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May 24, 2011

C 2011 American Chemical Society

A Boost for the Emerging Field ofRNA NanotechnologyReport on the First InternationalConference on RNA NanotechnologyGirish C. Shukla,†,3 Farzin Haque,‡,3 Yitzhak Tor,§ L. Marcus Wilhelmsson,^ Jean-Jacques Toulm�e, )

Herv�e Isambert,z Peixuan Guo,‡ John J. Rossi,# Scott A. Tenenbaum,4,* and Bruce A. Shapiro2,*

†Center for Gene Regulation in Health and Disease, Department of Biological Sciences, Cleveland State University, Cleveland, Ohio 44115, United States,‡Nanobiomedical Center, College of Engineering and Applied Science, and College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267, United States,§Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, California 92093, United States, ^Department of Chemical and BiologicalEngineering/Physical Chemistry, Chalmers University of Technology, Kemiv€agen 10, SE-412 96 G€oteborg, Sweden, )Universit�e Bordeaux Segalen, INSERM U869,B̂atiment 3A 1er�etage, 33076 Bordeaux Cedex, France, zInstitut Curie, Research Division, CNRS UMR 168, 11 rue P. & M. Curie, 75005 Paris, France, #Department ofMolecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California 91010, United States, 4College of Nanoscale Science & Engineering,University at Albany-SUNY, Albany, New York 12203, United States, and 2Center for Cancer Research Nanobiology Program, National Cancer Institute at Frederick,Frederick, Maryland 21702, United States. 3 These authors contributed equally to this work.

The meeting was launched with anintroductory keynote address byPeixuan Guo (University of Cincinnati),

the chair of the organizing committee. Dr. Guointroduced the topic of RNA nanotechnol-ogy, its history, approaches, current status,and future prospects, emphasizing that liv-ing organisms possess a wide variety ofnatural nanomachines, elegantly patternedarrays, and highly ordered structures per-forming diverse biological functions. Thereare numerous intriguing configurations thathave inspired biomimetic stategies. Henoted that macromolecules of DNA, RNA,and proteins have intrinsically defined fea-tures at the nanometer scale and can serveas powerful building blocks for bottom-upfabrication of nanostructures. The rapid ad-vances in DNA nanotechnology havecreated unexpected bridges between ma-terial engineering and synthetic structuralbiology.1�3 Dr. Guo emphasized that thefield of RNA nanotechnology is new andrapidly emerging. Over the last five years,there has been a burst of publications onRNA nanostructures, indicating increasinginterest in RNA nanotechnologies indiverse fields such as microbiology, bio-chemistry, biophysics, chemistry, struc-tural biology, nanomedicine, and cellbiology. RNA-based nanoscaffolds aretherefore expected to have great impactin the near future especially with regard todiagnostics and therapeutics.4

Guo noted that RNA, as a cousin of DNA,has recently emerged as an important nano-

technology platform due to its extraor-

dinary diversity in structure and function.

RNA nanoparticles can be fabricated with a

level of simplicity characteristic of DNA, plus

they possess versatile tertiary structure and

catalytic functions that can mimic some

forms of proteins.5�8 RNA is unique in

* Address correspondence tostenenbaum@albany.edu,shapirbr@mail.nih.gov.

Published online10.1021/nn200989r

ABSTRACT This Nano Focus article highlights recent advances in RNA nanotechnology as presented at

the First International Conference of RNA Nanotechnology and Therapeutics, which took place in Cleveland,

OH, USA (October 23�25, 2010) (http://www.eng.uc.edu/nanomedicine/RNA2010/), chaired by Peixuan

Guo and co-chaired by David Rueda and Scott Tenenbaum. The conference was the first of its kind to bring

together more than 30 invited speakers in the frontier of RNA nanotechnology from France, Sweden, South

Korea, China, and throughout the United States to discuss RNA nanotechnology and its applications. It

provided a platform for researchers from academia, government, and the pharmaceutical industry to share

existing knowledge, vision, technology, and challenges in the field and promoted collaborations among

researchers interested in advancing this emerging scientific discipline. The meeting covered a range of

topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures;

structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction,

andmodeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for

RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics. A special

invited talk on the well-established principles of DNA nanotechnology was arranged to provide models for

RNA nanotechnology. An Administrator from National Institutes of Health (NIH) National Cancer Institute

(NCI) Alliance for Nanotechnology in Cancer discussed the current nanocancer research directions and future

funding opportunities at NCI. As indicated by the feedback received from the invited speakers and the

meeting participants, this meeting was extremely successful, exciting, and informative, covering many

groundbreaking findings, pioneering ideas, and novel discoveries.

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comparison to DNA by virtue of itshigh thermodynamic stability,9,10 theformation of both canonical andnoncanonical base pairings,11�15 thecapability of base stacking,9,10 anddistinctive in vivo attributes.16�23

The remarkable modularity of RNAtertiary motifs can be encoded atthe level of an RNA sequence tospecify complex three-dimensional(3D) architectures exhibiting helices,loops, bulges, stems, hairpins, andpseudoknots. Further, a large varietyof single-stranded loops are suitablefor inter- and intramolecular inter-actions, serving as amounting dove-tail in self-assembly. Taking advan-tage of these unique characteristics,Dr. Guo presented highlights fromhis pioneering work in 1998, whichdemonstrated that RNA dimer, tri-mer, and hexamer nanopaticles canbe fabricated by re-engineering RNAmolecules using themodel of motorpRNA (packaging RNA), a compo-nent that gears the DNA packagingmotor of bacteriophage phi29.22 Heshowed that the pRNA can be usedas a building block or scaffold forconstructing a variety of RNA nano-structures with functional entities asdelivery vehicles or imaging tools.He further described the applicationof RNA nanomotors in various as-pects of cellular and molecular biol-ogy as a tool for potential thera-peutics. He pointed out that thesensitivity of RNA to RNase degrada-tion has previously made manyscientists shy away from RNA nano-technology. The demonstration thatRNA nanoparticles can be producedto drive viral DNA packagingmotorsusing chemically modified RNA sug-gests that it is possible to produceRNase-resistant, biologically active,and stable RNA for applications innanotechnology.24 Dr. Guo also dis-cussed the significant contributionsof Dr. Eric Westhoff, Dr. NeoclesLeontis, and Dr. Luc Jaeger for theirfundamental structural studies ofRNA motifs and important contribu-tions to RNA technology.13,25�28

