methods for construction and characterization of simple or

Methods xxx (2018) xxx–xxx

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Methods

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Methods for construction and characterization of simple or specialmultifunctional RNA nanoparticles based on the 3WJ of phi29 DNApackaging motor

https://doi.org/10.1016/j.ymeth.2018.02.0251046-2023/� 2018 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Sylvan G. Frank Endowed Chair in Pharmaceutics and Drug Delivery System, The Ohio State University, 912 Biomedical Research Tow460 W 12th Ave., Columbus, OH 43210, USA.

E-mail address: [email protected] (P. Guo).

Please cite this article in press as: S. Guo et al., Methods (2018), https://doi.org/10.1016/j.ymeth.2018.02.025

Sijin Guo a,b, Xijun Piao a,b, Hui Li a,b, Peixuan Guo a,b,c,d,⇑aCenter for RNA Nanobiotechnology and Nanomedicine, The Ohio State University, Columbus, OH 43210, USAbCollege of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH 43210, USAcCollege of Medicine, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USAd James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 December 2017Received in revised form 23 February 2018Accepted 28 February 2018Available online xxxx

The field of RNA nanotechnology has developed rapidly over the last decade, as more elaborate RNAnanoarchitectures and therapeutic RNA nanoparticles have been constructed, and their applications havebeen extensively explored. Now it is time to offer different levels of RNA construction methods for boththe beginners and the experienced researchers or enterprisers. The first and second parts of this articlewill provide instructions on basic and simple methods for the assembly and characterization of RNAnanoparticles, mainly based on the pRNA three-way junction (pRNA-3WJ) of phi29 DNA packagingmotor. The third part of this article will focus on specific methods for the construction of more sophisti-cated multivalent RNA nanoparticles for therapeutic applications. In these parts, some simple protocolsare provided to facilitate the initiation of the RNA nanoparticle construction in labs new to the field ofRNA nanotechnology. This article is intended to serve as a general reference aimed at both apprenticesand senior scientists for their future design, construction and characterization of RNA nanoparticles basedon the pRNA-3WJ of phi29 DNA packaging motor.

� 2018 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Methods for in vitro synthesis of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Production and purification of DNA templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. In vitro RNA transcription with T7 RNA polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. In vitro 20-F RNA transcription with Y639F mutant T7 RNA polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. Solid-phase chemical synthesis of short RNA by phosphoramidite chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Fundamental methods for the construction and characterization of simple pRNA-3WJ nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.1. Construction of pRNA-3WJ with high efficiency by bottom-up self-assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. Purification and gel assay of pRNA-3WJ nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. Evaluation of the thermodynamic stability of the resulting RNA nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.3.1. Temperature-gradient gel electrophoresis (TGGE) for Tm measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3.2. Real-time PCR for Tm measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.4. Enhanced enzymatic stability of pRNA-3WJ by chemical modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.5. High mechanical stability of pRNA-3WJ due to Mg clamps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.6. Evaluation of size and shape of pRNA-3WJ nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Specific methods for the construction of sophisticated pRNA-3WJ nanoparticles with various functionalities. . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.1. Incorporation of functional modules to pRNA-3WJ scaffold. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2. pRNA-3WJ conjugated with chemical ligands for specific tumor targeting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
er (BRT),

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2 S. Guo et al. /Methods xxx (2018) xxx–xxx

4.2.1. Labeling RNA with folate using AMP or GMP derivatives during in vitro transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2.2. Labeling RNA with folate by chemical conjugations of folate derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please

4.3. pRNA-3WJ incorporated with aptamers for specific tumor targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.3.1. In vitro evaluation of cell binding function of pRNA-3WJ incorporated with targeting ligands. . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.3.2. In vivo biodistribution study of pRNA-3WJ incorporated with targeting ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.4. pRNA-3WJ harboring siRNA for tumor regression and gene knockdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.5. pRNA-3WJ harboring anti-miRNA for tumor regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.6. pRNA-3WJ incorporated with ribozyme for catalytic functioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.7. pRNA-3WJ intercalated with doxorubicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.8. pRNA-3WJ labeled with fluorophores for imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5. Challenges and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

Nanotechnology involves the study, engineering, and applica-tion of nanoscale materials across a wide range of scientific fieldsthat include chemistry, biology, biophysics, materials sciences,and biomedical sciences. With defined structures and features, bio-molecules such as proteins, peptides, lipids, DNA, and RNA canserve as natural building blocks for constructing nanomaterialsfrom the bottom-up [1]. Among these molecules, RNA is an attrac-tive candidate, since it can be manipulated at a similar level of sim-plicity as DNA, while possessing the versatility of differentstructures, functions, and biological activities like proteins [2].RNA nanotechnology is conceptualized as the bottom-up self-assembly of nanometer scale RNA architectures, with their majorframe composed of RNA [2,3]. The scaffolds, ligands, therapeutics,and regulators of RNA nanoparticles can be made up of only RNAor a hybrid of RNA with other chemicals. The field of RNA nan-otechnology can be traced back to the first construction of RNAnanoparticles from the re-engineered phi29 packaging RNA (pRNA)molecules in 1998 [4]. The pRNAmolecules were originally derivedfrom bacteriophage phi29 DNA packaging motor [5,6]. They con-tain two interlocking loops that enable the formation of dimers, tri-mers, hexamers, and patterned superstructures via intermolecularinteractions [7,8]. Unlike conventional RNA biology, which mainlystudies RNA structures and functions, RNA nanotechnology is aunique field that focuses on the construction and application ofRNA nanomaterials.

Two major approaches have been used for the construction ofRNA nanoparticles. The first approach is the sequence-dependentself-folding of RNA nanoarchitectures, based on computationalalgorithms and prediction of secondary or tertiary structures[9,10]. The second approach employs the naturally-occurringRNA motifs as core building blocks, such as three-way junction,four-way junction, and kissing loops, to assemble RNA nanoparti-cles [11–22]. Particularly, the pRNA three-way junction (pRNA-3WJ) has been extensively used as a central core for the construc-tion of multifunctional RNA nanoparticles [12,16,23–28]. Thebranched feature of the 3WJ motif allows different functional mod-ules to be conveniently incorporated to the three helical regions,making it an ideal scaffold for targeted drug delivery. This multiva-lent property suggests that pRNA-3WJ nanoparticles have enor-mous potential for therapeutic, imaging and diagnosticapplications.

