https://orcid.org/0000-0002-5146-5970
Figures & data
Figure 1. The history of RNA origami. With the first studies on the structural design of ribozymes in the late 1990s, researchers began to understand how to re–use natural structural motifs in synthetic RNA, with the basic constructs published in 2001 by Jaeger et al. [Citation11]. Soon after, larger architectures were developed using “tectoRnas” – RNA with simple designs and interaction motifs based on natural RNA structures – to build arbitrary 2D and 3D shapes. In an early example of applications for synthetic RNA nanostructures, structures designed using pRNA were used to package siRNA therapeutics for antitumor therapy in mice. The complexity of RNA structures increased significantly with the application of fundamental principles from DNA nanotechnology, like multiway junctions and tile assembly, to create larger 3D structures. Nearly 20 years after the field’s beginnings, the first “RNA origami” co-transcriptionally folded from a single RNA molecule was developed by Geary et al. [Citation48]. Following this major leap in its evolution, the field of RNA origami soon had larger structures, with the largest origami thus far created in 2017 by Han et al. [Citation50]. Especially in recent years, great progress has been made in the bioproduction of RNA origami, with the first complex nanostructures produced in bacteria by Li et al. [Citation62] and in eukaryotic cells by Pothoulakis et al. [Citation63]. Not only production methods, but also general functionalization improved enormously, RNA origami were used as structural nanotubes and anticoagulants with clinical potential. Finally, with the introduction of ROAD by Geary et al. [Citation52], the field was revolutionized through the easily applicable design workflow capable of generating kilobase structures which could fold co–transcriptionally. As the complexity of design increased, so to did the complexity of applications. Høiberg et al. [Citation64] demonstrated delivery of multiple siRnas on a single structure and Pothoulakis et al. [Citation63] demonstrated RNA origami which controlled gene circuits in S. cerevisiae. Figures adapted with permission from [Citation9,Citation11,Citation12,Citation28,Citation48,Citation50,Citation52,Citation62–66] Adapted from RNA biology, 1/4, Eric Westhof, Benoit Masquida, Luc Jaeger, RNA tectonics: towards RNA design, R78-R88, Copyright [Citation9], with permission from Elsevier. 05 Adapted with permission from Khaled et al. [Citation20]. Copyright 2019 American Chemical Society. 18a Reprinted under a CC-BY license from M. Li et al. [Citation62]. 18b Adapted with permission. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 19a Adapted with permission. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 19b Adapted with permission from Stewart et al. [Citation65]. Copyright 2019 American Chemical Society. 22 Adapted under a CC by license from Pothoulakis et al. [Citation63].
![Figure 1. The history of RNA origami. With the first studies on the structural design of ribozymes in the late 1990s, researchers began to understand how to re–use natural structural motifs in synthetic RNA, with the basic constructs published in 2001 by Jaeger et al. [Citation11]. Soon after, larger architectures were developed using “tectoRnas” – RNA with simple designs and interaction motifs based on natural RNA structures – to build arbitrary 2D and 3D shapes. In an early example of applications for synthetic RNA nanostructures, structures designed using pRNA were used to package siRNA therapeutics for antitumor therapy in mice. The complexity of RNA structures increased significantly with the application of fundamental principles from DNA nanotechnology, like multiway junctions and tile assembly, to create larger 3D structures. Nearly 20 years after the field’s beginnings, the first “RNA origami” co-transcriptionally folded from a single RNA molecule was developed by Geary et al. [Citation48]. Following this major leap in its evolution, the field of RNA origami soon had larger structures, with the largest origami thus far created in 2017 by Han et al. [Citation50]. Especially in recent years, great progress has been made in the bioproduction of RNA origami, with the first complex nanostructures produced in bacteria by Li et al. [Citation62] and in eukaryotic cells by Pothoulakis et al. [Citation63]. Not only production methods, but also general functionalization