Nucleic Acid Engineering: RNA Following the Trail of DNA

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Nucleic Acid Engineering: RNA Following the Trail of DNA Hyejin KIm, Yongkuk Park, Jieun Kim, Jaepil Jeong, Sangwoo Han, Jae Sung Lee, and Jong Bum Lee ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.5b00108 • Publication Date (Web): 06 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016

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ACS Combinatorial Science

Nucleic Acid Engineering: RNA Following the Trail of DNA Hyejin Kim, Yongkuk Park, Jieun Kim, Jaepil Jeong, Sangwoo Han, Jae Sung Lee and Jong Bum Lee* Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemungu, Seoul 130-743, Korea KEYWORDS Structural DNA nanotechnology; RNA nanotechnology; materials science; bioengineering.

ABSTRACT

The self-assembly feature of the naturally occurring biopolymer, DNA, has fascinated researchers in the fields of materials science and bioengineering. With the improved understanding of the chemical and structural nature of DNA, DNA-based constructs have been designed and fabricated from two-dimensional arbitrary shapes to reconfigurable threedimensional nanodevices. Although DNA has been used successfully as a building block in a finely organized and controlled manner, its applications need to be explored. Hence, with the myriad of biological functions, RNA has recently attracted considerable attention to further the application of nucleic acid-based structures. This review categorizes different approaches of engineering nucleic acid-based structures, and introduces the concepts, principles and

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applications of each technique, focusing on how DNA engineering is applied as a guide to RNA engineering.

INTRODUCTION

DNA has long been appreciated for its vital role in the storing and carrying of information. Since the discovery of the molecular structure of DNA in the 1950s,1 DNA has become a versatile tool for materials engineering and has been studied extensively beyond the scope of genetics with the programmability enabled by the simple and powerful Watson-Crick basepairing. The reversibility of base-pairing provides another advantage that enables structural transformations and the organization of nano-sized structures.2 Furthermore, each nucleotide monomer can be modified easily from a chemical view, and nucleic acid chains can be elongated easily by enzymatic replication. These features have allowed the field of DNA nanotechnology to progress rapidly. In its early stages, the development of DNA structures focused mainly on two-dimensional (2-D) structures, such as crystal lattices3 and polygons.4 The fabrication of 2-D structures involved mainly simple hybridization,5 crossover6 and origami7 techniques. Since then, highly complex structures have become accessible with new techniques for design and construction being reported at a rapid pace. In only few decades, nucleic acid engineering has opened the way to the construction of three-dimensional (3-D) structures, such as nanotubes,8 polyhedrons9 and arbitrary shapes.10 Furthermore, DNA engineering techniques have been combined synergistically with other methods, such as enzymatic replication11 or ligation12. In


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addition, other materials, such as metal nanoparticles,13 proteins14 and chemical moieties,15 have been incorporated to provide extra functionality. Although ribonucleic acid (RNA) shares many aspects of DNA, the manipulation of RNA had been hampered by its structural complexity and instability arising from the extra hydroxyl group attached to the pentose ring in the 2’ position.16 RNA is more prone to the formation of intrastrand double helixes and diverse tertiary and quaternary structures than DNA. On the other hand, a myriad of biological functions of RNA have attracted the attention of researchers. In addition to its role as an intermediate in the informational pathway, RNA has the capacity to perform a wide range of non-coding functions, such as those performed by transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), clustered regularly interspaced short palindromic repeat (CRISPR) RNA, small interfering RNA (siRNA), microRNA (miRNA), and catalytically-active RNA (ribozyme).17 Inspired by the diverse functions of RNA, considerable efforts have been made to develop RNA nanotechnology, taking advantage of the insights gained from the development of advanced DNA architectures. On the other hand, RNA engineering still needs to be improved for practical applications because of its limitations, such as structural instability and relatively high cost.18 This review focuses on various technological approaches developed for engineering DNA, and how these technologies can be applied to RNA engineering, including practical applications.

1. Simple hybridization for nucleic acid-based structures Similar to the naturally occurring Holliday junction, which is a four-way junction that plays a key role as an intermediate in almost all recombination processes,19 artificial multi-arm DNA



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was fabricated as a building block.5 As shown in Figure 1a, the DNA building block was designed to bear a single-stranded overhang, called the ‘sticky end’ on its each arm. The DNA nanostructures were then constructed with the dangling sticky ends, which act as a glue for binding building blocks. The development of a stable DNA junction has potential use as building blocks to build-up complicated DNA structures. To construct a DNA network structure, oligomeric DNA sequences were designed rationally with a reliable prediction of the arrangement by the basepairing rule, and then assembled precisely as predicted. After the first demonstration of a fourway junction, multi-way junctions with a number of arms ranging from three to twelve have also become feasible.20 Each can serve an individual purpose. For example, Seeman’s group reported that three-arms can be used for a stick cube, four-arms for a stick octahedron, fivearms for a stick icosahedron, and six-arms for a simple cubic stick lattice.21 A good example of the utilization of multi-arm DNA is the multifunctional nanoarchitectures built up with ABC monomers.22 As shown in Figure 1b, Y-shaped DNA (Y-DNA; three-way junction) and X-shaped DNA (X-DNA; four-way junction) were used, and each arm was assigned a unique sequence to achieve anisotropy. These crosslinkable moieties have been used successfully to demonstrate target-driven polymerization, leading to highly sensitive pathogen detection. Furthermore, it could deliver both drugs and tracers simultaneously. Through the formation of a ligand-metal complex at the junction point, metal-locked DNA three-way junctions were also developed as a potential component of metal-triggered DNA nanomachines.23 While the branched DNA approach has gradually manifested its potential, attempts to manipulate RNA have followed this track. In particular, after the discovery of RNA interference (RNAi), which has a highly effective gene silencing effect in 1998,24 the demand


