self-assembly, makes it an attractive candidate for the development of nanoparticle drug delivery and biomaterial structures. RNA cubic scaffold, templated and non-templated assembly, and RNA origami are some of the approaches that have been developed in order to manufacture these RNA nanoparticles.

Aarhus University's Ebbe S. Andersen was the one who published the first work in the field of RNA origami, which was published in Science. For the purpose of designing individual RNA origami, researchers at Aarhus University utilized a variety of 3D models and computer software.  Following the encoding of a synthetic DNA gene, the incorporation of RNA polymerase led to the synthesis of RNA origami. Atomic force microscopy, a technique that enables researchers to examine molecules one thousand times closer than would ordinarily be possible with a standard light microscope, was the primary method that was utilized for the observation of RNA. In spite of the fact that they were able to build honeycomb shapes, they discovered that other shapes are also possible.

One of the most knowledgeable people in the subject of RNA origami, Cody Geary, provided a description of the distinctiveness of the RNA origami approach. According to what he said, the formula for its folding is recorded within the molecule itself, and its sequence is what determines its folding. The sequence is responsible for both the ultimate shape of the RNA origami as well as the motions of the structure which occur while it folds.  As a result of the fact that RNA folds on its own and can therefore quickly become tangled, the most significant difficulty that is linked with RNA origami is present.

The creation of the three-dimensional model, the writing of the two-dimensional structure, and the design of the sequence are the three primary operations that are required for computer-aided design of the RNA origami structure. To begin, a three-dimensional model is built by utilizing tertiary motifs that are currently stored in databases. For the purpose of ensuring that the newly formed structure has a reasonable geometry and strain, this is required. Creating the two-dimensional structure that describes the strand path and base pairs derived from the three-dimensional model is the next step in the process. Sequence constraints are introduced in this two-dimensional blueprint, which results in the creation of primary, secondary, and tertiary motifs. The last stage is to design sequences that are compatible with the structure that has been designed. The application of design algorithms allows for the creation of sequences that are capable of folding into a variety of configurations.

In the RNA origami method, double-crossovers (DX) are utilized to arrange the RNA helices in a manner that is parallel to one another in order to create a building block. This allows the approach to manufacture the required shape. Researchers were able to design a way for manufacturing DX molecules using only one strand of RNA, in contrast to the process of DNA origami, which requires the production of DNA molecules from many strands. The addition of kissing-loop complexes on the interior helices and hairpin motifs on the margins were the means by which this was accomplished.  The formation of a junction that is referred to as the dovetail seam is accomplished by stacking more DNA molecules on top of one another.  This dovetail seam features base pairs that cross between adjacent junctions; as a result, the structural seam that runs along the junction becomes sequence-specific. One of the most essential aspects of these folding interactions is the folding itself; the sequence in which contacts are formed has the potential to generate a situation in which one interaction blocks another, resulting in the formation of friction. These topological problems are not caused by the kissing-loop interactions or the dovetail interactions since they are either a half-turn or shorter than the other interactions available.

The organizing and coordination of significant molecular processes can be accomplished through the utilization of nanostructures of RNA and DNA.  Nevertheless, there are a number of significant distinctions between the two in terms of the core structure and applications associated with each.  The procedure of RNA origami is very different from the DNA origami techniques that were developed by Paul Rothemund, despite the fact that they were the source of inspiration.  RNA origami is a far more recent process than DNA origami; the study of DNA origami has been going on for close to a decade at this point, but the study of RNA origami has just recently started.

RNA origami, on the other hand, is produced by enzymes and then folds into pre-rendered designs. This is in contrast to DNA origami, which entails chemically synthesizing the DNA strands and arranging the strands to form any shape required with the assistance of staple strands.  Due to the presence of a number of secondary structural motifs, such as conserved motifs and short structural elements, RNA is able to fold into a variety of different ways inside complicated structures.  The secondary-structure interaction is a significant factor in determining the topology of RNA. This interaction includes motifs such as pseudoknots and kissing loops, adjacent helices stacking on top of one another, hairpin loops with bulge content, and coaxial stacks. This is mostly due to the presence of four distinct nucleotides, namely adenine (A), cytosine (C), guanine (G), and uracil (U), as well as the capacity to construct base pairs that are not considered to be canonical.