Lastly, Dr. Guo summarized thekey approaches in RNA nanoparti-cles construction4 starting with the

conception step, whereby the de-sired properties of the nanoparticlesare defined and the global structureof the particle and application areconsidered. A computational ap-proach is then applied to predictthe folding of the building blocksand the consequences of inter-RNAinteractions in the assembly of thefinal RNA quaternary structure. Thebuilding blocks are then generated(in vitro transcription or chemicalsynthesis), and the individual subu-nits are assembled (templated ornontemplated) into quaternary ar-chitectures. The resulting RNA nano-particles are characterized by theatomic force microscope (AFM),electron microscope (EM), gel elec-trophoresis, chromatography, orfluorescence experiments to ensureproper folding that is consistentwith the desired structural and func-tional capabilities. After thorough

assessment, RNA nanoparticles areused for a variety of applica-tions including the treatment anddiagnosis of diseases and the regu-lation of cellular functions. We havearranged the following sections ofthis article to match the six steps ofRNA nanoparticle construction, whichserved as the overarching themes ofthe meeting (Figure 1).

RNA Structures for Nanoparticle Con-struction. RNA typically contains varioussingle-stranded loops that interact toform 3D biologically important RNAstructures via inter- and intramolecularinteractions. Severalnaturallyoccurringdiverse RNA loops and motifs withdefined 3D architectures exist thatcan serve as structural scaffolds inRNA nanotechnology. One such exam-ple is based on the structural featuresof the pRNA of the bacteriophagephi29 DNA packaging motor,22,29

which uses a hexameric RNA ring to

Figure 1. Approaches in RNA nanotechnology. Figure modified with permissionfrom ref 4. Copyright 2010 Nature Publishing Group.

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gear themachine.30,31Dr. Guo's grouphas extensively re-engineered pRNAto form dimers, trimers, tetramers,hexamers (with proteins), and arraysviahand-in-hand or foot-to-foot inter-actions between two interlockingloops.32,33 Thedimer and trimer nano-particles have been used successfullyas polyvalent vehicles to deliver avariety of therapeutic molecules32,33

as well as for constructing RNA arrays(Figure 2).32

In his keynote address, Luc Jaeger(University of California, SantaBarbara) described two comple-mentary approaches for the designand engineering of versatile, pro-grammable, 3D RNA-based nano-scaffolds with precise control overtheir geometry, size, and composi-tion and possibility of further func-tionalization. The first approachtakes advantage of known RNAstructural elements. This approach,called “RNA architectonics”, achievesa remarkable degree of structuralcontrol by using prefolded RNA struc-

tural motifs as building blocks forbottom-up assemblies.34�37 Thisstrategy is based on the rational de-sign of artificial 3D RNA architecturesthat are formed by inverse foldingprocesses. The second approach,based only on canonical Watson�Crick interactions, utilizes relativelyshort (26�48 nt) single-strandedRNAs. This approach employs compu-ter-aided techniques to engineer3D RNA, RNA/DNA, and DNA nano-scaffolds.38 His group, in collaborationwith Bruce Shapiro (National CancerInstitute), developed a range of stra-tegies for obtaining self-assemblingRNA architectures with unprece-dented control and precise position-ingof functional groups in space, suchas3DRNAnanocubic scaffolds,38 tRNApolyhedrons,39 and nanorings.34

Another approach in RNA nano-particle construction is to utilize ex-isting RNA structures with knownfunctions as building blocks. Withinthe cell, there exist small RNAs, suchas riboswitches with regulatory func-

tions.40�43 Herve Isambert's group(Institut Curie�Center de Recherche,France) developed pioneering experi-mental and computational approachesfor the development of small regu-latory circuits based mainly on RNAinteractions.44 In addition, he pre-sented a specific example of a smallbacterial regulatory RNA, DsrA,which self-assembles into long fila-mentous-like structures, as well asmore extended 2D/3D nanostructureswith unique switching properties.45

This important finding suggests novelself-assemblyprinciples todesignRNAnanoparticles with structural switch-ing capabilities. An online RNA foldingserver (http://kinefold.curie.fr) hasmore than 40000 RNA folding simula-tion studies available.

In related work, Li Niu (State Uni-versity of New York, Albany) pre-sented an exciting structural fold-ing property of an RNA aptamersequence (identified by SELEX), whichcan assume two different structureswithdifferent functions. The twostruc-tures assemble during transcriptionand act collaboratively to inhibit theexpression of GluR2, one of the gluta-mate ion channel receptor subunits.46

The results highlight the adaptivenature of native RNA molecules toassume alternative stable structureswith different functions.

Nuclearmagnetic resonance (NMR)studies can elucidate the 3D struc-ture of RNA motifs based onsequence. Recently, the NMR struc-ture of a piece of the pRNA com-ponent (E-loop) was reported.47

Thomas Leeper's group (Universityof Akron) also presented solutionNMR analysis on the Ku motif de-rived from the telomerase RNA com-ponent, which is a highly flexiblescaffold that recruits multiple pro-tein components.48 Such architec-tural motifs can potentially be usedfor designing multifunctional RNAnanoparticles.