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for the synthesis of stable double-stranded RNA (dsRNA) structures has increased. One of the nanostructures for inducing a RNAi effect is dumbbell-shaped nanocircular RNAs.25 The double-helical stem acts as a substrate for the Dicer enzyme. The ends consist of two hairpin loops, and the nick was closed by T4 RNA ligase. As a result, dumbbell-shaped RNA could withstand enzymatic degradation, resulting in extended RNAi activity.25a Recently, branched RNA, called trimer RNA or tetramer RNA, was also adapted to trigger the RNAi effect (Figure 1c).26 Each arm acted as a substrate for the Dicer enzyme to generate siRNA for downregulation of the target gene. By benefitting from the sterically crowded nature of branched RNA, the prolonged release of siRNA or protection against nucleases was achievable. Dendrimeric siRNA was developed by the programmable molecular self-assembly of chimeric DNA/siRNA oligomers.27 As shown in Figure 1d, three individual single-stranded (ss-) DNA/siRNA chimeras produced ss-Y-siRNA (ss-Ya-siRNA). In this state, Y-DNA forms the core, leaving a sticky overhang bearing the antisense strand of siRNA in each arm. Nanostructures were generated by adding ss-Yb-siRNA bearing the sense strand of siRNA. This demonstration came out one decade after the first demonstration of the dendrimeric DNA assembly.28 Following the successful synthesis of multi-arm junction DNA structures, the first synthetic 3-D DNA nanostructures came into reality in the shape of a cube (Figure 2a).29 Each side was covered with a single-stranded cyclic molecule and catenated to four neighboring strands, with each vertex connected by an edge to three others. A few years later, a DNA-truncated octahedron was constructed, whose edges were double helices.9e These studies are expected to pave the way for the construction of more complex structures in the future. As synthetic processes are being improved continuously, the yield of DNA nanofabrication has also been improved greatly; from 1% at its advent to ~95% after more than a decade.30 In



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particular, the DNA tetrahedron, which is the simplest possible polyhedron, has been studied extensively. The DNA tetrahedron introduced by Turberfield’s group has a braced geometry, limiting the range of configurations it can adopt.9b In addition, the well-defined internal space of the polyhedral structure is suitable for hosting functional moieties. This way of nanofabrication has been adapted widely, including stable fluorescent DNA nanotag31 and electrochemical biosensing probe.32 Moreover, this DNA nanostructure was internalized successfully by mammalian cells without the aid of a transfection reagent, highlighting its potential as a drug delivery carrier33 and in vivo imaging agent34 owing to its small size (maximum dimension ~7 nm).35 Jaeger’s group reported a groundbreaking strategy for the engineering of programmable RNA nanocube scaffolds in 2010.36 First, sequence optimization was carried out for a rational design of the scaffolds using the advanced computational method of 3-D modeling.37 The computer-designed RNA sequences self-assemble into cubic-shaped scaffolds with or without the dangling ends at the middle of each side to bind to different moieties (Figure 2b). Prefolded RNA is not needed to assemble the RNA nanostructure, and it requires relatively short RNA sequences (28-54 nucleotides), which allows facile chemical synthesis and selective point modifications. More recently, this study was extended to maximize the versatility of the programmable RNA nanocube.38 As shown in Figure 2c, three different nanocubes were synthesized: i) RNA-RNA nanocubes, ii) the previously introduced RNA nanocubes decorated with RNA-DNA hybrids, and iii) DNA nanocubes decorated with dsRNAs to be processed by Dicer. Each nucleic acid nanocube scaffold served multiple functions, such as the activation of RNAi, type I IFNs and pro-inflammatory cytokine with an increased stability in human serum. By exploiting RNA-DNA hybrids, this demonstration also achieved the conditional activation of RNAi.


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Recently, structurally well-defined RNA tiles were developed by integrating a 90° bend of RNA and the artificially designed T-junction (Figure 2d).39 The RNA tiles have sufficient rigidity to homo-oligomerize into a large and uniform RNA architecture, an octameric cube. This achievement suggests that RNA nanotechnology took a step further to overcome the challenge in programmed RNA self-assembly and accelerated the practical applications of the RNA nanostructures.

2. Crossover techniques for the self-assembly of nucleic acids structures Although the branched DNA-based strategy is the simplest method, there are some drawbacks, such as the limited achievable rigidity owing to the inherent flexibility of the junction region.40 To overcome this hurdle, Seeman’s group introduced paired DNA junctions called ‘double-crossover (DX)’ molecules.6 To achieve more rigid building blocks, DX molecules were designed to contain two crossover sites between the helical domains. DX molecules were later modified with sticky ends, and were self-assembled successfully into DNA 2-D tiles. These 2-D tiles consisted of two or four antiparallel DX units (Figure 3a), with specific patterns on the nanometer scale.41 Following this demonstration, the crossover technique was extended to a triple crossover (TX).42 In TX molecules, four DNA strands form three double helices, providing cavities that can attract the guest macromolecules. Crossover-based structures have been studied continuously: Pseudohexagonal 2-D DNA crystals4a or double-double crossover (DDX)43 techniques have recently been developed. Unlike the previously introduced crossover DNA tiles with twofold symmetry, the DX triangle motif provides rigid trigonal arrays for the formation of pseudohexagonal 2-D DNA crystals (Figure 3b). More rigid and robust hexagonal 2-D structures could be formed by applying a