Over the past decade, the hurdle of enzymatic instability ofnative RNA has been overcome by various chemical modificationssuch as 20-fluoro (20-F) [29–34], and the concern of thermodynamicinstability has been addressed by the utilization of naturally-occurring highly stable RNA motifs such as pRNA-3WJ [12]. As aresult, the field of RNA nanotechnology has seen significant

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advances in biomedical applications, as evidenced by a substantialincrease of publications on RNA nanostructures. Compared tomany other well-developed nano-delivery systems, such as lipo-somes and polymers, RNA nanotechnology is relatively new, withunique features and various advantages [2]. For example, the fun-damental rule for RNA nanoparticle self-assembly relies on base-pairing, while many other synthetic nanoparticles are formed bycovalent polymerization and hydrophobic interactions [35,36]. Inaddition, RNA nanoparticles are assembled spontaneously usinghighly programmable and predictable building blocks in a prede-fined manner that is very different from traditional routes. Theconstruction of functional RNA nanoparticles integrates the knowl-edge of RNA chemistry, RNA biology, and computationalapproaches. Although the history and progress of RNA nanotech-nology have been well-reviewed [2,3,11,37,38], only few litera-tures summarized the methods for constructing andcharacterizing RNA nanoparticles [39,40]. Here, by using pRNA-3WJ platform as a model, we seek to summarize the most generalmethods for constructing and characterizing both simple, and rel-atively sophisticated multifunctional RNA nanoparticles for thera-peutic, imaging, and diagnostic applications. For other types ofRNA nanoparticles using different nanoscaffold designs, readercan refer to a previous protocol [40]. The goal of this article is tofoster the interdisciplinary research, and to encourage greater par-ticipation of new investigators into the field of RNAnanotechnology.

2. Methods for in vitro synthesis of RNA

RNA nanoparticles are typically constructed from severalsingle-stranded RNA (ssRNA) fragments shorter than 100-nucleotides (nt). These RNA strands can be synthesized eitherenzymatically or chemically, depending on the length of the RNAstrands and the types of chemical modifications or labeling. Thissection mainly introduces the most common methods for thein vitro synthesis of RNA.

2.1. Production and purification of DNA templates

The common enzymatic approaches of in vitro RNA synthesisinclude SP6, T7, and T3 RNA transcription that require highly puri-fied DNA templates with distinct promoter sequences. This articlewill mainly focus on the T7 RNA transcription. Prior to transcrip-tion, DNA template containing the T7 promoter (50-TAATACGACTCACTATA-30) at the 50-end can be obtained via standard poly-merase chain reaction (PCR) using a pair of DNA primers, ordirectly annealed from two complementary oligonucleotides. Thefirst two nucleotides after the T7 promoter should be GG, as theseare preferred by T7 RNA polymerase. To ensure the high quality of

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Table 1A T7 RNA transcription setup.

Components Amount Note

5 � Transcription Buffer 10 ll 400 mM HEPES-KOH pH 7.5,120 mMMgCl2, 10 mMspermidine, and 200 mM DTT.

0.1 M DTT 5 ll25 mM rNTPs 10 ll Added second to last; Made by

mixing equal amounts of 100mM ATP, GTP, CTP, and UTP.

0.5 lg/ll DNA Template 1 ll Purified prior to transcription.DEPC-treated H2O 14 llT7 RNA Polymerase 10 ll Added last.Total 50 ll

S. Guo et al. /Methods xxx (2018) xxx–xxx 3

RNA product, the DNA template must be of high purity. Therefore,the DNA template should be carefully purified to remove freenucleotides, salts, and any byproducts by commercially availablegel extraction kits or spin columns (e.g. QIAEX II Gel ExtractionKit; Ambion NucAway spin column), or by electro-separationsystem. Particularly, the electro-separation system consists of anelectrophoresis chamber and sample traps, driving the DNAsamples to migrate out of the gel slices and retain in membranetraps under the driving force of electric field (Fig. 1).

(i) Firstly, run DNA template products in 2% (wt/vol) synergel-agarose gel in 1 � TAE buffer (40 mM Tris-acetate and1 mM EDTA) at 120 V for 40 min at room temperature (RT).

(ii) Place the gel on a TLC plate, excise the gel slices containingDNA bands of interest under UV light (254 nm) on a TLC(Thin Layer Chromatography) plate.

(iii) Place the BT1 membranes (a dense matrix which buffer ionsand molecules less than 3–5 kD can pass through; availablefrom GE Whatman) at points A and C to form an elec-trophoresis chamber, and a BT2 membrane (a microporousprefilter that prevents gel pieces or other particulates frompassing through) at point B to form a trap.

(iv) Place the gel slices in the chamber. Add 1 � TAE running buf-fer, and apply100 V for 1 hour (h).

(v) The elution can be monitored by using UV light (254 nm) tovisualize the DNA samples. Purified DNA samples in the trapare collected, and 2.5 volumes of 100% ethanol and one-tenth volume of 3 M sodium acetate are added to precipitatethe DNA samples overnight at �20 �C.

(vi) Centrifuge the samples solution at 16,500 �g for 30 min at4 �C and discard the supernatant. Wash the pellet with 70%(vol/vol) ethanol and dry the pellet for 10 min with avacuum concentrator.

(vii) Dissolve the pellet in 1 � TE buffer (10 mM Tris-HCl and 1mM EDTA) and store at �20 �C until use. The suggestedDNA template concentration is 0.5 mg/mL.

2.2. In vitro RNA transcription with T7 RNA polymerase

In vitro T7 transcription is a standard method of synthesizingRNA molecules longer than 20-nt using a purified T7 RNA poly-merase. This method can produce up to several milligrams ofRNA with high quality. The purified DNA templates can be usedin the commercially available or homemade T7 transcription kitto synthesize RNA [39,41–43]. A minimum of 0.5 mg of DNA tem-plate is required per 50 ml transcription. A typical T7 RNA tran-scription setup can be found in Table 1, and a specific procedureis described below.

Fig. 1. Purification of DNA or RNA by electro-separation system. Negatively charged DNthrough.

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(i) Mix the samples well in a 1.5 ml RNase-free Eppendorf tube.The transcription reaction can be scaled up or down main-taining the same reagent concentration ratios.

(ii) Incubate the reactions at 37 �C for approximately 4 h.(iii) Terminate the reaction by adding 0.5 ml RNase-free DNase I

(1 mg/ml, Fermentas) and incubate at 37 �C for another15 min.

(iv) The transcription products are then prepared for purificationby adding equal volume of 2 � denaturing loading dye. Runthe samples on an 8% (wt/vol) polyacrylamide gel with 8 Murea in 1 � TBE buffer (89 mM Tris base, 200 mM boric acidand 2 mM EDTA) at 100 V at RT for 1–1.5 h.

(v) Place the gel on a TLC plate, excise the bands of interestunder UV light (254 nm), and cut the gel slices to smallpieces.

(vi) Elute the RNA from gel slices in 400 ml RNA elution buffer[0.5 M ammonium acetate, 10 mM EDTA and 0.1% (wt/vol)SDS in 0.05% (vol/vol) diethyl pyrocarbonate (DEPC)-treated water] at 37 �C for 2 h. Collect the supernatant andadd another 400 ml RNA elution buffer for a second elutionfor 1 h. Alternatively, the elution step can be replaced bythe electro-separation system, as described in Section 2.1(Fig. 1).