Importing from DNA Nanoarchitec-tures. Since DNA and RNA sharesome common structural and che-mical features, a special seminar onthe well-established principles ofDNA nanotechnology (for recent

Figure 2. Role of phi29 DNA packaging RNA (pRNA) in RNA nanotechnology. SixpRNA assemble into a hexameric ring to gear the DNA translocating machine. pRNAmonomers can be re-engineered to fold intowell-defined structures, such as dimers,hexamers, and arrays via hand-in-hand or foot-to-foot interactions between twointerlocking loops. pRNA-based nanoparticles have been successfully constructedwith functional modules and used as polyvalent vehicles to deliver a variety oftherapeutic/detection molecules to cancer- and viral-infected cells. Image courtesyof YinYin Guo. Figure adaptedwithpermission from ref 136. Copyright 2011Elsevier.

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reviews, see refs 1�3) was arrangedto provide viable models for thedeveloping RNA nanotechnologycommunity. DNA self-assembly isprogrammable and complex nano-structures and patterns can be cre-ated with full addressability. DNA-directed self-assembly is an efficientand precise way to organize nano-materials on a molecular level, asdemonstrated elegantly by branchedDNA tiles, tensegrity triangles (rigidstructures in perioidic array form),49

algorithmic self-assembled Sierpinskitriangles (aperiodic arrays of fractalpatterns),50 nanotubes, helix bun-dles,51 polycatenated DNA lad-ders,52 and 3D cubes, polyhedrons,prisms, and buckyballs.3,53

A striking demonstration of theaddressable and programmable pro-perties of DNA is Paul Rothemund'sDNA origami.54 More recently,Yan Liu's group (Arizona StateUniversity) in collaboration withHao Yan's group (Arizona StateUniversity) was able to use DNA tobuild a reconfigurable M€obius strip(topological surface with only oneside and only one boundary) basedon DNA fold-and-cut methodology(DNA kirigami).55 Catenane (inter-locking ring-like structures) was as-sembled efficiently without enzy-matic ligation. In other work, thetwo groups in collaboration withWilliam Shih's group (HarvardUniversity) were able to design andconstructmultilayered, 3DDNAnano-structures (DNA helices packed onsquare-lattice geometries) using thescaffolded-DNA-origami strategyprocess.56 Their goal is to utilizeprogrammable, 3D DNA nano-assemblies to direct the assembly ofmaterials, such as proteins arrays, as atool in proteomics, crystallography,and bioagent screening processes.

Although the folding propertiesof RNA and DNA differ to somedegree, the fundamental principlesin DNA nanotechnology are applic-able to RNA nanotechnology. Theuse of three-way junctions (3WJ)and four-way junctions (4WJ)13,37

to build novel and diverse RNA ar-chitectures is very similar to the

branching approaches in DNA.1,3

Both RNA and DNA polymers canbe developed into bundles32,45,57

and jigsaw puzzles.36,58

RNA Computation and Modeling. Un-like DNA, RNA molecules displaydiverse structures mediated by bothcanonical and noncanonical basepairing and further stabilized by ter-tiary interactions, pseudoknots, kis-sing loops, stem stacking, etc. Theuse of computationalmethodologiescan significantly lessen the time andexpense required to bring the designof RNA-based nanoconstructs to ex-perimental fruition. However, pre-diction of RNA structure or fold-ing for particle assembly remains agreat challenge, due to the unusualfolding properties involving non-canonical interactions. Single basemodifications can result in foldingalterations and functional loss. Cur-rently, using the RNA two-dimen-sional (2D) prediction program byZuker, typically only 70% of the 2Dfolding prediction is accurate basedon experimental data.59,60 Clearly,predicting the RNA 3D and 4D struc-tures is even more elusive. Overthe past decade, several groupshave developed computational al-gorithms to predict the secondaryand tertiary structures of RNA.

Christoph Flamm (UniversitaetWien, Austria) presented theoreticalbackground based on the graph-coloring problem for the design of

RNA switches and an approach thatenables analysis of the kinetic beha-vior of the RNA folding energy land-scapes. The main focus of thepresentation was a computationalapproach for the design of RNAsequences that can fold into two ormore metastable secondary struc-tures that can further be inducedto fold into one of the designedconformations based on an externaltrigger (e.g., temperature).61,62

Computational strategies that per-mit the design of RNA nanoarchitec-tures were detailed by Bruce Shapiro(National Cancer Institute). He dis-cussed novel strategies that predictnucleotide sequences that can self-assemble into3DRNAnanocomplexes(also see http://www-ccrnp.ncifcrf.gov/∼bshapiro/).34,38 Dr. Shapiro pre-sented elements of a computationalRNA nanodesign pipeline,63,64 dis-cussing the RNAJunction Database,19

NanoTiler,65 RNA2D3D algorithms,66

and RNA Dynamics,67,68 which areused to build RNA nanoconstructsthat incorporate individual RNAmo-tifs adhering to defined user speci-fications (Figure 3).69 Several ofthe designed constructs, such asnanocubes and hexagonal nano-rings (Figure 3B,C), have been shownto self-assemble in vitro and havetherapeutic potentials.34,38

A computational coarse-grainedapproach for predicting RNA 3Dstructure was presented by NikolayDokholyan (University of NorthCarolina). His group was able topredict the native folding of largeRNA molecules by combining ex-perimental and computationalapproaches70,71 and also predictedthe folding of small RNAs using dis-crete coarse-grained molecular dy-namics (DMD).72,73

Irina Novokova from NeoclesLeontis's group (Bowling GreenState University) described a com-bined experimental and modelingapproach for the assembly of tecto-RNAs using pairs of GNRA loop/loop-receptor interaction motifs.Closed programmable assembledcomplexes were shown to form di-mers, trimers, and tetramers as well

Unlike DNA, RNA

molecules display

diverse structures

mediated by both

canonical and

noncanonical base

pairing and further

stabilized by tertiary

interactions,

pseudoknots, kissing

loops, stem stacking, etc.

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as open forms. Experimental evi-dence supports the formation ofthese complexes with high resis-tance to nucleases.74 Additionalfunctionalities can be incorporatedin the helical regions, thus renderingthese nanocomplexes as potentialtherapeutic delivery agents.