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crossover interaction. In addition, DDX molecules could form more densely packed tiles due to one DNA strand going through four double helixes in DDX. Therefore, it is suitable for the compact packing of nanomaterials, such as metal particles or biomolecules. These crossover DNA molecules have been applied for a variety of purposes, such as molecular arrays14a, 44 and templates for wires.45 In accordance with the technological advances of DNA crossover techniques, similar approaches for manufacturing RNA structures have been introduced. One of these approaches for the fabrication of RNA structures was to design the self-assembly of RNA strands with DNA strands. In this manner, a self-assembled RNA-DNA hybrid architecture was constructed with the precise programmability of DNA along with the rich functionality of RNA.46 On the other hand, the assembly of RNA was generally developed under a slightly different perspective due to the secondary interactions in a RNA strand. For this reason, RNA tectonics based on tertiary interactions have been introduced for the self-assembly of RNA. In particular, hairpin-hairpin or hairpin-receptor interactions have been used widely to construct RNA structures. Recently, the constructions of RNA structures have been exploited with tectoRNAs, which were suggested by Jaeger’s group.47 TectoRNAs can be connected to each other through a tetraloop-receptor interaction. Because tertiary interactions exhibit strong binding affinity under cation-rich conditions (e.g., Mg2+),48 RNA tectonics can be a powerful tool for the controlled self-assembly of RNA. Similarly, Harada’s group introduced RNA building blocks containing two hairpin loops using the dimerization initiation site (DIS) of HIV RNA as an extension of the tectoRNA.49 RNA building blocks were connected to each other based on the stable kissing hairpin (loop-loop) interactions. Mg2+ can be used as a switch for RNA assembly because this interaction is also sensitive to the Mg2+ concentration. By adjusting the stem


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sequence, each building block can be assigned a different pair; thus, simple formations, such as circular and linear forms can be assembled. In addition, Jaeger’s group introduced the formation of RNA jigsaw puzzles with tectoRNAs in line with a 2-D DNA crossover lattice.50 Tectosquares are comprised of four non-identical tectoRNAs, and are assembled in the presence of Mg2+. The 3’ sticky tails located at each corner of the tectosquares have also been used as a glue to connect them to form large tiles (Figure 3c). Therefore, a range of RNA nano-patterns could be generated (Figure 3d). Furthermore, 3-D polyhedron particles (Figure 3e)51 and closed-ring structures52 made of tectoRNA units have also been introduced. Despite this, the broad applications of RNA tectonics were hindered by the limited conjugation method. Therefore, the development of a motif was needed for the extensive use of RNA molecules. To resolve this issue, paranemic crossover techniques were introduced utilizing the loop-receptor interactions.53 In this technique, DNA crossover with four strands was replaced with only two RNA strands possessing loops that crossover in the paranemic motif (Figure 3f). The RNA strands are more stable when the strands form paranemic binding than when each strand is folded alone. As the design of the paranemic crossover motif can be changed easily, it can be used as a versatile building block with stability and tunability. TectoRNA molecules also have medical applications. For example, tectoRNA molecules in the shape of nanorings were used to deliver siRNAs for clinical applications (Figure 3g).54 In this study, RNA nanorings were synthesized, based on the kissing-loop interaction. By encoding the precursor siRNA into the RNA nanostructures, siRNA can be delivered more efficiently with a resistance to exonuclease.55 Moreover, the recently introduced thermoresistant RNA nanoparticles have shown promising potential for a range of applications in



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industry as a polymer (Figure 3h).56 This could also be used as a sensing material in a cellular system.

3. Scaffolded origami The traditional crossover method involves complicated interactions among numerous short DNA strands. Therefore, the synthetic process requires multiple steps, and additional purification is essential to increase the yield of the completed structures.41 To overcome these limitations, Rothemund et al. suggested the ‘scaffolded DNA origami’ technique.4b The bottom line involves folding a long single scaffold DNA to assemble complex 2-D structures in the same way as paper-folding by clipping with numerous short staple strands. In a single step, the staple strands bind to the selected parts of the scaffold DNA in a sequence-specific manner, which can produce the desired products in high yield with increased accuracy. Moreover, the arrangement of DNA helices in a stable 3-D lattice became easier using 3-D origami design software, caDNAno (http://cadnano.org/).57 As the DNA origami technique is enhanced greatly with the precise controllability, it has been applied as a platform for chemical reactions58 or 3D arrays of nanoparticles.59 In addition, by designing DNA origami to have an inner vacancy or inter-arm angle, 2-D origami structures were facilitated as a frame for the observation of a reversible conformational change in DNA60 and DNA modification reaction.61 Because the frames are comprised of DNA, a ssDNA can be attached easily to the frame, so that it can facilitate the monitoring of nanoscale changes. Recently, reconfigurable origami sheets were developed by inserting a flexible hinge region, and applied to molecular detection 62 and drug delivery.63