(vii) Add 2.5 volumes of 100% ethanol and one-tenth volume of3 M sodium acetate to the supernatant, and precipitateRNA samples overnight at �20 �C.

(viii) Centrifuge the samples solution at 16,500 �g for 30 min at4 �C and discard the supernatant. Wash the pellet with 70%(vol/vol) ethanol and dry the pellet for 10 min with avacuum concentrator.

(ix) Dissolve the pellet in 0.05% (vol/vol) DEPC-treated water andstore at �20 �C until use.

A or RNA migrate into and are retained in a membrane trap, while salt ions pass

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2.3. In vitro 20-F RNA transcription with Y639F mutant T7 RNApolymerase

20-F modified RNA can be synthesized by in vitro transcriptionusing 20-F CTP, 20-F UTP and Y639F mutant T7 RNA polymerase[44]. Compared to the normal transcription in Section 2.2, thein vitro 20-F RNA transcription requires a higher input of purifiedDNA template, an optimized 20-F transcription buffer, and anextended reaction time (6–12 h) to ensure sufficient yield (Table 2),because the Y639F mutant T7 RNA polymerase has a lower effi-ciency of incorporating ribonucleotides. It is generally recom-mended that over 2.5 lg of purified DNA templates are used fora 50 ll transcription reaction. After treatment with RNase-freeDNase I, the 20-F RNA transcripts must be purified by polyacry-lamide gel electrophoresis (PAGE) or electro-separation systemusing the same procedure described in Section 2.2.

2.4. Solid-phase chemical synthesis of short RNA by phosphoramiditechemistry

Solid-phase synthesis via standard phosphoramidite chemistryis a universal method for large scale production of short RNAoligonucleotides (<80-nt). The phosphoramidites are commerciallyavailable nucleoside derivatives with protecting groups. Notably,this technique allows the incorporation of various chemical modi-fications or labels to RNA oligonucleotides for different applica-tions [45]. Opposite to enzymatic RNA synthesis, the solid-phasechemical synthesis proceeds in the 30 ? 50 direction. It begins withthe first phosphoramidite linked to controlled pore glass (CPG)beads as the 30-end, followed by a series of successive reactions(detritylation, coupling, oxidation, and capping) to elongate theRNA strand by adding phosphoramidites based on the inputsequence [46]. These reactions will continuously run in cycles untilthe last 50-end nucleotide is attached. Next, the protecting groupsat each base are cleaved by treatment with ammonia, and the syn-thesized RNA strand will be cleaved from the CPG simultaneously.The 20-protecting group will then be removed by treatment withtriethylamine trihydrofluoride (TEA.3HF) to yield a final RNA prod-uct. During synthesis, chemically-modified phosphoramidites suchas 20-F modified nucleotides or locked nucleic acid (LNA) can beincorporated into the RNA strand, and functional groups such asamine (ANH2), alkyne (AC„CH), thiol (ASH), biotin, or fluo-rophores can be incorporated at the 50-, 30-end, or other specificsite within the sequence. After synthesis, the RNA products shouldbe purified by a desalting column to remove free nucleotides andsalts. High-performance liquid chromatography (HPLC) or denatur-ing PAGE is recommended to yield RNA products with higherpurity.

Table 2A 20-F T7 RNA transcription setup.

Components Amount Note

10 � Transcription Buffer 5 ll 400 mM Tris-acetatepH8.0, 10 mM EDTA, 100mMMg-acetate, 5 mMMnCl2, 80 mMspermidine, and 50 mMDTT.

0.1 M DTT 5 ll50 mM ATP, GTP, 20-F CTP,

and 20-F UTP5 ll for each(5 ll � 4 = 20 ll)

Added second to last.

0.5 lg/ll DNA Template 5 ll Purified prior totranscription.

DEPC-treated H2O 10 llY639F T7 RNA Polymerase 5 ll Added last.Total 50 ll


3. Fundamental methods for the construction andcharacterization of simple pRNA-3WJ nanoparticles

3.1. Construction of pRNA-3WJ with high efficiency by bottom-up self-assembly

The pRNA-3WJ motif was derived from the central domain ofthe natural pRNA of bacteriophage phi29 (Fig. 2A) [12,47]. It canbe constructed from three short ssRNA fragments (3WJ-a, 3WJ-b,and 3WJ-c) with unusually high efficiency by a bottom-up self-assembly approach (Fig. 2B). Briefly, these three fragments aremixed together at equal molar concentration in TMS buffer(50 mM Tris pH 8.0, 100 mM NaCl, 10 mM MgCl2), followed byheating to 85 �C for 5 min and slowly cooled over 40 min to 4 �Con a Thermal Cycler. It has been reported that the three separatestrands co-assemble into pRNA-3WJ via a two-step mechanism, 3WJ-b + 3WJ-cM 3WJ-bc + 3WJ-aM 3WJ-abc [48]. The first stepbetween 3WJ-b and 3WJ-c is highly dynamic since these two frag-ments only contain 8 complementary base pairs. Upon the secondassociation with 3WJ-a, which contains 17 complementary basepairs to the 3WJ-bc dimer complex, the unstable dimer was lockedinto a highly stable 3WJ. Generally, this assembly process is highlyefficient even in the absence of Mg2+, as long as the three RNAfragments are mixed at equal molar ratio.

3.2. Purification and gel assay of pRNA-3WJ nanoparticles

The assembled pRNA-3WJ nanoparticles are generally purifiedon 12% native PAGE at 120 V for 2 h in TBM running buffer (89mM Tris, 200 mM borate acid, and 5 mM MgCl2) at 4 �C. The majorband of pRNA-3WJ samples is then excised from the gel under UVlight (254 nm), and two elution approaches (see Section 2.2) can beapplied to recover the pRNA-3WJ samples. However, the RNA elu-tion buffer should be supplied with 10 mMMg2+, if the assembly ofRNA nanoparticles requires Mg2+. After precipitation, the pelletshould be dried and dissolved in 0.05% DEPC-treated water orTMS buffer. The purity of pRNA-3WJ can be confirmed by perform-ing a step-wise assembly of pRNA-3WJ monomers (3WJ-a, 3WJ-b,or 3WJ-c), dimers (3WJ-a + 3WJ-b, 3WJ-b + 3WJ-c, or 3WJ-a + 3WJ-c), and pRNA-3WJ trimer (3WJ-a + 3WJ-b + 3WJ-c) on a nativePAGE, while the unpurified pRNA-3WJ nanoparticle can be usedas control for comparison. Approximate 200 ng RNA nanoparticlesin gel can be greatly stained by ethidium bromide (EtBr), and visu-alized on a common gel imaging system.