Rhiju Das (Stanford University)made a case for a novel algorithmfor high-resolution 3D structuremodeling of RNA. He presented afull-atom refinement framework forpredicting and designing noncano-nical RNA motifs that stabilize theoverall conformation via tertiary

interactions.75 He further presentedan alternative RNA modeling meth-od, StepWise Ansatz (SWA), that canidentify noncanonical RNA motifsand has the potential to characte-rize conformation landscapes of thiscomplex biomolecule.

A novel computational methodwasdevelopedbySvetlanaShabalina'sgroup (National Institutes of Health,Bethesda, USA) addressing a signifi-cant challenge in the RNAi field,namely, the prediction of efficientoligonucleotides for RNA interfer-ence. The algorithm can designand validate siRNA and shRNA

associated with high silencing effi-ciency, and targeted specificity canbe achieved by optimizing param-eters such as terminal duplexasymmetry and duplex stability.76

This novel approach considers thepredicted local secondary struc-ture of the target and antisenseRNAs, and it is powerful enough forgenome-wide si/shRNA screening.The program and numerous RNAiexperiments with various siRNAand shRNA constructs are publiclyavailable at ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/siRNA/si_shRNA_selector/.

Figure 3. (A) Representation of part of the computational RNA nanodesign pipeline associated with NanoTiler.63�65 What isdepicted is one result, ofmany,whichproduces a closed-ring structure generatedwith theNanoTiler software thatwas derivedin a totally automated fashion from a combinatorial search of motifs found in the RNAJunction database.19 Sequences thatwere predicted for the shown structure were ultimately shown to be able to experimentally self-assemble into the depictedform. Panel A adapted with permission from ref 65. Copyright 2008 Elsevier. (B) Depiction of 10 stranded RNA cubes that werecomputer-designed with NanoTiler and later shown to be able to self-assemble.38,65 The layouts of the interacting strands areshown on the right. It should also be noted that the 10 stranded cubes, as shown here, were assembled in three forms: withouta malachite green aptamer, with one malachite green aptamer, and with two malachite green aptamers. A properly formedaptamer fluoresces in the presence of a triarylmethane dye. The formation of the aptamers was used to confirm the cubes' self-assembly and designed functionalization. Panel B adapted with permission from ref 38. Copyright 2011 Nature PublishingGroup. (C) Depiction of the computer-designed, and later shown to be able to self-assemble, RNA hexagonal ring.34,68,69 Eachcorner of the ring contains themotif derived from the RNAIi/RNAIIi kissing loop interaction that forms an angle of about 120�,which is conducive to hexagonal ring formation. Sides and dangling ends can be further functionalized. Panel C adapted fromref 69. Copyright 2011 American Chemical Society.

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Finally, protein�RNA interactionsare common in protein synthesis,binding transfer RNA (tRNA), andribosomal RNA (rRNA), as well asbinding messenger RNA (mRNA)and small nuclear RNA (snRNA),whichare involved in RNA modification.Jarek Meller (University of Cincinnati)discussed the potential of sequence-based solvent accessibility predictionfor mapping RNA interaction sites inproteins.

Biophysical and Single-Molecule Ap-proaches in RNA Nanotechnology. RNAas an intermediary molecule in thecomplex cascade of gene expressionmanifests diverse arrays of structuresand participates in numerous tertiaryinteractions. To synthesize function-ally relevant and deliverable RNAnanoparticles, an understanding ofhow RNA structures are formed andstabilized via higher order second-ary structure and tertiary interac-tions is of paramount importance.

Single-molecule imaging of RNAmolecules is a powerful method thatcan significantly contribute to betterunderstanding of RNA structures,their folding properties, and asso-ciated interactions within the qua-ternary complexes. Peixuan Guo(University of Cincinnati) demon-strated the utility of single-moleculefluorescence resonance energytransfer (smFRET) in understandingpRNA-based nanostructures. His

groupwas able to predict conforma-tional changes of pRNA upon bind-ing to the phage procapsid.77 Further-more, the application of single-molecule high-resolution imagingwith photobleaching (SHRImp)78 indistancemeasurementsof twopRNAsboundtoprocapsidwasalsodiscussed.

Elvin Aleman from David Rueda'slab (Wayne State University) discussedthe use of 2-aminopurine (2AP), afluorescent nucleotide, to study localconformational changes using the sin-gle-molecule approach. They devel-oped a click-chemistry-based surfaceimmobilization approach79 to study2AP fluorescence dynamics when it isincorporated in a ssDNA and a dsDNA.This method can also be applied toRNA.

Advances in single-particle track-ing will serve as a promising tool inDNA and RNA nanotechnology. NilsWalter'sgroup (UniversityofMichigan),in collaboration with Eric Winfree's(Caltech), Hao Yan's (Arizona StateUniversity), and Milan Stojanovic's(Columbia University) laboratories,developed experimental approachesto study the molecular motion ofnucleic-acid-based nanoscale mole-cular assemblies, called “spiders”,using real-time single-particle fluo-rescence microscopy.80,81 Theywere able to observe directionalmotion of the molecular robotalong a 2D DNA origami substrate(Figure 4).

Mass spectrometry (MS)-basedapproaches are an elegant way tocharacterize oligonucleotides, RNA-mediated interactions, and effectsof common ligands in these inter-actions. Patrick Limbach's group(University of Cincinnati) is applyinghigh-resolution, high-mass-accu-racy MS to characterize oligonucleo-tides and RNA-based nanoparticlebuilding blocks as a means for diag-nostic applications and as a drugdevelopment platform.82,83 DanieleFabris's group (State University ofNew York, Albany) is also apply-ing MS-based approaches to de-cipher RNA structures and investi-gate tertiary/quaternary interac-tions and modulatory effects of

small ligands as potential thera-peutic targets.84

Scott Tenenbaum's group (StateUniversity of New York, Albany) ispioneering an RNA-based nano-switch technology, called “structu-rally interacting RNAs” (sxRNA) forreal-time detection of specific micro-RNAs (miRNA) in cells; this technol-ogy has tremendous potential to bedeveloped as a method in diagnos-tics and therapeutics.

