TectoRNA: A Blueprint for Programmable RNA Nanostructures

Chapter 1: TectoRNA

The modular RNA units known as tectoRNAs have the ability to self-assemble into bigger nanostructures following a predetermined set of instructions. They are produced by rational design through a method known as RNA architectonics, which makes use of RNA structural modules that have been identified in natural (or sometimes manufactured) RNA molecules in order to spontaneously build pre-defined three-dimensional structures.

An appealing biomolecule for design is RNA because of its capabilities, which include the ability to catalyze reactions and to link bases in a manner that is not conventional. In order to shape RNA into specific geometries and perform a variety of tasks, it is necessary to utilize the knowledge of computational modeling and biological characterisation. As a result, tectoRNA is also capable of carrying functions that allow for the construction of huge functional nanostructures. These nanostructures can be utilized for applications in synthetic biology and nanotechnology.

Nadrian Seeman was the first person to suggest that DNA may be utilized as a material for the purpose of manufacturing nanoscopic structures that are capable of self-assembling. This concept was extended to RNA by Jaeger and collaborators in the year 2000. They did this by utilizing the concept of RNA tectonics, which was previously presented by Jaeger and Westhof and collaborators in the year 1996.

In order to construct a tectoRNA, one must have a comprehensive understanding of the tertiary structure of RNA. The X-ray and nuclear magnetic resonance (NMR) structures that are already known serve as the foundation for the logical design of tectoRNA. It is possible to compare tectoRNAs to words, and by making use of the natural syntax of RNA structural motifs, it is possible to rationally design and synthesis a wide variety of thermodynamically stable structures. The sequence that specifies for stable, recurrent, and modular structural motifs, such as the GNRA tetraloop, kissing loops, kink turns, A-minor interaction, and so on, can be encoded inside tectoRNAs in order to govern the geometry of the nanostructures and their ability to self-assemble. TectoRNA, on the other hand, is capable of including flexible junctions as well as RNA modules (also known as RNA aptamers) that are responsive of ligands.

At this point in time, the construction of sequences of tectoRNAs can be facilitated by the utilization of powerful algorithms and huge databases. With the goal of maximizing their thermodynamic stability and decreasing their free energy, tectoRNAs can be folded in the most efficient manner possible. While the majority of the RNA sequences are transcribed in vitro, the folding state for RNA is also a significant factor to consider. In order to fold RNA in the correct manner, it is necessary to introduce Mg2+ and other ions into the solution, and the concentration must be carefully monitored. A wide variety of biochemical methods are used to characterize the folding and self-assembly capabilities that scientists anticipate they would possess. When determining the Kd of self-assembled tectoRNAs, native poly-acrylamide gel electrophoresis (PAGE) is the diagnostic method of choice. Quantification of the thermodynamic stability of nanostructures can be accomplished by the utilization of temperature gradient gel electrophoresis (TGGE). By using chemical probing techniques, such as DMS probing, we are able to gain an indirect understanding of the structure of RNA folding. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and cryo-EM are all powerful techniques that provide us with a direct indication of the appearance of RNA nanostructures. By a significant margin, fragile structures such as squares or hearts have been effectively proven during the course of various study.

In the field of RNA architecture, the fundamental self-assembling unit is known as tectoRNAs. The length of the sequence of tectoRNA is often less than 200 nucleotides when it comes to RNA architectonics. Since tectoRNAs are primarily derived from single-stranded RNA molecules, once they have been folded, they function similarly to LEGO bricks in that they are used to construct higher order designs. While transcription is taking place under isothermal conditions, they are capable of being synthesised, folded, and self-assembled into multimeric nanostructures simultaneously. In light of this, the approach known as RNA architectonics can be interpreted as RNA modular origami. The synthesis of larger self-assembling units that were more than 400 nanometers in length was accomplished by extending this method. More recently, the concept of RNA origami has been expanded to include the construction of lengthy sequences of single-stranded RNA that are capable of folding into enormous nanostructures that have been pre-defined. Therefore, RNA modular origami, also known as RNA architectonics, RNA origami, and RNA single-stranded origami are all derived from the same notion, which states that RNA sequences can be designed to fold themselves and assemble themselves into predetermined designs. Take note that, from a conceptual standpoint, DNA single-stranded origami is more closely connected to RNA origami than it is to DNA