improved enormously, RNA origami were used as structural nanotubes and anticoagulants with clinical potential. Finally, with the introduction of ROAD by Geary et al. [Citation52], the field was revolutionized through the easily applicable design workflow capable of generating kilobase structures which could fold co–transcriptionally. As the complexity of design increased, so to did the complexity of applications. Høiberg et al. [Citation64] demonstrated delivery of multiple siRnas on a single structure and Pothoulakis et al. [Citation63] demonstrated RNA origami which controlled gene circuits in S. cerevisiae. Figures adapted with permission from [Citation9,Citation11,Citation12,Citation28,Citation48,Citation50,Citation52,Citation62–66] Adapted from RNA biology, 1/4, Eric Westhof, Benoit Masquida, Luc Jaeger, RNA tectonics: towards RNA design, R78-R88, Copyright [Citation9], with permission from Elsevier. 05 Adapted with permission from Khaled et al. [Citation20]. Copyright 2019 American Chemical Society. 18a Reprinted under a CC-BY license from M. Li et al. [Citation62]. 18b Adapted with permission. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 19a Adapted with permission. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 19b Adapted with permission from Stewart et al. [Citation65]. Copyright 2019 American Chemical Society. 22 Adapted under a CC by license from Pothoulakis et al. [Citation63].](/cms/asset/6fc2754e-5d1a-464b-b57e-46997babdb39/krnb_a_2237719_f0001_oc.jpg)
Figure 2. RNA origami design. A) Early software optimization of RNA ring structures was performed with tools like Nanotiler [Citation76]). B-D) the ROAD software enables the design of co–transcriptional RNA origami. B) ROAD implements several structural RNA motifs. C) the combination of these motifs allows for the design of complex architectures. D) in particular, dovetail seams, in which crossovers are offset by numbers of bases differing from a full A-helical turn enable the generation of 3D structures. A) Reprinted from Journal of Molecular Graphics and Modelling, Vol 27, E. Bindewald et al., Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler, Pages 299–308, Copyright (2008), with permission from Elsevier. B-D) Figures adapted with permission from [Citation52].
![Figure 2. RNA origami design. A) Early software optimization of RNA ring structures was performed with tools like Nanotiler [Citation76]). B-D) the ROAD software enables the design of co–transcriptional RNA origami. B) ROAD implements several structural RNA motifs. C) the combination of these motifs allows for the design of complex architectures. D) in particular, dovetail seams, in which crossovers are offset by numbers of bases differing from a full A-helical turn enable the generation of 3D structures. A) Reprinted from Journal of Molecular Graphics and Modelling, Vol 27, E. Bindewald et al., Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler, Pages 299–308, Copyright (2008), with permission from Elsevier. B-D) Figures adapted with permission from [Citation52].](/cms/asset/cd18d8a6-25be-40e1-b8d8-49d06cdbd953/krnb_a_2237719_f0002_oc.jpg)
Figure 3. Computational modelling of RNA origami with different levels of coarse-graining. A) All-atom force fields specify interaction potentials between each atom in the RNA molecule. B) the general-purpose MARTINI force field coarse-grains each RNA nucleotide into six pseudoatoms. C) HiRE-RNA uses either six or seven coarse-grained beads per nucleotide. D) TIS (three interaction site) places beads at the centers of mass of the sugar, the phosphate and the base site. E) oxRNA uses a single anisotropic bead per nucleotide with empirically–derived interaction potentials between the beads. A) Adapted with permission from Zgarbová et al. [Citation96] Copyright 2013 American Chemical Society. Further reprint permission inquiries should be directed to ACS. B) Reprinted from Biophysical Journal, 113/2, Jaakko J. Uusitalo, Helgi I. Ingólfsson, Siewert J. Marrink, Ignacio Faustino, Martini Coarse-Grained Force Field: Extension to RNA, 246–256, Copyright [Citation97], with permission from Elsevier. C) Adapted with permission from Pasquali and Derreumaux [Citation98] Copyright 2010 American Chemical Society. D) Adapted with permission from Denesyuk and Thirumalai [Citation99] Copyright 2013 American Chemical Society. E) Reprinted from Šulc et al. [Citation100], with the permission of AIP publishing.