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In addition to the DNA origami technique, the synthesis of RNA structures via scaffold and staple strands have also attracted considerable attention. Although RNA origami techniques are underdeveloped because of their high cost, several RNA origami structures are being developed continually by synthesizing long RNA strands with enzymatic transcription. In the initial attempt, a long ssRNA was produced by transcription and folded into the desired structures using DNA staples (Figure 4a).64 With the guidance of the staples, simple shapes, such as a ribbon, rectangle and triangle, were synthesized successfully with RNA in high yield, and the results were confirmed by atomic force microscopy (AFM, Figure 4b). In addition, RNA template-based square tiles and seven-helix bundled tiles were generated using DNA staple strands (Figure 4c) and confirmed by AFM (Figure 4d).65 With only the substitution of bases, thymine to uracil, DNA-RNA hybrid tiles have a smaller size than DNADNA tiles because of the different height of the base-pair between the A-form helix and Bform helix.66 Through the RNA origami technique, RNA-RNA origami structures were generated using a long RNA as a scaffold strand (Figure 4e) and confirmed by AFM (Figure 4f).67 Similar to the DNA origami structures, RNA origami structures can be produced considering the helical turn of the A-form structures. In contrast to the sensitivity of the DNARNA structures to number of base-parings per turn pitch, RNA-RNA origami structures are insensitive to the over-wound helical pitch.65 Although there have been a few applications of RNA origami up to now, the precisely controlled nano-sized RNA origami structures have great potential for biomedical applications with their resistance to nuclease and precise controllability.

4. Self-assembly of nucleic acid structures with tandem repeated sequences



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Among a myriad of enzymes, polymerase is one of the most promising enzymes for the costeffective production of nucleic acids. DNA and RNA can be synthesized using an in vitro system by mimicking the natural process of nucleic acid replication by polymerases in a cellular system. With incorporation of a mixture of nucleotide triphosphates, polymerase continuously synthesizes single DNA or RNA strands complementary to the template DNA. Unlike the chemical synthesis of nucleic acids, polymerization is carried out in a one-pot process, which can greatly reduce the cost and time for synthesizing nucleic acids. Benefiting from this advantage, DNA polymerase was used mainly for the polymerase chain reaction (PCR), which amplifies the desired DNA strands from template DNA.68 The PCR method has been adapted widely for the detection of target genes because polymerase can amplify a small quantity of nucleic acids.69 Recently, the rolling circle replication (RCR) approach,70 including rolling circle amplification (RCA) and rolling circle transcription (RCT), was used for the isothermal enzymatic production of nucleic acids. Through the following processes, a range of structures bearing multiple copies of functional nucleic acids can be constructed by the RCR approach: First, the template DNA was constructed in the shape of a circle by ligation before the polymerization process. Subsequently, polymerase continuously generates long linear nucleic acids, rotating around this circular DNA. The nucleic acid strands replicated from the circular DNA have repetitive complementary sequences to the template DNA. For example, a bulkscale DNA hydrogel was developed by RCA followed by a multi-primed chain amplification (MCA) process.71 By removing and reintroducing water, the hydrogel could self-recover its original shape. As shown in Figure 5a, the massive replication of template DNA also enabled the synthesis of a DNA three-way junction72 as well as nanotubes73 and nano-flowers.11a


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Another contribution of polymerization was made regarding the synthesis of RNA-based structures to overcome the limitation of manipulating RNA. As mentioned previously, it is generally difficult to exploit RNA for the construction of artificial structures because of the structural instability and high cost of RNA. On the other hand, RNA polymerization enables the construction of complex structures with RNA at relatively low cost.74 For example, long RNA strands replicated by RNA polymerase could be designed rationally to be folded into a RNA tile structure.75 Based on the RNA sequences, the tiles can be connected to each other by tile-tile interactions, resulting in stable tile architectures. Furthermore, self-assembled RNA structures were assigned with specific biological functions, such as RNAi. In particular, RNA strands with predetermined sequences for RNAi were used to synthesize a functional RNA-based structure. According to a previous study, long replicated RNA strands from RCT by T7 RNA polymerase showed similar behavior to traditional synthetic polymers; they condense into sheet-like structures at the critical concentration.11b,

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By exploiting this physical property, a sponge-like spherical structure

consisting of multiple siRNA precursors (RNAi microsponge) was synthesized by RCT (Figure 5b). Furthermore, the self-assembled messenger RNA nanoparticles were introduced recently.77 The mRNA-NPs were generated by RCT from plasmid DNA, and were used to demonstrate cellular gene expression. These studies revealed a new platform for the synthesis of self-assembled functional RNA particles. An advanced technique, complementary rolling circle transcription (cRCT), was also developed. 78 With this approach, two complementary circular DNAs were used to replicate long RNA strands continuously. The resulting two complementary RNA strands bind together by hybridization and self-assemble into a robust and colossal RNA architecture. For example, a robust RNA membrane comprised solely of RNA was created through a two-step process,



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cRCT and evaporation-induced self-assembly (EISA), as shown in Figure 5c.78 A number of RNA strands were produced by cRCT, and the surface evaporation of the reaction solution induced the binding and entangling of the RNA strands due to the increased local concentration of RNA strands in the meniscus of the reaction tube. This is the first demonstration of the synthesis of a macroscopic RNA structure that can be observed readily by the naked eye. In addition, the RNA membrane is stable under high salt conditions or in the presence of serum, suggesting that it can be applied to siRNA release or drug delivery in the human body provided that the size can be optimized. Moreover, size-controllable RNAi nanoparticles for efficient cellular uptake were also developed by manipulating the concentration of RNA polymerase (Figure 5d).79 Although the RNA nanoparticles contained a large amount of potential siRNA molecules, they were synthesized at much lower cost than that of commercially available RNA molecules.