3.3. Evaluation of the thermodynamic stability of the resulting RNAnanoparticles

The pRNA-3WJ scaffold was demonstrated to be highly thermo-dynamically stable, as evidenced by its high melting temperatures(Tm), resistance to denaturation at high urea concentration, and itsability to remain intact at ultra-low concentrations (picomolar)[12]. The thermodynamic parameters of pRNA-3WJs composed ofdifferent types of oligonucleotides (DNA, RNA, and 20-F RNA) havebeen studied previously [49]. It was found that the formation of3WJRNA displays the least favorable enthalpy change (DH) butthe most favorable entropy change (DS), as compared to 3WJDNAand 3WJ20-F. The Gibbs free energy (DG) of 3WJRNA formation,however, was higher than 3WJDNA but lower than 3WJ20-F. Thiscomparison indicated a mechanism of entropy-driven assemblyof pRNA-3WJ. The thermodynamic parameters for pRNA-3WJswere typically obtained from a Van’t Hoff curve by plotting theTm versus the concentrations of the assembled pRNA-3WJs. Thereare two independent methods for the accurate measurements ofTm values of pRNA-3WJ, namely, temperature-gradient gelelectrophoresis (TGGE) and quantitative real-time PCR (rtPCR).

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Fig. 2. Structure, assembly and characterization of pRNA-3WJ. (A) Secondary sequence of pRNA-3WJ (H1, H2, and H3 represent helical regions). (B) 3D computationalmodeling of the bottom-up self-assembly of pRNA-3WJ. (C) Tm measurement of 20-F pRNA-3WJ by TGGE (M means monomer). (D) Tm measurement of 10 mM 20-F pRNA-3WJby real-time PCR. (E) Enzymatic stability assay of pRNA-3WJ with 20-F modification on different strands in 10% FBS supplemented cell culture medium. (F) AFM image of theextended pRNA-3WJ. (G) Size distribution of pRNA-3WJ measured by DLS (n = 3).


3.3.1. Temperature-gradient gel electrophoresis (TGGE) for Tmmeasurement

TGGE (Biometra GmbH) applies a linearly increasing tempera-ture gradient perpendicular to the electrical current on a polyacry-lamide gel. As a result, each separate lane of RNA sample will besubjected to heating at a gradually-increased temperature acrossthe gel, driving the nanoparticles to undergo a transition fromthe assembled to denatured conformation [50] (Fig. 2C). Samplein each lane is then quantitatively analyzed to generate a meltingcurve, and the Tm is defined as the specific temperature when50% of the RNA samples are dissociated [49]. Normally, the


increasing temperature gradient will result in the swelling of gelpores, leading to the gradual increasing migration rate of theRNA nanoparticles. The dissociation of RNA nanoparticles can bevisualized by a relatively sharp transition of the band migration.A specific procedure is described below.

(i) Load approximate 200 ng assembled pRNA-3WJ samples ineach well of a 12% native PAGE, and run under 100 V inTBM buffer for 10 min to allow the samples to enter the gel.

(ii) Disassemble gel sandwich, clean and dry the backside of thegel support film.

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(iii) Apply 150 ll of thermal coupling solution (0.01% Triton) onthe thermal block to clean the surface, and place the gel onthe thermal block after drying the surface. Avoid leavingany bubbles trapped under the gel support film.

(iv) Cover the gel with a cover film. Attach pre-soaked bufferwicks on the top and bottom of the gel.

(v) Attach gel cover plate, and set up the temperature gradientprogram. Run the gel at 100 V for 1 h.

TGGE possesses some advantages over other common methodsfor Tm measurements, such as UV melting. It’s especially useful fornucleic acid samples composed of multiple oligonucleotidestrands, as each strand can be conveniently detected with differentlabeled fluorophores. Consequently, the dissociation of each strandfrom the complex can be monitored simultaneously. Another nota-ble advantage of TGGE is that it’s suitable for a wide range of con-centrations of nucleic acid samples. By applying radiolabeling,fluorophores, and Ethidium Bromide (EtBr) staining, TGGE candetect the process of melting from low nanomolar to micromolarconcentrations [49–51].

3.3.2. Real-time PCR for Tm measurementReal-time PCR provides another technique for Tm measurement

of RNA nanoparticles (Fig. 2D). It is especially favorable for high-throughput Tm screening of nucleic acid samples. Up to 96 samplescan be measured simultaneously within 1 h. A specific procedure isdescribed below.

(i) 18 ml of purified pRNA-3WJ samples are mixed well with 2 ml10 � SYBR Green II. Generally, the final concentration ofpRNA-3WJ can be 1–10 mM.

(ii) Load the samples into an optical 96-well plate and seal theplate with a sealing film.

(iii) Samples are heated at 95 �C for 5 min in the Roche LightCy-cler 480 real-time PCR system, followed by slowly cooling ata rate of 0.11 �C/s to 20 �C for several cycles. The heatingprocess generates an initial denaturing period for thenanoparticles, while the cooling process generates anassembling period.

(iv) The melting curves are obtained by real-time monitoring thefluorescence levels of SYBR Green II. The Tm is determined asthe specific temperature when 50% of the fluorescenceintensity is observed.

It has been well-confirmed that the two methods discussedabove have a high degree of consistency for Tm measurements of3WJDNA, 3WJRNA, and 3WJ20-F [49]. However, the real-time PCRmethod relies heavily on dye intercalation into base pairs, andmay not be suitable for other highly chemically-modified oligonu-cleotide samples due to the decreased intercalation rate and effi-ciency. It’s always recommended to use more than one methodto confirm the Tm measurements.

3.4. Enhanced enzymatic stability of pRNA-3WJ by chemicalmodifications

Natural RNA molecules are susceptible to RNase-mediateddegradation, extremely limiting their usefulness for in vivo applica-tions. Over the past years, many chemical modification studiesaiming to improve the enzymatic stability and the in vivo proper-ties of RNA have been reported [29–34,52]. The common modifica-tions include the modification of phosphodiester backbone,substitution of 20-OH group, base modification, and ribose modifi-cation [38,52–54]. Particularly, 20-F modification has been widelyused to construct RNase-resistant pRNA-3WJ nanoparticles[12,16,23–28,54]. This modification typically replaces the 20-OH


group of cytidines (C) and uridines (U) with a 20-fluoro, withoutaffecting the authentic folding and assembly of the pRNA-3WJ[12]. The enhanced enzymatic stability of 20-F RNA nanoparticlescan be greatly evaluated by a serum stability assay [12,51,55],where 20-F RNA nanoparticles are incubated with 10–50% fetalbovine serum at 37 �C to mimic an in vivo environment for differ-ent time points (Fig. 2E). The resulting nanoparticles are run innative PAGE, and the percentage of intact nanoparticles can bequantitatively analyzed to generate a degradation curve. It wasfound that 20-F modification further enhanced the thermodynamicstability of RNA nanoparticles [49,51]. Another advantage of 20-FRNA is that it can be readily created by either in vitro transcriptionor solid-phase chemical synthesis (see Section 2). The in vitro 20-Ftranscription enables the production of long strands of 20-F RNA.In addition, LNA modification on C and U can dramatically improvethe half-life of RNA molecules in vivo [51]. The recent progress ofchemical modifications has considerably facilitated the in vivoapplications of RNA nanotechnology.