Meni Wanunu in Marija Drndic'sgroup (University of Pennsylvania),in collaboration with New EnglandBiolabs, presented an electronic-de-tection platform using syntheticnanopores for detecting and quanti-fying miRNA enriched from biologi-cal tissue.85 This single-moleculeapproach provides an attractivelow-cost alternative and, combinedwith ultralow sample volumes (nL),has the potential to exceed the de-tection limits of conventional meth-ods of microarray-based miRNAprofiling, fluorescence, and radioac-tive gel electrophoresis assays.

Nicolas Spinelli (Universit�e JosephFourier, France) presented an ap-proach to direct the assembly ofoligonucleotides into G-quadruplexarchitectures (four-stranded struc-ture of stacked guanine tetrads) usinga peptide scaffold. Surface plasmaresonance (SPR) was then used toevaluate the affinity, selectivity, andbinding mode of various ligandswith the immobilized G-quadruplexcomplexes of various topologies,86

as ameans for screening viable drugtargets.87

Blaine Moore's group (Universityof Oklahoma Health Sciences Center)is working toward determining thecrystal structure of a fragment ofguide RNAs (gRNA) that are involvedin post-transcriptional editing ofmRNA (uridine insertion/deletion),a requirement for the subsequentexpression of several mitochondrialproteins. The results provide valu-able insight on the conformationand dynamics of the U-helix, whichis common among all gRNA/mRNAduplexes and is a valuable drugtarget.88

To synthesize

functionally relevant and

deliverable RNA

nanoparticles, an

understanding of how

RNA structures are

formed and stabilized via

higher order secondary

structure and tertiary

interactions is of

paramount importance.

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RNA Nanoparticle Assembly. Self-as-sembly of RNA building blocks in apredefined manner to form largermultidimensional structures is a pro-minent bottom-up approach andrepresents an important means bywhich biological techniques andbiomacromolecules can be success-fully integrated into nanotechnol-ogy.32,89,90 Within the realm ofself-assembly, there are two mainsubcategories: templated and non-templated assembly. Templated as-

sembly involves the interaction ofRNAs with one another under theinfluence of a specific external force,structure, or spatial constraint. RNAtranscription, hybridization, replica-tion, molding, and phi29 pRNA hex-americ ring formation are within thiscategory. Nontemplated assembly

involves the formation of a largerstructure by individual componentswithout any external influence. Ex-amples include ligation, chemicalconjugation, covalent linkages, loop/loop interactions of RNA such as theHIV kissing loop, and phi29 pRNAdimer or trimer formation.25,32,33,89,90

Natural RNA molecules formunique and intriguingmultimers withspecial functionalities. Examples in-clude retroviral kissing loops thatfacilitate dimerization,91,92 pRNAof the bacteriophage phi29 DNApackaging motor that assemblesinto dimers and hexamers via hand-in-hand interactions between right-and left-interlocking loops,21 andbicoid mRNA of Drosophila embryosthat form dimers via hand-in-arminteractions.93 In addition, numer-ous RNA molecules contain motifs

with fixed structure and tightlyfolded domains. These natural prop-erties have been utilized to assem-ble varieties of RNA nanoparticleswith diverse biological propertiesand wide-ranging functions.

Peixuan Guo's group (Universityof Cincinnati) reported the construc-tion of chemically modified stablepRNA-based nanoparticles thatwere RNase-resistant, while retain-ing their biological activity in pro-capsid binding and gearing thephi29 DNA packaging motor.24 Hefurther demonstrated the scale-upsynthesis of stable RNA nanoparti-cles greater than 100 nt,94 whichovercomes the limitation of cur-rently available commercial synth-esis of RNA oligonulceotides (up to80 nt with low yield). His groupgenerated bipartite chimeric pRNAconstructs that can assemble intothe full-length functional pRNA,while retaining their biological activ-ity and proficiency for incorporatingtherapeutic and diagnostic modules.

RNA aptamers are a class ofoligonucleotides that can recog-nize specific ligands through theformation of binding pockets.95�97

SELEX is typically used to screenfor the aptamers from randomizedRNA pools.95,96,98 Chaoping Chen(Colorado State University) has beendeveloping innovative approachesto tackle Flaviviral infections includ-ing Dengue, West Nile, and YellowFever, for which there are no effec-tive therapeutics currently available.Using SELEX, her group was ableto identify RNA aptamers that werefound to bind with RNA-capping

enzymes of Flaviviruses. Using in vitroand in vivo experiments, she demon-strated the therapeutic potential ofnovel tRNA�aptamer fusions in treat-ment of viral infections. In relatedwork, Hua Shi (State University ofNew York, Albany) discussed bot-tom-up approaches that utilize RNAaptamers to construct protein-likemolecules with predetermined func-tions.99,100

Porphyrin macrocycles have beenstudied for their roles in oxygentransport in blood andphotosynthe-sis. By virtue of porphines' inter-action with light, this class ofmolecules has been exploited inmedicine. Magnus Bergkvist (StateUniversity of New York, Albany) pre-sented work demonstrating theoligonucleotide-mediated packag-ing of photoactive porphyrin intobacteriophage MS2 capsid. Hisgroup has been working on virion-derived nanocontainers that arecapable of delivering nonviral RNA/DNA for therapeutics.