5. Nanofabrication with functionalized nucleic acids To explore further applications of nucleic acid-based structures, many attempts have been made to functionalize the structures with functional moieties. Functionalized nucleic acids exhibit a range of properties, such as amphiphilic properties,80 enhanced nuclease resistance81 and rapid target-specific binding.44a,

58, 82

For example, a new type of DNA junction was

synthesized by conjugating DNA strands with a Y-shaped chemical.83 In addition to simple 2D structures, multiple DNA-branched structures were also generated by manipulating the number of linkers and used for the synthesis of oligonucleotide dendrimers.84 Recently, chemically modified dendrimer materials were also used for functional siRNA delivery.85


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Furthermore, lipid-functionalized DNA was used to construct multifunctional nanocarriers with the intrinsic chemical properties of a lipid.80 For the construction of nanocarriers, three differently modified DNA strands (fluorescence dye-modified DNA, long ssDNA and lipidfunctionalized DNA) were self-assembled into a Y-shaped monomer. With amphiphilic properties from the lipid-DNA, Y-shaped monomers were assembled into a liposome-like structure called DNAsomes (Figure 6a). Chemical modification has also been developed for the exploitation of RNA. Generally, RNA is modified to improve their stability under in vivo conditions and to effectively utilize a range of biological functions of RNA. For example, the 2’ modification of ribonucleotides with 2’-fluoro RNA, 2’-O-methoxyethyl RNA or 2’-O-methyl RNA showed that these modifications exhibit improved thermal stability and nuclease resistance of the RNA oligomer.86 In particular, using the 2’ modification on RNA, Sullenger group reported that 2’modified siRNA could inhibit the expression of a target gene in vivo with increased stability and retention times in human plasma.87 Furthermore, the position-specific 2’-O-methyl modification of RNA could reduce the off-target effect of siRNA, and more effective siRNA delivery could be achieved without side effects.88 The advances in the techniques for modifying ribonucleotides has contributed to the utilization of functional RNA molecules, such as RNA aptamer89 and ribozyme.90 With chemical base modification, the stability of the RNA aptamer can be improved in human serum, and the modified aptamer can maintain its binding affinity to target molecules.91 Moreover, the modified RNA aptamer can be conjugated with other materials via a covalent linkage. Because of the specificity and binding affinity of the aptamer, the chimeric molecules synthesized by conjugating RNA aptamer and other materials have been developed for biosensing92 (Figure 6b) or target-specific therapeutics93 (Figure 6c). Chemical modification has also been applied



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to ribozymes to increase their stability. These ribozymes showed higher stability to nuclease while maintaining their catalytic activities when modified appropriately in the catalytic core.90 In addition, with the improved stability, cleavage of the target gene in the cells could be achieved.90b Among the number of approaches for the modification of nucleobase, sugar and phosphate, the bridged or locked nucleic acids (BNA/LNA) approach has attracted considerable attention. LNA has the unique ribose unit, which is locked through an oxymethylene bridge connecting the 2’-oxygen and 4’-carbon of the ribose, and these analogues form stable oligonucleotide duplexes in synthetic DNA, as well as RNA. With their high stability and enhanced binding affinity, LNA-based nanostructures have been applied widely to therapeutics.94 Based on synthetic chemistry, the field of DNA nanotechnology has been expanded further into the conjugation of nucleic acids with metallic materials. Characteristically, gold nanoparticles (AuNPs) are used widely as a scaffold for the fabrication of DNA-based structures because of their unique advantages, such as the ease of modification, low cytotoxicity, ease of size control, and well-developed surface chemistry. To take advantage of AuNPs, thiol groups are attached to DNA strands, and the functionalized DNA strands are then assembled on AuNPs by forming a self-assembled monolayer (SAM).95 Interestingly, DNAAuNP conjugates have special abilities, such as transfection into cells without the assistance of co-carriers, nuclease resistance and hybridization with complementary nucleic acids.96 In particular, the ability to hybridize with complementary sequences enables the construction delicate nucleic acid-based nanostructures. In addition to DNA-AuNP conjugates, a range of DNA-metal complex conjugates have been demonstrated. With the different chemical properties of metals, such as nickel(II) (NiII) and ruthenium(II) (RuII),97 the assembly of DNA strands could be controlled through a metal-complex junction. These methods are promising


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approaches to the synthesis of building blocks for the precise construction of supra molecular structures. Nucleic acids-metal conjugates have also contributed to the development of RNA technology. For example, thiolated RNA strands were used to synthesize the RNA-AuNP conjugates. By utilizing these RNA-AuNP conjugates, the specific and sensitive detection of target DNA was investigated with RNase.98 As shown in Figure 6d, the target DNA could form a DNA-RNA heteroduplex with fluorescently-labeled RNA strands on the AuNPs. After the formation of the heteroduplex, RNase H was used as a trigger for the diffusion of the fluorescence dye because RNase H specifically degrades the RNA strands within the heteroduplex. As a result, the specific sequences of DNA could be detected sensitively by detecting the change in fluorescence resulting from RNA degradation. Moreover, RNA-metal conjugates have been exploited for siRNA delivery or the fabrication of nanostructures.99

CONCLUSION The field of structural DNA engineering has evolved rapidly, and the immense potential of synthesizing DNA-based architectures in a highly controlled and finely organized manner has been shown. Moreover, a range of techniques with synergistic effects have been developed for the advancement of other fields, such as materials engineering, computer programming, chemistry and physics. Inducing the transformation of nanoarchitectures has also become available, benefitting from the programmability of DNA. Although RNA has molecular similarity to DNA, RNA nanotechnology is not as advanced due mainly to the high cost of synthesis and intrinsic structural complexity. On the other hand, manipulating RNA as a building block for nano- and macro- structures has continuously



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followed the trail DNA engineering, and opened its own route by utilizing specific intermolecular interactions, such as kissing loops and interlocking loops. Moreover, a myriad of biological functions of RNA has prompted researchers to explore RNA engineering even further. Accordingly, elaborate RNA structures have been synthesized successfully by predicting the behavior of RNA molecules and rational design. With the advances in other fields, DNA fabrication tactics are believed to be further developed. Together with the development of DNA technology, RNA has become another option for nucleic acid technology. With its biological functions, it is expected that RNA nanotechnology, with the baton handed over from DNA engineering, will be advanced further.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the Global Innovative Research Center (GiRC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012K1A1A2A01056093) and was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2013R1A2A2A04015829) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1A1A1A05001174).