3.5. High mechanical stability of pRNA-3WJ due to Mg clamps

The pRNA-3WJ structure has been recently reported to be anovel mechanically anisotropic structure and shows potentialapplications in biomaterials. Single-molecule atomic force micro-scopy (AFM) can be used to investigate the mechanical propertyof pRNA-3WJ [47,56,57]. To study this mechanical stability,pRNA-3WJ was modified with amine groups at 50- and 30-end ofdifferent helical arms, and linked to the cantilever tip and substratevia two bi-functionalized PEG linkers [N-hydroxyl succinimide(NHS)–PEG–NHS], respectively. AFM pulling experiment was per-formed by retracting the cantilever tip at a constant velocity thatgradually unfolded the pRNA-3WJ structure at a defined pullingdirection. In the same way, amine groups can be modified at otherhelical arms to establish different pulling directions. The forcespectroscopy curves were recorded to reveal the mechanical prop-erty of pRNA-3WJ. It was found that pRNA-3WJ structure displaysstrong resistance to stretching along its coaxial helices. In thepresence of Mg2+, pRNA-3WJ could resist the rupture force of about219 pN along the H1-H3 coaxial direction. This robust resistance iscomparable to some mechanically strong elastomeric proteinssuch as titin immunoglobulin domain. In the absence of Mg2+,pRNA-3WJ exhibited weaker resistance to rupture forces alongall directions, demonstrating a mechanism of Mg2+-dependentmechanical property. This Mg2+-dependent mechanical anisotropyis attributed to two Mg clamps between the phosphates of G23 andA90 as well as C24 and A89 (Fig. 2A). These two Mg clamps resistcoaxial stretching in a cooperative manner and contribute to theextraordinary mechanical stability of pRNA-3WJ, making it arobust scaffold for constructing more sophisticated RNAnanoparticles.

3.6. Evaluation of size and shape of pRNA-3WJ nanoparticles

Size and shape are important parameters to evaluate the struc-tural property of RNA nanoparticles. AFM has been developed as apowerful tool for the structural characterization of nanoparticles.The high resolution and compatible sample examination condi-tions make it suitable for RNA nanostructures [12,17,58–60]. Toclearly visualize the branched structure of the pRNA-3WJ, threehelical arms were extended with a 60 or 58 base pairs RNA helix[23,24]. After purification by PAGE, the size and shape of theextended pRNA-3WJ can be observed by AFM (Fig. 2F). WhileAFM is more favorable for analyzing the surface morphology ofRNA nanoparticles, cryo-electron microscopy (cryo-EM) is anotherpowerful technique to characterize their three-dimensional fea-tures [60,61]. In addition, dynamic light scattering (DLS) is useful

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to evaluate the size of pRNA-3WJ nanoparticles through the assess-ment of average hydrodynamic diameter (Fig. 2G). As alternatives,gel electrophoresis, including agarose gel and polyacrylamide gel,can be conveniently used to characterize the step-wise formationof RNA nanoparticles [12,49]. The combination of gel electrophore-sis, DLS, AFM, and cryo-EM creates a general characterization sys-tem to evaluate the size and shape properties of RNA nanoparticles.

4. Specific methods for the construction of sophisticated pRNA-3WJ nanoparticles with various functionalities

4.1. Incorporation of functional modules to pRNA-3WJ scaffold

Apart from the high enzymatic and thermal stability, the multi-valent nature is another favorable attribute of pRNA-3WJ nanopar-ticles. The pRNA-3WJ can be functionalized simultaneously withdifferent modules to achieve synergistic effects. Generally, in orderto construct multifunctional pRNA-3WJ nanoparticles, the types offunctional modules and functionalities need to be well defined, andthe global structure needs to be established before the RNA synthe-sis and construction (Fig. 3). The common functional modulesinclude targeting ligands (chemical ligands or aptamers), thera-peutic molecules [short interfering RNA (siRNA) or anti-microRNA (anti-miRNA)], catalytic ribozymes [e.g. anti-hepatitis B virushammerhead (anti-HBV) ribozyme], chemotherapeutic drugs (e.g.doxorubicin), and imaging probes (fluorophores or radionuclide).The general strategies for the multifunctionalization of RNAnanoparticles include both covalent and non-covalent methods,such as 50- or 30-end extensions, hybridization, and chemical con-jugation or labeling. The rigidity of the pRNA-3WJ scaffold allowsall incorporated modules to be spaced apart from each other,retaining their authentic folding and independent functionalities[62]. Recently, the integration of multiple functional modules into

Fig. 3. Strategies for RNA nanotechnology. The construction of RNA nanoparticlesincludes several distinct steps: conception, computation, synthesis, assembly,assessment and characterization, and applications.


one nanoparticle has been used for the treatments of various can-cer models [16,23–28,63,64]. The following subsections will focuson the well-established methods for design, incorporation and con-struction of pRNA-3WJ nanoparticles with different functionalmodules.

4.2. pRNA-3WJ conjugated with chemical ligands for specific tumortargeting

Chemical ligands such as folate can be conjugated to pRNA-3WJnanoparticles for specific cancer targeting [16,23,27,64]. Folatereceptors are highly overexpressed on �40% of tumor types, butshow only limited expression in healthy organs and tissues [65].This makes the folate ligand-receptor pair an efficient strategyfor targeted drug delivery. Recent studies have shown that thefolate-conjugated pRNA-3WJ nanoparticle can specifically bind tocancer cells in vivo, including glioblastoma [27], gastric cancer[23] and colorectal cancer metastasis in the liver, lungs and lymphnode [64], with little or no accumulation in vital organs and tis-sues. The most common chemical strategies for the conjugationof folate to synthetic RNA can be summarized into two followingcategories.