There are other tertiary interactions consisting of RNA that are more sophisticated and have a greater range. DNA is unable to produce these tertiary patterns, and as a result, it is unable to match the functional capacity of RNA in terms of performing jobs that are potentially more diverse. As a result of the placement of metal ions at their active sites, RNA molecules that have been folded appropriately have the potential to function as enzymes. This provides the molecules with a wide range of catalytic capabilities. RNA structures have the ability to be produced within living cells and utilized for the purpose of organizing cellular enzymes into discrete groups. This is due to the fact that RNA structures are related to enzymes.

There is also the fact that the molecular disintegration of DNA origami is difficult to assimilate into the genetic material of an organism.  On the other hand, RNA origami can be written directly as a DNA gene and then transcribed with the help of RNA polymerase.  Consequently, while the production of DNA origami involves the cultivation of cells outside of the cell, the production of RNA origami may be done in large quantities and at a low cost directly within the cell by simply cultivating bacteria. The additional functionality of RNA structure, in conjunction with the fact that it is feasible and cost-effective to manufacture RNA in living cells, provides a promising prospect for the creation of RNA origami.

The notion of RNA origami is relatively new, and it has the potential to be applied in a variety of fields, including nanomedicine and synthetic biology. A new approach was created in order to facilitate the construction of massive RNA nanostructures that can be used to establish defined scaffolds for the purpose of combining RNA-based functions. Due to the fact that RNA origami is still in its infancy, a significant number of its possible applications are still in the process of being discovered. Because of its architecture, it is able to offer a stable foundation that enables RNA components to demonstrate their usefulness. In addition to riboswitches and ribozymes, these structures also consist of interaction sites and aptamers. Aptamer structures enables the binding of tiny molecules, which opens the door to the possibility of the fabrication of RNA-based nanodevices in the future. Further applications of RNA origami include cell identification and binding for diagnostic purposes, among other applications. Additionally, research has been conducted on targeted delivery as well as the passage of the blood-brain barrier. RNA origami could have a number of important applications in the future, one of which is the construction of scaffolds that can be used to arrange other tiny proteins and enable them to collaborate with one another.

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Chapter 2: Native state

According to the principles of biochemistry, the native state of a protein or nucleic acid is the form in which it is folded and/or assembled in the correct manner, and it is both functional and operative. It is possible for a biomolecule to have all four levels of biomolecular structure in its original state. The secondary through quaternary structure is created via weak interactions along the covalently-bonded backbone of the biomolecule. This is in contrast to the denatured state, which is characterized by the disruption of these weak interactions, which ultimately results in the loss of these forms of structure and the retention of only the core structure of the biomolecule involved.

In spite of the fact that all protein molecules start out as straightforward chains of amino acids that are not branched, once they are finished, they take on extremely specialized three-dimensional forms. This ultimate shape, which is additionally referred to as the tertiary structure, is the folded shape that holds the least amount of free energy.  The tertiary, folded structure of a protein is what enables it to carry out the biological function that it was designed to do. In point of fact, the major cause of a number of neurodegenerative disorders, such as those caused by prions and amyloid (i.e., mad cow disease, kuru, and Creutzfeldt–Jakob disease), is the alteration of the form of proteins.

Many enzymes and other non-structural proteins have more than one native state, and they operate or undergo regulation by transitioning between multiple states. This is the case for many enzymes and proteins. On the other hand, the term native state is virtually always used in the single version, and it is often employed to differentiate properly folded proteins from those that are denatured or unfolded. One of the most common terms used to describe the folded shape of a protein is its native conformation or structure. This term is also used in other situations.

Given that many proteins become insoluble upon denaturation, it is frequently possible to differentiate between folded and unfolded proteins based on the water solubilities of the two types of proteins. It is possible to detect the secondary structure of proteins in their original state using spectroscopic methods, such as circular dichroism and nuclear magnetic resonance (NMR). A specified secondary structure will be present in proteins.

Among other things, the distances that are detected by nuclear magnetic resonance (NMR) can be used to differentiate between the natural state of a protein and a molten globule. Within a protein that is folded steadily, amino acids that are far apart in the sequence of the protein may come into contact with one another or lie quite close to one another. However, when they are in a molten globule, their time-averaged distances are likely to be higher than they would be otherwise.

It is essential to acquire knowledge regarding the manufacturing process of native state proteins, as previous attempts to produce proteins from scratch have resulted in the formation of molten globules rather than genuine native state products. In order to achieve success in protein engineering, it is essential to have a solid grasp of the native state.