ABBREVIATIONS 2-D, two-dimensional; 3-D, three-dimensional; AFM, atomic force microscopy; Apt, aptamer; AuNP, gold nanoparticle; BNA, bridged nucleic acids; cRCT, complementary rolling circle transcription; CRISPR, clustered regularly interspaced short palindromic repeat; DIS,



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dimerization initiation site; DDX, double-double crossover; DNA, deoxyribonucleic acid; ds-, double-stranded; DOX, doxorubicin; DX, double-crossover; EISA, evaporation-induced selfassembly; -HB, -helix bundled; LNA, locked nucleic acids; MCA, multi-primed amplification; NiII, nickel(II); PCR, polymerase chain reaction; QD, quantum dot; RCA, rolling circle amplification; RCR, rolling circle replication; RCT, rolling circle transcription; RNA, ribonucleic acid; RNAi, RNA interference; rRNA, ribosomal RNA; RuII, ruthenium(II); siRNA, short-interfering RNA; snRNA, small nuclear RNA; SAM, self-assembled monolayer; SEM, scanning electron microscopy; ss-, single-stranded; tRNA, transfer RNA; TX, triple-crossover; -WJ, -way junction; X-DNA, X-shaped DNA; Y-DNA, Y-shaped DNA.

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nanomaterials with alkyl-chain-substituted amines for tunable siRNA delivery to the liver endothelium in vivo. Angew. Chem., Int. Ed. Engl. 2014, 53, 14397-14401. (86) Corey, D. R. Chemical modification: The key to clinical application of RNA interference? J. Clin. Invest. 2007, 117, 3615-3622. (87) Layzer, J. M.; McCaffrey, A. P.; Tanner, A. K.; Huang, Z.; Kay, M. A.; Sullenger, B. A. In vivo activity of nuclease-resistant siRNAs. RNA 2004, 10, 766-771. (88) Jackson, A. L.; Burchard, J.; Leake, D.; Reynolds, A.; Schelter, J.; Guo, J.; Johnson, J. M.; Lim, L.; Karpilow, J.; Nichols, K., et al. Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing. RNA 2006, 12, 1197-1205. (89) Mayer, G. The chemical biology of aptamers. Angew. Chem., Int. Ed. Engl. 2009, 48, 2672-2689. (90) (a) Beigelman, L.; McSwiggen, J. A.; Draper, K. G.; Gonzalez, C.; Jensen, K.; Karpeisky, A. M.; Modak, A. S.; Matulic-Adamic, J.; DiRenzo, A. B.; Haeberli, P., et al. Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance. J. Biol. Chem. 1995, 270, 25702-25708. (b) Takahashi, M.; Funato, T.; Suzuki, Y.; Fujii, H.; Ishii, K. K.; Kaku, M.; Sasaki, T. Chemically modified ribozyme targeting TNF-alpha mRNA regulates TNF-alpha and IL-6 synthesis in synovial fibroblasts of patients with rheumatoid arthritis. J. Clin. Immunol. 2002, 22, 228-236. (91) Lorger, M.; Engstler, M.; Homann, M.; Goringer, H. U. Targeting the variable surface of African trypanosomes with variant surface glycoprotein-specific, serum-stable RNA aptamers. Eukaryotic Cell 2003, 2, 84-94. (92) (a) Choi, J. H.; Chen, K. H.; Strano, M. S. Aptamer-capped nanocrystal quantum dots: A new method for label-free protein detection. J. Am. Chem. Soc. 2006, 128, 15584-15585. (b) Schoukroun-Barnes, L. R.; Wagan, S.; White, R. J. Enhancing the analytical performance of electrochemical RNA aptamer-based sensors for sensitive detection of aminoglycoside antibiotics. Anal. Chem. 2014, 86, 1131-1137. (93) (a) Bagalkot, V.; Zhang, L.; Levy-Nissenbaum, E.; Jon, S.; Kantoff, P. W.; Langer, R.; Farokhzad, O. C. Quantum dot-aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer. Nano Lett. 2007, 7, 3065-3070. (b) Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad, O. C. Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery. Biomaterials 2007, 28, 869-876. (c) Farokhzad, O. C.; Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.; Richie, J. P.; Langer, R. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 6315-6320. (94) Kokil, G. R.; Veedu, R. N.; Ramm, G. A.; Prins, J. B.; Parekh, H. S. Type 2 diabetes mellitus: Limitations of conventional therapies and intervention with nucleic acid-based therapeutics. Chem. Rev. 2015, 115, 4719-4743. (95) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 1996, 382, 607-609. (96) Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical nucleic acids. J. Am. Chem. Soc. 2012, 134, 1376-1391. (97) (a) Stewart, K. M.; McLaughlin, L. W. Four-arm oligonucleotide Ni(II)-cyclamcentered complexes as precursors for the generation of supramolecular periodic assemblies. J. Am. Chem. Soc. 2004, 126, 2050-2057. (b) Stewart, K. M.; Rojo, J.; McLaughlin, L. W. Ru(II) tris(bipyridyl) complexes with six oligonucleotide arms as precursors for the generation of supramolecular assemblies. Angew. Chem., Int. Ed. Engl. 2004, 43, 5808-5811.