4.2.1. Labeling RNA with folate using AMP or GMP derivatives duringin vitro transcription

AMP and GMP derivatives refer to the chemically-modified ade-nosine monophosphate and guanosine monophosphate with reac-tive groups, respectively. A variety of AMP and GMP derivativeshave been synthesized by using linker molecules through well-established chemistry [66–68]. These AMP or GMP derivativescan be used for single labeling of chemical ligands at the 50-endof RNA because they are efficient initiators of T7 RNA transcriptionbut cannot be used in the chain elongation step. By utilizing theAMP or GMP derivatives, RNA strands can be efficiently labeledwith folate by a co-transcription procedure. A well-studied folatelabeling procedure was performed by adding a AMP-hexanediamine (HDA)-folate derivative together with normalribonucleoside triphosphates (rNTPs) during the in vitro RNA tran-scription [66]. Specifically, an AMP-HDA derivative is firstly syn-thesized through the direct coupling of HDA to AMP in thepresence of condensing reagent N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). On the other hand, folatemolecules can be modified into folate-NHS ester derivative in thepresence of N,N’-dicyclohexylcarbodiimide (DCC), NHS, and tri-ethylamine (TEA) in dimethylsulfoxide (DMSO). Next, couplingfolate-NHS ester with the free amino group of AMP-HDA resultsin the AMP-HDA-folate derivative product (Fig. 4A). To ensure ahigh yield of RNA production as well as folate labeling efficiency,an excess amount of AMP-HDA-folate should be used, comparedto normal ATP. A 16:1 ratio of AMP-HDA-folate to ATP wasreported to be the best ratio for this co-transcriptional folate label-ing method [66].

4.2.2. Labeling RNA with folate by chemical conjugations of folatederivatives

Besides the co-transcriptional method, RNA can be labeled withfolate via a wide range of chemical conjugations. These chemicalconjugation strategies require that both the RNA and folate mole-cule are modified with a pair of reactive groups. For RNA, the reac-tive groups are more favorably attached at the 50- or 30-ends toavoid interfering the base pairing. The well-established reactionchemistry includes (Fig. 4B): 1) Reaction of a NHS ester, an acti-vated carboxylic group, on the folate derivative with a free -NH2

on the RNA strand. This reaction can yield a stable amide linkageunder slightly alkaline conditions (pH 7.2 to 9); 2) Click reactionof an alkyne- or azide-modified folate derivative with an

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Fig. 4. Chemical conjugations and structures for RNA nanotechnology. (A) The chemical structure of synthesized AMP-HDA-folate derivative. (B) Chemical conjugationmethods for labeling RNA with chemical ligands.


azide- or alkyne-labeled RNA strand, respectively. The copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is an out-standing reaction for folate labeling of RNA due to its high reactionefficiency, mild reaction conditions, and high compatibility withother functional groups; 3) Reaction of a maleimide group on thefolate derivative with a free -SH group labeled on the RNA strand.This reaction occurs under the pH 6.5–7.5, and results in a non-reversible stable thioether linkage. It should be noticed that allthe folate derivatives above are commercially available. The RNAstrands labeled with functional groups at the 50- or 30-end can beeither chemically synthesized using the modified phosphoramiditederivatives, or directly purchased commercially (see Section 2.4).After the chemical conjugations, the RNA-folate product shouldbe purified by reverse phase HPLC or PAGE to remove unreactedmolecules prior to the nanoparticle assembly.

4.3. pRNA-3WJ incorporated with aptamers for specific tumortargeting

RNA aptamers are RNA oligonucleotides selected via the sys-tematic evolution of ligands by exponential enrichment (SELEX)to bind targets with high affinity and selectivity, similar to an anti-body binding to an antigen. Many RNA aptamers have been exten-sively applied to cancer targeting, diagnosis, and therapeutics[69,70]. Here, this section will not focus on the generation of apta-mers by SELEX technology, but the methods for incorporating well-developed aptamers to pRNA-3WJ scaffold. The first step is todetermine a suitable aptamer for the specific objective. Thesequence of aptamers can be rationally linked to the 50- or 30-endof one helical arm of 3WJ. For example, a 39-nt epidermal growthfactor receptor aptamer (EGFRapt) was incorporated to the pRNA-3WJ scaffold for efficient tumor targeting in the triple negativebreast cancer model (Fig. 5A) [24]. The EGFR aptamer was directlyextended from the 30-end of 20-F 3WJ-b, resulting in a 59-nt 20-F


EGFRapt-displaying 3WJ-b strand. This strand can be synthesizedby either in vitro 20-F RNA transcription, or solid-phase chemicalsynthesis (see Section 2), and mixed with 20-F 3WJ-a and 20-F3WJ-c at equal molar concentration to assemble the 20-F 3WJ-EGFRapt nanoparticle. Instead of being extended from one strandof 3WJ, RNA aptamers can also be inserted between two strandsof 3WJ to obtain a higher cell binding efficiency. For example, a43-nt anti-prostate-specific membrane antigen A9g aptamer(PSMAapt) was used to connect 3WJ-a and 3WJ-c, and specificallytargeted prostate cancer after the assembly of 20-F 3WJ-PSMAapt

(Fig. 5B) [25]. These aptamers retained their authentic foldingand specific cell binding activities after incorporation to 3WJ. Typ-ically, their in vitro and in vivo specific cancer cell binding capacitycan be evaluated by flow cytometry analysis, confocal microscopyimaging, and in vivo biodistribution study.

4.3.1. In vitro evaluation of cell binding function of pRNA-3WJincorporated with targeting ligands

After the construction of pRNA-3WJ with targeting ligands(folate or aptamers), their specific cell binding function should beevaluated in vitro prior to animal trials. The most commonapproaches include flow cytometry analysis and confocal micro-scopy imaging. In these cases, the RNA nanoparticles must be addi-tionally labeled with fluorophores for detection and imaging (seeSection 4.8). The types of cell line vary according to the specificincorporated ligand or aptamer that recognizes the specific cell-surface marker. To evaluate the cell binding efficiency by flowcytometry, a standard procedure will be:

(i) Trypsinize the cells and wash themwith PBS buffer (137 mMNaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4 pH7.4).

(ii) Resuspend the cells to a concentration of 2 � 105 cells in50 ml ice-cold PBS buffer.

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Fig. 5. Multifunctional RNA nanoparticles based on pRNA-3WJ. (A, B) pRNA-3WJ incorporated with anti-miRNA and aptamers. (C, D) pRNA-3WJ incorporated with siRNA andfolate.


(iii) Add fluorophore-labeled RNA nanoparticles incorporatedwith targeting ligand to cell suspension at a final concentra-tion of 50–100 nM, and incubate at 37 �C for 1–2 h. A typicalcontrol group will be a fluorophore-labeled RNA nanoparti-cle without targeting ligand.

(iv) Rinse the cells twice with PBS buffer to remove free RNAnanoparticles, resuspend the cells and assay them by flowcytometer.

As an alternative, a standard confocal microscopy experimentfor RNA nanoparticles will be:

(i) Seed 5 � 104 cells on glass coverslips in cell culture mediumovernight.

(ii) Incubate the cells with either fluorophore-labeled RNAnanoparticles incorporated with targeting ligand or controlgroups at 37 �C for 2 h.


(iii) Gently wash the cells with PBS buffer twice, fix the cells with4% (vol/vol) paraformaldehyde at RT for 30 min, and washcells three times with PBS buffer.

(iv) Stain the cells cytoskeleton using Alexa Fluor 488 phalloidinfor 30 min at RT, and rinse them with PBS buffer for threetimes.