Base pairing and, to a lesser extent, other interactions such as coaxial stacking are the means by which nucleic acids arrive at their natural state.  The majority of the time, biological DNA is found in the form of long linear double helices that are coupled to proteins in chromatin. On the other hand, biological RNA, such as tRNA, frequently forms complicated native configurations that are comparable to complex folded proteins.  In addition, the artificial nucleic acid structures that are utilized in DNA nanotechnology are designed to have particular native configurations. These configurations involve the assembly of many nucleic acid strands into a single complex.

There are situations in which the natural state of biological DNA is able to carry out its tasks without being influenced by any additional regulatory units.

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Chapter 3: Biomolecular structure

The intricate folded, three-dimensional shape that is generated by a molecule of protein, DNA, or RNA and that is essential to the function of the molecule is referred to as the biomolecular structure.  It is possible to examine the structure of these molecules at any one of a number of different length scales, ranging from the level of individual atoms to the connections among complete protein subunits. It is common practice to characterize this helpful distinction between scales as a deconstruction of molecular structure into four levels: primary, secondary, tertiary, and quaternary. The secondary level is where the scaffold for this multiscale structure of the molecule is formed. At this level, the fundamental structural constituents of the molecule are the different hydrogen bonds that it contains. This results in the formation of a number of distinct domains of protein structure and nucleic acid structure. These domains include secondary-structure elements such as alpha helixes and beta sheets for proteins, and hairpin loops, bulges, and internal loops for nucleic acids.

The concepts of primary, secondary, tertiary, and quaternary structure were initially presented by Kaj Ulrik Linderstrøm-Lang in his Lane Medical Lectures delivered at Stanford University in the year 1951.

A biopolymer's fundamental structure is the precise specification of its atomic composition and the chemical bonds connecting those atoms (including stereochemistry). This structure is also known as the atomic structure.  The main structure of a typical unbranched and uncrosslinked biopolymer (such a molecule of a typical intracellular protein, or of DNA or RNA) is similar to specifying the sequence of its monomeric components, such as amino acids or nucleotides. This is because the biopolymer is not branched and does not have any crosslinks.

It is common practice to describe the primary structure of a protein beginning with the amino N-terminus and ending with the carboxyl C-terminus. On the other hand, the primary structure of a DNA or RNA molecule is referred to as the nucleic acid sequence, which is described beginning with the 5' end and ending with the 3' end.

The nucleic acid sequence is the precise order in which the nucleotides that make up the whole molecule are arranged. A significant number of sequence motifs that are of functional significance are frequently encoded by the main structure. Some examples of such motifs include the C/D and H/ACA boxes that are found in snoRNAs, the LSm binding site that is present in spliceosomal RNAs like U1, U2, U4, U5, U6, U12, and U3, the Shine-Dalgarno sequence, the Kozak consensus sequence, and the RNA polymerase III terminator.

The pattern of hydrogen bonds that are present in a biopolymer is what constitutes the secondary structure of a protein.  These are responsible for determining the broad three-dimensional form of the local segments of the biopolymers; nevertheless, they do not explain the global structure of individual atomic positions in three-dimensional space, which are referred to as tertiary structure.  In a formal sense, secondary structure is characterized by the hydrogen bonds that are present in the biopolymer, as seen in an atomic-resolution structure.  Patterns of hydrogen bonds between backbone amine and carboxyl groups are what define the secondary structure of proteins. Sidechain–mainchain and sidechain–sidechain hydrogen links are unimportant, and the DSSP definition of a hydrogen bond is used to describe these types of relationships.

The formation of hydrogen bonds between nitrogenous bases is what determines the secondary structure of a nucleic acid from a structural standpoint.

When it comes to proteins, on the other hand, the hydrogen bonding is connected with other structural properties, which has led to the development of less formal definitions of secondary structure.  Helices, for instance, are capable of adopting backbone dihedral angles in certain regions of the Ramachandran plot. As a result, a segment of residues that exhibits such dihedral angles is sometimes referred to as a helix, regardless of whether or not it possesses the appropriate hydrogen bonds.  Curvature and torsion are two examples of ideas that are frequently applied in the context of differential geometry of curves, which are among the many other less formal definitions that have been proposed.  In the process of solving a new atomic-resolution structure, structural biologists will occasionally assign the secondary structure of the structure by eye and then record their assignments in the Protein Data Bank (PDB) file that corresponds to the