RNA Nanoparticles in Therapeutics.Over the past decade, RNA-basedtherapeutics have been investigatedextensively for treatment and detec-tion of cancer, viral ailments, andgenetic diseases. Several naturaland synthetic RNA molecules arebeing actively pursued, including,but not limited to (1) design andconstruction of antisense and siRNAfor silencing genes with high effi-ciency and specificity; (2) assemblyof multivalent RNA nanoparticleswith receptor-binding aptamers fortargeted delivery; (3) incorporationof ribozymes for intercepting and

Figure 4. Watching the movement of single-molecular spiders at super-resolution (Nils Walter group). A multi-leggedmolecular nanoassembly walks along a prescribed substrate track on a DNA origami (A), where its fluorophore label (F1) istracked at super-resolution relative to a label on the end of the track (F2) by total internal reflection fluorescence (TIRF)microscopy (B).80,81 The resulting trajectory compares well with a scale-drawn schematic of the origami (C). Figure modifiedwith permission from ref 81. Copyright 2010 Elsevier.

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cleaving target RNA substrates; and(4) conjugation of drugs and chemi-cal ligands for therapy and detection.

Ningsheng Shao's group (BeijingInstitute of Basic Medical Sciences,China) is developing a wide range ofSELEX techniques for identifying tu-mor markers as a means for earlydiagnosis and therapy. Subtractivecell SELEX procedure, developed inhis lab in 2003, can select aptamersthat can distinguish differentiatedcells (e.g., carcinoma cells) from un-differentiated epithelial cells.101 Asimilar strategy, using whole-bacter-ium-based SELEX, can select ssDNAaptamers for detection of patho-gens, such as the Staphylococcus

aureus.102 The group has also devel-oped an in situ tissue-slide-basedSELEX method that can select foraptamers specific to all tissue frac-tions (such as extracellular matrix,membrane components, and intra-cellular targets) and can distinguishcancer cells from normal cells inclinical specimens.103

Jean-Jacques Toulm�e (Universit�ede Bordeaux, France) presentedwork on a novel aptamer identifiedvia SELEX that folds into a hairpin

structure and can target viral RNA(HIV and HCV) structural elementsby forming stable kissing com-plexes.104 Such a kissing aptamertargeted to the trans-activating re-sponsive (TAR) element of HIV-1 dis-played both high affinity and ex-quisite specificity (Figure 5). RNAaptamers produced in situ in HeLaP4cells from an expression vector in-hibit TAR-dependent expression of areporter gene. Chemically modifiedaptamers also reduce TAR-depen-dent expression upon transfectionof recipient cells.

RNA interference (RNAi)-basedtherapeutics using siRNA have thepotential to silence disease-causinggenes. However, significant chal-lenges remain, especially with re-gard to in vivo delivery of siRNA.Muthiah Manoharan (Alnylam Phar-maceutical, USA) delineated theprospects of novel lipid-like nano-particles (LNP) mediated in vivo de-livery of RNAi therapeutics.105 Healso discussed the potential of che-mically modified RNA and varioussiRNA-based therapeutics currentlyin clinical trials or under develop-ment. Likewise, Weikang Tao (Merck

Research Laboratories, USA) pre-sented current work on in vivo siRNAtherapeutics, their potential toxici-ties, and therapeutic efficacies. Heprovided guidance on how to opti-mize the development of LNP-for-mulated siRNA for therapeutics andways to minimize immuno-stimula-tory activities for improving theirsafety profiles.106

Thomas Hermann's (University ofCalifornia, San Diego) group hascharacterized a new RNA drug tar-get in the hepatitis C virus's internalribosome entry site (IRES) and estab-lished a conformational mechanismof action for a small molecule inhi-bitor that binds to this RNA.107

He was able to obtain the crystalstructure at 2.2 Å resolution of asquare-shaped, double-stranded RNAconstruct derived from HCV IRESdomain. He also presented a novelsupramolecular approach for cellu-lar delivery of siRNAs using a spe-cially designed β-cyclodextrin peptideconjugate, which acts as a ligand forcomplex formationwith theRNA (RGOBiosciences LLC).

Use of multimerized siRNA con-jugate complexes for cell delivery

Figure 5. Complex between the trans-activating responsive RNA element TAR, viral (Tat), and cell proteins (cyclin T1, CDK-9) isrequired for efficient transcription of the HIV-1 genome. Disruption of this complex leads to abortive transcription of theretroviral DNA. A high-affinity RNA hairpin aptamer (R06) specifically recognizes TAR. The transcription of a R06 construct inrecipient cells reduces the production of β-galactosidase whose expression is driven by the HIV-1 promoter containing TAR, inresponse to retroviral infection. Therefore, targeting RNA hairpins offers an alternative to targeting proteins for the design ofartificial modulators of gene expression.

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and gene silencing was presentedby Tae Gwan Park (Korea AdvancedInstitute of Science and Technology,South Korea). He reviewed the use ofcharged cationic polymers, lipids,and peptides for intracellular deliv-ery of siRNAs. He also presentedwork from his laboratory focusedon the development and deliveryof self-cross-linked and multimer-ized siRNAs via cleavable chemicallinkages. Efficacy of these multimer-ized siRNAs in gene silencing with-out invoking immune induction waspresented.108

Dong-Ki Lee's group (SungkyunkwanUniversity, South Korea) presentedpro-misingnewresultsonthedevelopmentof a branched RNA duplex structurethat has the potential to target and tosilence multiple genes implicated incancer cell proliferation and survival.109

Henry Li (Kylin Therapeutics,USA) presented compelling evi-dence on the therapeutic prospectsof pRNA-based RNA nanoparticles.pRNA nanoparticles have a definedstructure (modular design that en-ables multiple functionalities), arethermodynamically and metaboli-cally stable, have an optimum dia-meter of 10�50 nm, and displaysuperior in vivo pharmacologicalprofiles.110 Malak Kotb's group(University of Cincinnati) is usingpRNA as a scaffold for constructingchimeric pRNA-siRNA-CD4 aptamernanoparticles to block the growth oflymphocytic leukemia and lympho-ma cells without affecting the survi-val of normal lymphocytes.