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(98) Kim, J. H.; Estabrook, R. A.; Braun, G.; Lee, B. R.; Reich, N. O. Specific and sensitive detection of nucleic acids and RNases using gold nanoparticle-RNA-fluorescent dye conjugates. Chem. Commun. (Camb) 2007, 4342-4344. (99) (a) Bates, A. D.; Callen, B. P.; Cooper, J. M.; Cosstick, R.; Geary, C.; Glidle, A.; Jaeger, L.; Pearson, J. L.; Proupin-Perez, M.; Xu, C., et al. Construction and characterization of a gold nanoparticle wire assembled using Mg2+-dependent RNA-RNA interactions. Nano Lett. 2006, 6, 445-448. (b) Lee, J. S.; Green, J. J.; Love, K. T.; Sunshine, J.; Langer, R.; Anderson, D. G. Gold, poly(beta-amino ester) nanoparticles for small interfering RNA delivery. Nano Lett. 2009, 9, 2402-2406.



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FIGURES

Figure 1. (a) Formation of a DNA 2-D crystal structure by hybridizing branched DNA molecules. Each arm has a sticky end that can bind another arm. (b) Schematic illustration of the target-driven polymerization of multi-arm DNA nanoarchitecture. (c) Design (top) and AFM images with a schematic diagram (bottom) of multifunctional RNA nanoparticles that


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can trigger the RNAi effect based on 3WJ. (d) Schematic illustration of constructing dendrimeric siRNA self-assembled via programmability of chimeric DNA/siRNA oligomers.

(a) Reprinted from Journal of Theoretical Biology, 99, Nadrian C. Seeman, Nucleic acid junctions and lattices, 237, Ref. [3] Copyright 1982, with permission from Elsevier. (b) Reprinted by permission from Macmillan Publishers Ltd: Nature Nanotechnology Ref. [22], copyright (2009). (c) Reprinted with permission from Ref. [26]. Copyright 2015 American Chemical Society. (d) Reprinted with permission from Ref. [27], Dendrimeric siRNA for efficient gene silencing/ Hong, C. A.; Eltoukhy, A. A.; Lee, H.; Langer, R.; Anderson, D. G.; Nam, Y. S./ Angewandte Chemie International Edition 54. Copyright © 2015 John Wiley and Sons, Inc.



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Figure 2. (a) 3-D diagram of DNA nanocube with each side covered with a single-stranded cyclic DNA. (b) 3-D structures and plane figures of three cubes containing 6-stranded RNA and 10-stranded RNA with or without dangling ends. Each strand is represented by a distinctive color. (c) Three different types of multifunctional nucleic acid nanocubes composed of RNARNA, RNA-DNA, and DNA-RNA. (d) Schematic diagram of the synthesis of RNA homooctameric nanoprism. The rationally designed RNA strands form RNA tiles via T-junction


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cohesion, and the tiles homo-oligomerize into the octameric cube structure (Pa, Pb, Pc; duplex regions, Ta, Tc; tails, Lab, Lbc; internal loops).

(a) Reprinted by permission from Macmillan Publishers Ltd: Nature Ref. [29], copyright (1991). (b) Reprinted by permission from Macmillan Publishers Ltd: Nature Nanotechnology Ref. [36], copyright (2010). (c) Reprinted with permission from Ref. [38]. Copyright 2014 American Chemical Society. (d) Reprinted by permission from Macmillan Publishers Ltd: Nature Communications Ref. [39], copyright (2015).



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Figure 3. (a) Schematic illustrations of DNA double-crossover (DX) structures. DAE (left) and DAO (right) refer to the DX molecules having antiparallel domains with an even number


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or odd number of helical half-turns between branch points, respectively. (b) Schematic illustration of pseudohexagonal 2-D DNA crystals synthesized by combining the DX motifs with the bulged triangle motifs (left), and the AFM image of pesudohexagonal 2-D DNA crystals (right). (c) Schematic illustration of tectosquare assembled from tectoRNA, which contains two interacting sites (top). The front and side views of 3-D model of tectosquare (bottom). Each kissing loop is represented by four colors (red, blue, green, and purple). (d) AFM images of tectosquare patterns with the predicted diagram. Scale bars: 20 nm. (e) 3-D model of the tRNA unit with angle between each arm, particularly the amino acid (aa) arm, anticodon (ac) arm and variable arm indicated (left). Upper view of 3-D model of the octameric tRNA particle (right). (f) Design of RNA paranemic crossover structures that interacted over three double-helical half turns. (g) RNA nanoring synthesized by kissing-loop interaction for the delivery of siRNA. The nanoring can be synthesized with double-stranded siRNAs (top), or nanoring monomers can be joined complementarily with siRNAs at the extended strands at their 3’ ends. (h) Schematic illustration of the construction of hexagons and hexagonal arrays which consist of thermo-resistant RNA triangular nanoscaffold (top). AFM images of RNA triangular nanoscaffold, hexagons and hexagonal arrays (bottom).