(v) Mount the cells with Prolong Gold antifade reagent to staincell nucleus.

(vi) Assess cell binding and entry event with a Zeiss LSM 510laser-scanning confocal microscope.

4.3.2. In vivo biodistribution study of pRNA-3WJ incorporated withtargeting ligands

Apart from in vitro cell binding experiments, the tumor target-ing of pRNA-3WJ incorporated with folate or aptamers should befurther evaluated by in vivo biodistribution. It should be noted thatmice must be on a folate-deficient diet to avoid the competitive

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binding from free folate molecules, if folate is used as the targetingligand. All RNA nanoparticles are recommended to be 20-F modifiedto increase their enzymatic stability in vivo. A far-red or near-IRdye, such as Alexa 647, will be preferred for biodistribution imag-ing of RNA nanoparticles due to their better fluorescent signal pen-etration and low background noise. Tumor model varies dependingon the types of incorporated targeting ligands. A detailed proce-dure is described below.

(i) Inject 4–6 weeks-old nude mice (nu/nu) with approximate1 � 106 cancer cells in PBS buffer to generate tumor model(if using folate as the ligand, keep mice on a folate-deficient diet for 2 weeks before cancer cells injection).

(ii) Once the tumors grow to approximate 500 mm3, IV injectfluorophore-labeled RNA nanoparticles at a single dose of20 mM in 100 ml PBS buffer, or PBS buffer as a negative con-trol through the tail vein.

(iii) Anesthetize the mice and perform whole-body imaging withan IVIS Spectrum Station at different time points (e.g. 1, 4, 8,12, 24 h) after injection.

(iv) To further evaluate the biodistribution in organs and tumors,sacrifice the mice by CO2 asphyxiation followed by cervicaldislocation. Major organs including heart, lung, spleen, liver,and kidney as well as tumor are collected and subjected forfluorescence imaging using the IVIS Spectrum Station.

4.4. pRNA-3WJ harboring siRNA for tumor regression and geneknockdown

RNA interference (RNAi) is an important post-transcriptionalmethod of gene regulation in many living organisms [71,72]. A pre-vious protocol offers a well-established method for the design andassembly of siRNA-functionalized RNA nanoring and RNA nano-cube [40]. Here, we mainly introduce the fast and simple methodto construct siRNA-functionalized pRNA-3WJ nanoparticles forspecific targeted delivery and tumor regression. Due to the samechemical nature, a typical 21–25 bp siRNA can be readily incorpo-rated into pRNA-3WJ nanoparticles. The primary siRNA sensesequence can be extended from the 50- or 30-end of 3WJ, whilethe antisense strand is fused to sense strand by complementarybase pairing. To ensure the subsequent Dicer processing, the siRNAcan be spaced apart from the helical region of 3WJ by the introduc-tion of a -UU- or -AA- bulge between these two modules, and the2-nt overhang at 30-end of the siRNA must be retained for Dicerrecognition. It is important to note that the antisense strands aregenerally retained unmodified to avoid compromising the activesilencing potency. It has been demonstrated that the incorporationof siRNA to pRNA-3WJ nanoparticles didn’t affect the authenticfolding and activities of the individual units [12,62]. A typicalexample is the incorporation of BRCAA1 siRNA to pRNA-3WJ forgastric cancer regression (Fig. 5C) [23]. The BRCAA1 sense strandwas linked to the 50-end of 20-F 3WJ-b. The entire nanoparticlewas designed to be constructed from four ssRNA fragments, includ-ing folate-displaying 20-F 3WJ-a, BRCAA1-harboring 20-F 3WJ-bsense strand, 20-F 3WJ-c, and BRCAA1 antisense strand. Thisnanoparticle actively targeted gastric cancer and showed a signifi-cant inhibitory effect on gastric tumor growth in vivo. Anotherexample is the incorporation of luciferase siRNA to pRNA-3WJ(Fig. 5D) [27]. Different from the previous design, the luciferasesiRNA sense strand was linked at the 30-end of 20-F 3WJ-a. Afterthe construction, 20-F 3WJ nanoparticles harboring luciferasesiRNA efficiently targeted human patient-derived glioblastomastem cells and knocked down luciferase reporter gene expression.By incorporating siRNA to pRNA-3WJ platform, the specific tumortargeting strategy can potentially overcome the obstacles for siRNAtherapeutics, such as improve the in vivo stability and half-life of


the siRNA, prevent undesirable side effects, and increase the silenc-ing potency of a given dose.

4.5. pRNA-3WJ harboring anti-miRNA for tumor regression

MiRNAs are another important part of RNAi therapeutics [73].Typically, mature miRNAs are naturally-occurring small noncodingRNA molecules composed of around 22 nucleotides. They can reg-ulate target genes by inhibiting the translation of specific mRNA ordegrading the mRNA. Therefore, miRNAs play important roles inthe regulation of cell cycle, differentiation, metabolism, apoptosis,oncogenesis, and tumor growth. In recent years, anti-miRNAs havebeen developed as therapeutics for tumor regression [74]. Similarto siRNA, small miRNA or anti-miRNA can be incorporated to the50- or 30-end of pRNA-3WJ for targeted delivery. For example, arecent study showed that the specific delivery of anti-miRNA-21by pRNA-3WJ to triple negative breast cancer can successfullyachieve tumor regression (Fig. 5A) [24]. The anti-miRNA-21 usedin this study was an 8-nt LNA sequence (50-GATAAGCT-30) that iscomplementary to the oncogenic miRNA-21 seed region. The LNAmodification significantly improves the binding affinity and speci-ficity. To incorporate anti-miRNA-21 to the pRNA-3WJ, the anti-miRNA-21 was linked to a 25-nt DNA sequence which can behybridized to 20-F 3WJ-a with a 25-nt extended complementarysequence, leaving the 8-nt LNA as an overhang for miRNA target-ing. The synthesis of this anti-miRNA-21 LNA strand can be readilyaccomplished by solid-phase chemical synthesis using LNA phos-phoramidites (see Section 2.4), or directly purchased commer-cially. Next, the 20-F 3WJ harboring anti-miRNA-21 can beassembled from four fragments, using the same bottom-up self-assembly procedure as described above. Besides breast cancer,3WJ nanoparticles harboring anti-miRNA-21 were also used forthe treatment of glioblastoma and prostate cancer in animal mod-els, and showed enhanced anti-tumor effect (Fig. 5B) [25,75].