![Figure 3. Computational modelling of RNA origami with different levels of coarse-graining. A) All-atom force fields specify interaction potentials between each atom in the RNA molecule. B) the general-purpose MARTINI force field coarse-grains each RNA nucleotide into six pseudoatoms. C) HiRE-RNA uses either six or seven coarse-grained beads per nucleotide. D) TIS (three interaction site) places beads at the centers of mass of the sugar, the phosphate and the base site. E) oxRNA uses a single anisotropic bead per nucleotide with empirically–derived interaction potentials between the beads. A) Adapted with permission from Zgarbová et al. [Citation96] Copyright 2013 American Chemical Society. Further reprint permission inquiries should be directed to ACS. B) Reprinted from Biophysical Journal, 113/2, Jaakko J. Uusitalo, Helgi I. Ingólfsson, Siewert J. Marrink, Ignacio Faustino, Martini Coarse-Grained Force Field: Extension to RNA, 246–256, Copyright [Citation97], with permission from Elsevier. C) Adapted with permission from Pasquali and Derreumaux [Citation98] Copyright 2010 American Chemical Society. D) Adapted with permission from Denesyuk and Thirumalai [Citation99] Copyright 2013 American Chemical Society. E) Reprinted from Šulc et al. [Citation100], with the permission of AIP publishing.](/cms/asset/b77b713d-0cc8-4d78-8c0c-f40ec54f15b3/krnb_a_2237719_f0003_oc.jpg)
Figure 4. Molecular hardware. A) Schematics of an RNA octahedron design (top) and gene knockdown experiment (bottom) showing the high activity of the RNA octahedron in a dual luciferase assay in H1299 cells [Citation64]. B) 3D model of a 2–helix RNA origami (2HF–RNA) with thrombin aptamer binding sites highlighted in yellow (top); schematic from the crystal structure of an exosite 2-binding RNA aptamer bound to thrombin and PTTe assay showing the increased anticoagulant activity upon action of 2HF-RNA displaying four aptamers (2HF-RNA2211) (bottom) [Citation115]. C) Design (left), AFM (top right) and epifluorescence microscopy characterization (bottom right) of micrometer-sized nanotubes without and with toehold [Citation65]. D) design and characterization of RNA filaments showing AFM images of filaments (top right), and its binding either to proteins (bottom left) and, in presence of biotin aptamers, to biotinylated lipid membranes (bottom right) [Citation116] A) Adapted with permission. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. B) Adapted with permission. © 2021 Wiley-VCH GmbH. C) Adapted with permission from Stewart et al. [Citation65]. Copyright 2019 American Chemical Society.
![Figure 4. Molecular hardware. A) Schematics of an RNA octahedron design (top) and gene knockdown experiment (bottom) showing the high activity of the RNA octahedron in a dual luciferase assay in H1299 cells [Citation64]. B) 3D model of a 2–helix RNA origami (2HF–RNA) with thrombin aptamer binding sites highlighted in yellow (top); schematic from the crystal structure of an exosite 2-binding RNA aptamer bound to thrombin and PTTe assay showing the increased anticoagulant activity upon action of 2HF-RNA displaying four aptamers (2HF-RNA2211) (bottom) [Citation115]. C) Design (left), AFM (top right) and epifluorescence microscopy characterization (bottom right) of micrometer-sized nanotubes without and with toehold [Citation65]. D) design and characterization of RNA filaments showing AFM images of filaments (top right), and its binding either to proteins (bottom left) and, in presence of biotin aptamers, to biotinylated lipid membranes (bottom right) [Citation116] A) Adapted with permission. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. B) Adapted with permission. © 2021 Wiley-VCH GmbH. C) Adapted with permission from Stewart et al. [Citation65]. Copyright 2019 American Chemical Society.](/cms/asset/97a9f520-0fa3-4516-a9f9-75a2c8d45830/krnb_a_2237719_f0004_oc.jpg)