(a) Reprinted with permission from Ref. [6]. Copyright 1993 American Chemical Society. (b) Reprinted with permission from Ref. [4a]. Copyright 2004 American Chemical Society. (c-d) From Ref. [50], Chworos, A.; Severcan, I.; Koyfman, A. Y.; Weinkam, P.; Oroudjev, E.; Hansma, H. G.; Jaeger, L. Building programmable jigsaw puzzles with RNA. Science 2004, 306, 2068-2072. Reprinted with permission from AAAS. (e) Reprinted by permission from Macmillan Publishers Ltd: Nature Chemistry Ref. [51], copyright (2010). (f) Reprinted with permission from Ref. [53]. Copyright 2008 American Chemical Society. (g) Reprinted with permission from Ref. [54]. Copyright 2011 American Chemical Society. (h) Reprinted with permission from Ref. [56]. Copyright 2014 American Chemical Society.



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Figure 4. (a) Scheme of fabricating RNA-DNA hybrid origami. The long ssRNA was transcribed from a duplex plasmid (pUC19) and folded into three different RNA-DNA origami structures with the help of DNA staple strands. (b) AFM images of the RNA-DNA hybrid origami structures with the shapes of ribbon (top), rectangle (middle) and triangle (bottom). (c) The RNA-templated origami structures, the seven-helix bundled (7HB) tile and the six-helix bundled (6HB) tube, constructed by combining DNA staples and unmodified or modified RNA transcripts from the template dsDNA. (d) AFM images of 7HB-tiles (top) and 6HB-tubes (bottom). (e) Schematic illustration of the construction of RNA origami structures. The 7HBtiles and 6HB-tubes were fabricated by annealing RNA scaffold with staple RNAs. (f) AFM images of the 7HB-tiles (top) and 6HB-tubes (bottom). Schematic interpretation of the AFM image of the 7HB-tile (top) and the cross-sectional analysis of the AFM image to examine the height and width of the 6HB-tube (bottom).

(a-b) Reproduced from Ref. [64] with permission of The Royal Society of Chemistry. (c-d) Reproduced from Ref. [65] with permission of The Royal Society of Chemistry. (e-f) Reprinted with permission from Ref. [67], Preparation of chemically modified RNA origami nanostructures/Endo, M.; Takeuchi, Y.; Emura, T.; Hidaka, K.; Sugiyama, H./Chemistry-A European Journal 20. Copyright © 2014 John Wiley and Sons, Inc.



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Figure 5. (a) Schematic illustration of the enzymatic synthetic approach to produce threedimensional Y-shaped DNA (Y-DNA) nanostructures. (b) Synthesis of RNAi microsponges consisted of numerous hairpin structures containing siRNA units generated by T7 RNA polymerase. A linear DNA is circularized via ligation and hybridized with the promoter DNA. With T7 RNA polymerase, long RNA strands are produced and self-assembled into RNAi microsponge. (c) Schematic illustration of the fabrication of RNA membrane; cRCT with two complementary circular DNA and T7 RNA polymerase, followed by EISA-mediated condensing of RNA strands. (d) Synthesis of size controllable RNA particles by adjusting enzyme concentration (left), and SEM images of RNA nanoparticles (right).

(a) Reproduced from Ref. [72] with permission of The Royal Society of Chemistry. (b) Reprinted by permission from Macmillan Publishers Ltd: Nature Materials Ref. [11b], copyright (2012). (c) Reprinted by permission from Macmillan Publishers Ltd: Nature Communications Ref. [78], copyright (2014). (d) Reproduced from Ref. [79] with permission of The Royal Society of Chemistry.



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Figure 6. (a) Self-assembly of DNAsomes consisting of DNA-lipid hybrid molecules, which was induced by the amphiphilic property. DNAsomes can be applied to biosensing and drug delivery, simultaneously. (b) RNA aptamer-based sensor with the target-induced changing of the conformation and flexibility. The high signal gain sensor (right) transforms more extensively than the low signal gain sensor (left) upon target binding. (c) Schematic illustration


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outlining the workflow of the doxorubicin (DOX)-loaded quantum dot-aptamer (QD-Apt) conjugates for sensing of drug delivery based on Bi-FRET: DOX intercalated in the aptamer results in quenching of the fluorescence from QD and DOX. After internalization to the target cancer cell, DOX can be released from the QD-Apt(DOX) conjugates, and then the fluorescence from QD and DOX can be recovered. (d) Schematic diagram of the fluorescentlylabeled RNA-gold conjugates for the sensitive detection of target DNA. By examining specific degradation of the target DNA-RNA heteroduplex by RNase H, a sensitive detection of the target DNA is possible.

(a) Reprinted with permission from Ref. [80], DNAsomes: Multifunctional DNA‐based nanocarriers/ Roh, Y. H.; Lee, J. B.; Kiatwuthinon, P.; Hartman, M. R.; Cha, J. J.; Um, S. H.; Muller, D. A.; Luo, D./Small 7. Copyright © 2011 John Wiley and Sons, Inc. (b) Reprinted with permission from Ref. [93b]. Copyright 2014 American Chemical Society. (c) Reprinted with permission from Ref. [94a]. Copyright 2007 American Chemical Society. (d) Reproduced from Ref. [99] with permission of The Royal Society of Chemistry.



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For Table of Contents Use Only

Nucleic Acid Engineering: RNA Following the Trail of DNA Hyejin Kim, Yongkuk Park, Jieun Kim, Jaepil Jeong, Sangwoo Han, Jae Sung Lee and Jong Bum Lee*


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Nucleic Acid Engineering: RNA Following the Trail of DNA

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