John Rossi (Beckman ResearchInstitute, USA) presented an elegantstudy in which three endogenoustargets were silenced by siRNA cock-tails in both in vitro cell cultures andin vivo animal models. This ap-proach utilized a combination ofsiRNAs targeting expression of hostand viral genes. Dr. Rossi's researchdemonstrates that siRNAs are ableto inhibit HIV replication in a huma-nized HSC-CD34 mouse model andprevent HIV-mediated T-cell deple-tion characteristic of AIDS.111,112 MeiZhang (Case Western Reserve Uni-versity, USA) also presented the

efficacy of siRNA-mediated inhibitionof mammary tumor growth andmetastasis in a 4T1 mouse model.

Numerous proteins are associatedwith innate immunity (initial immuneresponse to pathogens), includingRNA-activated protein kinase (PKR).These classes of proteins have theability to discriminate self-RNA fromforeign, pathogenic RNA via patho-gen-associatedmolecular patterns.113

Philip Bevilacqua's group (Pennsyl-vania State University) is developingmRNA-based therapeutics by design-ing nucleoside modifications for reg-ulating the activity of PKR.114

RNA Chemistry for Nanoparticle Synthe-sis, Conjugation, and Labeling. For thefield of RNA nanotechnology andtherapeutics, it is vital to developefficient methods for (1) preparinglarge amounts of pure RNA oligonu-cleotide sequences and (2) incorpor-ating a large diversity of conjugatesand labels for functionalizing andprobing the RNA construct at specificpositions. Fundamentally, RNA oligo-nucleotides can be prepared byeither enzymatic transcription reac-tions or automated solid-phasesynthesis. Enzymatic approachescan provide access to relativelylong transcripts in significant quan-tities; however, their outcome isfrequently sequence-dependent.The heterogeneity of the 30-endhas been an issue,115 which can beaddressed by extending the tran-scribed sequence beyond the in-tended end and then cleavingthe RNA at the desired site usingribozymes, DNAzymes, or RNaseH.115�117 Transcription reactionsare also limited in their ability tofurnish labeled or sequence specifi-cally modified RNA transcripts. Tomeet this challenge, GMP or AMPderivatives, which can only be usedfor transcription initiation but notfor chain elongation, have beenused. Fluorescent RNA can also beeasily synthesized in vitro with T7RNA polymerase using a new agenttCTP.118 To generate longer RNA,two short, synthetic RNA fragmentscan be further ligated using RNaseligase II or T4 DNA ligases.116

Standard solid-phase phosphor-amidite chemistry for the synthesisof DNA and RNA oligonucleotideshas advanced significantly in recentyears, particularly when it comes tothe synthesis of relatively long RNAoligonucleotides.119,120 In additionto “classical” approaches relying onthe tert-butyldimethylsiloxy (TBDMS)protecting group for the 20-hydroxyl,which were somewhat limited intheir ability to provide rather longsequences, two “modern” approach-es have been introduced forRNA synthesis: (1) the 50-O-DMT-20-O-[(trisisopropylsilyl)oxy]methyl(20-O-TOM) protecting scheme de-veloped by Pitsch,121 and (2) the 50-O-silyl-20-O-orthoester (20-ACE) pro-tecting group combination, intro-duced by Scaringe and Caruthers.122

These protection schemes facilitatethe synthesis of labeled and mod-ified RNA oligonucleotides for di-verse applications. However, chal-lenges remain when it comes tothe preparation and incorporationof novel nucleosides, linkers, andlabels, as their compatibility withcurrent synthetic and protectionschemes needs to be empiricallydetermined.

Natural RNA is sensitive to RNaseand is especially unstable in serum.The relative instability of RNA haslong hindered its application as aconstruction material. Improve-ments in RNA stability have pro-gressed rapidly, including (1)chemical modification of the base(e.g., 5-Br-Ura and 5-I-Ura); (2) mod-ification of the phosphate linkages(e.g., phosphothioate, boranophos-phate); (3) C20 (e.g., 20-fluorine, 20-O-methyl or 20-amine);123 (4) peptidenucleic acids (PNA), locked nucleicacids (LNA) and derivatives;124,125 (5)30-end-capping;126 and use of cross-linking agents (psoralen, nitrogenmustard derivatives, and transitionmetal compounds).127 Adam Mazur(Girindus America Inc., USA) dis-cussed the commercial availabilityof a wide variety of these chemicallymodified RNAs and their advan-tages in RNA-based therapeutics.He also discussed the challenges in

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manufacturing processes, from op-timizing small-scale sequence-direc-ted synthesis to purification schemesand large-scale production of theoligonucleotides.

In probing and monitoring thestructure and structural changes ofRNA nanoconstructs and therapeu-tics, the use of external and/or inter-nal fluorescence reporter molecules(Figure 6) is frequently central. Fluo-rescent labels are also important fordetecting and determining thestrength of the interaction betweenRNA and, for example, other macro-molecules and ligands. There is con-tinuous development of this groupof reporter molecules and a rapidlygrowing subgroup is the fluorescentnucleic acid base analogues. MarcusWilhelmsson (Chalmers Universityof Technology, Sweden) discussedthe advantage of fluorescent ana-logues of the tricyclic cytosinefamily128,129 in calculating accuratedistances and orientation changesin nucleic acid systems using FRET.130

Since nucleic acids are practicallynonfluorescent in nature, artificialfluorescent base analogues are im-portant for investigating DNA orRNA systems. Fluorescent base ana-logues are morphologically similarto natural nucleobases and have theability to form hydrogen bonds witha natural nucleobase in the comple-mentary strand. Furthermore, theseartificial bases retain the structure of

DNA (or RNA) and can be incorpo-rated internally and therefore havean advantage over covalently at-tached end-labeled dyes (fluorescein,rhodamines, Cy or Alexa dyes).