Figure 5. Expression and function of RNA origami in cells; A) (top) Synthetic RNA molecules assemble into functional discrete, 1D, and 2D scaffolds in vivo to control the spatial organization of ferredoxin and hydrogenase enzymes for hydrogen production. Single RNA molecules were folded into a duplex with aptamer loops which bound with PP7 and MS2 adaptor domains on the enzymes. The proteins and RNA scaffold were co-expressed in E. coli and the biosynthesis of hydrogen gas was monitored using gas chromatography. (Bottom) Hydrogen biosynthesis as a function of RNA scaffold organization. Hydrogen production increased up to 48–fold when the cells expressed the 2D scaffold [Citation118]. B) Cloning and expression of RNA nanostructures in vivo. An expression vector (orange) carrying a DNA sequence (green) is transformed into E. coli. Upon IPTG induction, the DNA was transcribed into RNAs, which self–assemble into the designed double-square structure inside the cell [Citation62]. C) RNA scaffolds were designed with several aptamers for the produced protein, which acted as a negative feedback self–regulating system. Expression could be recovered by expression of an RNA scaffold with competing aptamer domains [Citation119]. D) Apta–FRET constructs were genetically encoded and transformed E. coli cells. When the RNA origami folded correctly, two fluorescent RNA aptamers were organized in close proximity, as a result, high FRET observed between the two fluorophores, DFHBI-1T and YO3-biotin [Citation120]. E) Synthetic RNA scaffolds expressed in mammalian cells used to control cell-death pathways by regulating the assembly and oligomerization of apoptosis-regulatory proteins [Citation121]. A) from Delebecque et al. [Citation118]. Reprinted with permission from AAAS. B Reprinted under a CC-BY-NC-ND licence from Nguyen et al. [Citation119]. C) Reprinted under a CC-BY license from M. Li et al. [Citation62] D) Reprinted under a CC-BY license from Jepsen et al. [Citation120] E) Reprinted under a CC-BY license from Shibata et al. [Citation121].
![Figure 5. Expression and function of RNA origami in cells; A) (top) Synthetic RNA molecules assemble into functional discrete, 1D, and 2D scaffolds in vivo to control the spatial organization of ferredoxin and hydrogenase enzymes for hydrogen production. Single RNA molecules were folded into a duplex with aptamer loops which bound with PP7 and MS2 adaptor domains on the enzymes. The proteins and RNA scaffold were co-expressed in E. coli and the biosynthesis of hydrogen gas was monitored using gas chromatography. (Bottom) Hydrogen biosynthesis as a function of RNA scaffold organization. Hydrogen production increased up to 48–fold when the cells expressed the 2D scaffold [Citation118]. B) Cloning and expression of RNA nanostructures in vivo. An expression vector (orange) carrying a DNA sequence (green) is transformed into E. coli. Upon IPTG induction, the DNA was transcribed into RNAs, which self–assemble into the designed double-square structure inside the cell [Citation62]. C) RNA scaffolds were designed with several aptamers for the produced protein, which acted as a negative feedback self–regulating system. Expression could be recovered by expression of an RNA scaffold with competing aptamer domains [Citation119]. D) Apta–FRET constructs were genetically encoded and transformed E. coli cells. When the RNA origami folded correctly, two fluorescent RNA aptamers were organized in close proximity, as a result, high FRET observed between the two fluorophores, DFHBI-1T and YO3-biotin [Citation120]. E) Synthetic RNA scaffolds expressed in mammalian cells used to control cell-death pathways by regulating the assembly and oligomerization of apoptosis-regulatory proteins [Citation121]. A) from Delebecque et al. [Citation118]. Reprinted with permission from AAAS. B Reprinted under a CC-BY-NC-ND licence from Nguyen et al. [Citation119]. C) Reprinted under a CC-BY license from M. Li et al. [Citation62] D) Reprinted under a CC-BY license from Jepsen et al. [Citation120] E) Reprinted under a CC-BY license from Shibata et al. [Citation121].](/cms/asset/1ffcf74e-bd17-436c-a463-3c1b8396ec9c/krnb_a_2237719_f0005_oc.jpg)