4.6. pRNA-3WJ incorporated with ribozyme for catalytic functioning

Ribozymes are a class of RNAmolecules with enzymatic activitysimilar to proteins [76,77]. These catalytic RNAs possess significanttherapeutic potential because they can regulate protein transla-tion, RNA splicing process, and viral replication. Like RNA apta-mers, siRNA, and miRNA, ribozymes are another importantfunctional module that can be readily incorporated to pRNA-3WJnanoparticles. For example, the anti-HBV ribozyme is a RNAenzyme that cleaves the genomic RNA of HBV genome [12]. Itwas incorporated to a pRNA-3WJ scaffold by joining it to the helicalregion of 3WJ-a/3WJ-b branch (Fig. 6A). The 3WJ-a/b harboringanti-HBV ribozyme sequence can be synthesized by in vitro RNAtranscription (see Section 2), followed by mixing with 3WJ-c toform the 3WJ nanoparticle harboring anti-HBV ribozyme. Afterthe construction, the original catalytic activity of the anti-HBVribozyme was confirmed by its ability to cleave its 135-nt RNAgenome substrates into two small fragments of 60-nt and 75-nt,respectively [12].

4.7. pRNA-3WJ intercalated with doxorubicin

Traditional chemotherapy still plays an essential role in cancertreatment. However, routine systemic administration of smallchemotherapeutic drugs always requires a high dose to achievetherapeutic effects, leading to non-desirable side effects and non-specific toxicity. RNA nanotechnology shows great potential inovercoming the challenges of traditional chemotherapy, bylowering off-target effects, enhancing circulation time, loweringinjection dose, and improving drug efficacy. Anti-cancer drugs suchas doxorubicin (Dox) have been incorporated to pRNA-3WJ

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Fig. 6. Illustration of functional pRNA-3WJ nanoparticles incorporated with (A) anti-HBV ribozyme, and (B) intercalated with doxorubicin.


nanoparticles for targeted delivery to ovarian cancer [28]. To inter-calate Dox within pRNA-3WJ, a 26-bp GC rich sequence wasextended from the 3WJ-b/3WJ-c helical branch to serve as adrug-loading region (Fig. 6B). By gently mixing Dox with pRNA-3WJ in intercalation buffer (0.1 M sodium acetate, 0.05 M NaCl,0.01 MMgCl2) at RT for 1 h, the planar Dox will preferentially inter-calate between neighboring GC base pairs within the RNA helix.Excess Dox can be readily removed from the reaction mixture bypassing it through a Sephadex G50 spin column. After the conjuga-tion, the drug loading efficiency can be determined by measuringthe fluorescence intensity of dox with a fluorescence spectropho-tometer. The non-covalent intercalation allows Dox to be releasedover time during cancer treatment. The incorporation of Dox topRNA-3WJ has the potential to enhance its therapeutic efficiencyat low doses through targeted drug delivery for cancer treatment.

4.8. pRNA-3WJ labeled with fluorophores for imaging

Fluorophores are useful for the in vivo localization of pRNA-3WJnanoparticles. Covalent labeling of RNA with fluorophores can beaccomplished or obtained in several ways, including commonchemical conjugations (see Section 4.2.2 and Fig. 4B), solid-phasechemical synthesis using fluorophores-modified phosphoramidites(see Section 2.4), and directly purchase from commercial vendors.It is strongly recommended to purify the fluorescently labeled RNAstrand from unlabeled RNA and excess fluorophores by reversephase HPLC. After the pRNA-3WJ assembly, the fluorescentpRNA-3WJ nanoparticles, functionalized with targeting groups,can be used for in vivo imaging and detection (Fig. 5) [16,64,78].

5. Challenges and perspectives

RNA nanotechnology is an innovative and novel platform andits rapid progress in recent years has rendered tremendous poten-tial for the treatment of various diseases, especially cancer treat-ments. The construction of functional RNA nanoparticles hasbeen well-established, but the following challenges need to beaddressed to facilitate the field toward clinic and industry:

1) Accurate prediction of the global folding of RNA structuresand assembly of RNA nanoparticles continues to be challeng-ing. The computational modeling of RNA nanoparticle con-struction requires the understanding of boththermodynamic and kinetic aspects of RNA folding.Although some user friendly RNA computation resourcesare available online, such as Mfold [79], RNA designer [80],Sfold [81], NUPACK [82], Nanofolder [20], and Hyperfold[83], the development of RNA structural computation is stillat the early stage.


2) The drug loading capacity and controlled drug release ofpRNA-3WJ needs to be improved. The branched featureenables the pRNA-3WJ scaffold to carry multiple functionalmodules or small therapeutic agents; however, the amountof payload is still limited. Different solutions have beendeveloped, such as the construction of RNA dendrimersand RNA nanocages [59–61]. Nevertheless, some importantissues, such as the capacity of drug loading, effective strate-gies for controlled drug release, await improvement.

3) The knowledge of intracellular processing and endosomalescape of RNA nanoparticles is still restricted. After incorpo-ration of targeting ligands, pRNA-3WJ nanoparticles canenter cells by receptor-mediated endocytosis. However, itis possible that the RNA nanoparticles are transferred toand trapped in the late endosomes, causing them to fail toreach their targets, especially for siRNA delivery. Somemethods have been reported to assist endosome escape.For example, the use of pH-responsive chemical groups, suchas acetal, hydrazone, and maleic acid amides, or acid proto-nating groups like imidazole, sulfonamide can help disruptthe endosome by proton sponge effects [84,85]. More stud-ies are needed to apply these methods to the RNA nanopar-ticle platform.

4) The large-scale production and high cost issue of RNAnanoparticles remain one of the bottlenecks for their useful-ness in clinical and industrial applications. Nevertheless, sig-nificant progress has been made to improve the chemicalsynthesis efficiency of RNA and the availability of chemicalmodifications. Furthermore, the rapid development of RNAproduction facilities and technologies are expected todecrease the cost of RNA nanoparticle production in thefuture.

6. Concluding remarks

The field of RNA nanotechnology has achieved many excitingdevelopments in recent years, especially its application in targetedcancer therapy. The 3WJ motif derived from phi29 pRNA providesan ideal platform for constructing multifunctional RNA nanoparti-cles. By incorporating different functional modules to the branchedregion of 3WJ, RNA nanoparticles can be constructed via a bottom-up self-assembly approach with controllable structures, definedsizes, precise stoichiometries, and polyvalent functionalities. TheseRNA nanoparticles will play a significant role in biomedicalsciences. This article covers the fundamental methods for thedesign, construction and characterization of both simple andsophisticated pRNA-3WJ nanoparticles. The methods discussedabove are expected to help readers understand this field betterand be used in their own research.

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Acknowledgements

This research was supported by NIH grants R01EB019036,U01CA151648 and U01CA207946. P.G.’s Sylvan G. Frank EndowedChair position in Pharmaceutics and Drug Delivery is funded by theCM Chen Foundation. P.G. is the consultant of Oxford NanoporeTechnologies and Nanobio Delivery Pharmaceutical Co. Ltd; he isthe cofounder of Shenzhen P&Z Bio-medical Co. Ltd and its sub-sidiary US P&Z Biological Technology LLC, as well as ExonanoRNALLC.

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