Yitzhak Tor's group (University ofCalifornia, San Diego) is working onRNA folding and RNA drug binding.He discussed the design, synthesis,and incorporation of novel fluorescentnucleobase analogues and describedthe advantages in developing fluo-rescence-based discovery and detec-tion systems.131�133 For differentclasses of fluorophores, their charac-teristic profiles, and applications, seerecent reviews.134,135

Funding Opportunities in Nanotechnol-ogy for Therapeutics. Sara Hook(National Cancer Institute, USA) pre-sented the tremendous resourcesand infrastructure available throughthe Alliance for Nanotechnology inCancer established by the NationalCancer Institute (NCI) to supportcancer-relevant nanotechnologyendeavors. She described how thealliance supports interdisciplinaryresearch efforts between biologists,clinicians, and physical scientistswith the goal of advancing preven-tion, diagnosis, and treatment efforts.The NCI alliance will continue tosupport collaborations by providinginfrastructure as well as research andtraining grants for establishing a di-verse portfolio of new technologiessupporting a range of applications.

In addition, several centers of cancernanotechnology across the UnitedStates will be established. Detailedinformation regarding the NCI sup-ported Alliance for Nanotechnologyin Cancer and other funding oppor-tunities is available at the NCI Website (http://nano.cancer.gov). A fullyequipped Nanotechnology Char-acterization Laboratory (http://ncl.cancer.gov) is also available forcharacterizing potential therapeuticnanoparticles, such as their physicalattributes (e.g., size, shape, surfacechemistry, and solubility), in vitro bio-logical properties (e.g., pharmacologyand cytotoxicity), and in vivo compat-ibility assays (e.g., efficacy and safety).At present, the challenging areas in-clude molecular imaging and earlydetection (high spatial and temporalresolution, aswell as sensitivity), in vivonanotechnology imaging systems(minimal or noninvasive methods),and multifunctional therapeutics,especially with regard to cancerswith low survival rates such as brain,lung, pancreas, and ovarian cancers.

FUTURE OUTLOOK

Significant challenges remainwith regard to delivery of RNA-based nanoparticles. From a com-mercial perspective, the manufac-turing process can be complex andinconsistent. The production/synth-esis of oligonucleotides with func-tional moieties has to be a scalable

Figure 6. (A) Internal nucleic acid reporter group, represented here by the fluorescent base analogue tC in pair with guaninelooking down the long axis of a DNA duplex. (B) Same internal reporter group looking along the short axis of a DNA duplex.

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process (chemically). The RNA nano-scaffold has to retain its folding pro-perties and display a stable nature.The RNA construct needs to have amodular design, such that the com-plex can be self-assembled from thebasic building blocks. Moreover, thecost ofmanufacturing and associatedanalytical characterizations of nano-particles needs to be reasonable.From a structural point of view,

there are concerns over the stoichi-ometry and particle size, robustnessof functional modules, and stability(thermodynamic, chemical, and meta-bolic) of the RNA nanoparticles. Ef-fective computational approaches ac-counting for noncanonical basepairing, long-range tertiary interac-tions, and equilibrium folding of thefinal complex are in great demand forthe design and construction of RNAnanoparticles (using interacting, archi-tectural, and ligand-binding motifs)with functional modalities (therapeu-tic, targeting, anddiagnostic). Theflexi-bility of RNAmolecules and unpredict-ability of RNA folding presents achallenge for simulationandmodeling.From an in vivo delivery point of

view, the nanoparticles need to dis-play favorable pharmacological pro-files, biodistribution, pharmokineticprofiles, minimum immunogenicity,relatively high efficiency of diseasetissue/cell targeting, and remain in-tact at extremely low concentra-tions. Furthermore, the RNAi-basedtherapeutics need to be internalizedby the target cell and escape fromthe endosomes if they enter via

receptor-mediated endocytosis. En-dosomal escape for release of thetherapeutic nanoparticles remains achallenge in the field.As outlined in this Nano Focus

article, several groups have madesignificant strides with regard toaptamer- and dendrimer-based de-livery approaches to therapeuticRNAs. Combinatorial therapy usingaptamers and siRNA has proved use-ful for achieving maximal efficiencyand efficacy in targeting. In addition,structural and functional aspects ofRNA nanoparticles have made rapidprogresswith respect to their design

and delivery. Finally, chemical label-ing of RNA using fluorescent analo-gues has progressed remarkably.The meeting highlighted current re-search and future prospects of RNAnanotechnology. Applications andthe impact of RNA nanotechnologyin the assembly and delivery ofsiRNAs, pRNA, therapeutic ribozymes,RNA aptamers, and riboswitches arebecoming a reality. RNA nanotech-nology is already making a signifi-cant impact in drug delivery andtherapeutics as many siRNA-basedproducts are in the pipeline for po-tential treatment of respiratorysynchitial viral infections, hepatitisC virus, liver cancer, and Hunting-ton's Disease. Collaborative endea-vors between academia, government,and industry are likely necessary toadvance RNA nanotechnology into apractical tool for drug discovery anddelivery in the near future.

Acknowledgment. We thank the parti-cipants and sponsors of the meeting.We also wish to thank Thomas Hermann,Mark Laskovics, Christoph Flamm, ChadSchwartz, Randall Reif, and Daniel Binzelfor their insightful comments. We apolo-gize to participants whose work could notbe reviewed in this article due to spacelimitations. The meeting was supportedand sponsored by funding or donations inpart from Kylin Therapeutics, Inc., Girin-dus, Trilink BioTechnologies, Agilent Tech-nologies, NIH Nanomedicine DevelopmentCenter of Phi29 DNA Packaging Motor forNanomedicine (PN2 EY 018230), NIH grantsEB003730, GM059944, CA151648, and fund-ing from the laboratories of the invitedspeakers and the co-authors. P.G. is a co-founderandcontractor ofKylin. This researchwas supported [in part] by the IntramuralResearch Program of the NIH, National Can-cer Institute, Center for Cancer Research.

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