Dual functional RNA nanoparticles containing phi29 motor...

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Methods xxx (2011) xxx–xxx

YMETH 2708 No. of Pages 11, Model 5G

28 January 2011

Contents lists available at ScienceDirect

Methods

journal homepage: www.elsevier .com/locate /ymeth

Dual functional RNA nanoparticles containing phi29 motor pRNA and anti-gp120aptamer for cell-type specific delivery and HIV-1 Inhibition

Jiehua Zhou a, Yi Shu b, Peixuan Guo b, David D. Smith c, John J. Rossi a,d,⇑a Division of Molecular and Cellular Biology, City of Hope, Duarte, CA, USAb Nanobiomedical Center, SEEBME, University of Cincinnati, Cincinnati, OH 45267, USAc Division of Biostatistics, Beckman Research Institute of the City of Hope, Duarte, CA, USAd Graduate School of Biological Sciences, City of Hope, 1500 East Duarte Rd., Duarte, CA 91010, USA

a r t i c l e i n f o

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Article history:Available online xxxx

Keywords:RNAiAnti-gp120 aptamerNanobiotechnologyBionanotechnologyNanotechnologyAIDS treatmentViral DNA packagingNanomotors

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1046-2023/$ - see front matter � 2011 Published bydoi:10.1016/j.ymeth.2010.12.039

Abbreviations: RNAi, RNA interference; siRNA , smphi29 motor packaging RNA; HIV, human immunodeglycoprotein 120.⇑ Corresponding author at: Graduate School of Biol

1500 East Duarte Rd., Duarte, CA 91010, USA. Fax: +1E-mail addresses: [email protected] (J. Zhou), s

[email protected], [email protected] (P. Guo), [email protected] (J.J. Rossi).

Please cite this article in press as: J. Zhou et al.

a b s t r a c t

The potent ability of small interfering RNA (siRNA) to inhibit the expression of complementary RNA tran-scripts is being exploited as a new class of therapeutics for diseases including HIV. However, efficientdelivery of siRNAs remains a key obstacle to successful application. A targeted intracellular deliveryapproach for siRNAs to specific cell types is highly desirable. HIV-1 infection is initiated by the interac-tions between viral glycoprotein gp120 and cell surface receptor CD4, leading to fusion of the viral mem-brane with the target cell membrane. Once HIV infects a cell it produces gp120 which is displayed at thecell surface. We previously described a novel dual inhibitory anti-gp120 aptamer–siRNA chimera inwhich both the aptamer and the siRNA portions have potent anti-HIV activities. We also demonstratedthat gp120 can be used for aptamer mediated delivery of anti-HIV siRNAs.

Here we report the design, construction and evaluation of chimerical RNA nanoparticles containing aHIV gp120-binding aptamer escorted by the pRNA of bacteriophage phi29 DNA-packaging motor. Wedemonstrate that pRNA–aptamer chimeras specifically bind to and are internalized into cells expressingHIV gp120. Moreover, the pRNA–aptamer chimeras alone also provide HIV inhibitory function by block-ing viral infectivity. The Ab0 pRNA–siRNA chimera with 20-F modified pyrimidines in the sense strand notonly improved the RNA stability in serum, but also was functionally processed by Dicer, resulting inspecific target gene silencing. Therefore, this dual functional pRNA–aptamer not only represents a poten-tial HIV-1 inhibitor, but also provides a cell-type specific siRNA delivery vehicle, showing promise forsystemic anti-HIV therapy.

� 2011 Published by Elsevier Inc.

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1. Introduction ular, a targeted intracellular delivery approach for siRNAs to spe- 58
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The global epidemic of infection by HIV-1 has created a contin-uing need for new classes of antiretroviral agents [1]. The potentability of siRNAs to inhibit the expression of complementary RNAtranscripts is being exploited as a new class of therapeutics for avariety of diseases [2,3] including HIV [4–6]. Although novel ther-apeutic strategies to combat HIV/AIDS by siRNAs show consider-able promise as shown in many previous studies [7–10], efficientdelivery of siRNAs still remains a key obstacle to its successfultherapeutic application and clinical development [11,12]. In partic-

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Elsevier Inc.

all interfering RNA; pRNA ,ficiency virus; HIV-1 gp120,

ogical Sciences, City of Hope,626 301 8271.

[email protected] (Y. Shu),[email protected] (D.D. Smith),

, Methods (2011), doi:10.1016/

cific cell populations or tissues is highly desirable for the safetyand efficacy of RNAi-based therapeutics. Moreover, the advent ofnanotechnology has greatly accelerated the development of drugdelivery, and the field of RNA-based nanotechnology has beenemerging [13].

HIV-1 infection is initiated by the interactions between theexternal envelope glycoprotein gp120 of HIV and the human cellsurface receptor CD4, subsequently leading to fusion of the viralmembrane with the target cell membrane [14–16]. Thus HIV-1 en-try into its target cells represents an attractive target for new anti-HIV-1 drug development [17–21]. We previously described a noveldual inhibitory function anti-gp120 aptamer–siRNA chimera inwhich both the aptamer and the siRNA portions have potentanti-HIV activities [22]. We also demonstrated that gp120 ex-pressed on the surface of HIV infected cells can be used for aptamermediated delivery of anti-HIV siRNAs [23]. Therefore theanti-gp120 aptamers represent a promising class of antiviralagents which can also function as siRNA delivery vehicles.

j.ymeth.2010.12.039

http://dx.doi.org/10.1016/j.ymeth.2010.12.039

mailto:[email protected]







http://www.sciencedirect.com/science/journal/10462023

http://www.elsevier.com/locate/ymeth


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Packaging RNA (pRNA), one component of the bacteriophagephi29 DNA-packaging motor [24,25], has been developed andmanipulated to produce chimeric RNAs that form dimers via inter-locking right- and left-hand loops. pRNA monomers can fold into astable and unique secondary structure that serve as the buildingblocks to form nanostructures via bottom-up assembly [13,26–29]. Fusion of the pRNA with a variety of therapeutic and chemicalcompounds did not impede the formation of dimers or interferewith function [30,31]. Incubation of cells with dimers, one subunitof which carried a gene-silencing molecule and the other a recep-tor-binding moiety, resulted in successful binding, entry, andsilencing of target genes [28,31,32]. Recently, it has been reportedthat the 20-F modified pRNA retained its property for correct fold-ing in dimer and hexamer formation, appropriate structure phi29procapsid binding, authentic function in driving the DNA-packag-ing motors, and biological activity in producing infectious virions[33]. The results suggest that it is possible to construct stable pRNAas siRNA or aptamer carrier.

We have designed, synthesized and evaluated the potential ofpRNA-gp120 aptamer chimeras as effective HIV-1 inhibitors andcell-type specific delivery vehicles. In order to ensure the dualfunctions of both cell-specific targeting and efficient siRNA deliv-ery, our delivery systems contained two structural entities: (1) ABa0 pRNA-gp120 aptamer portion (Fig. 1A) to specifically targetthe HIV infected cells and (2) An Ab0 pRNA–siRNA portion(Fig. 1B) to efficiently transport efficiently anti-HIV siRNAs. Our re-sults demonstrate that the pRNA–aptamer chimeras specificallybind to and are internalized into cells expressing HIV gp120. More-over, the pRNA–aptamer chimeras provide an HIV inhibitory func-tion which is as effective as the anti-gp120 aptamer. On the otherhand, the Ab0 pRNA–siRNA chimera with a 20-F modified sensestrand not only improves the RNA stability in serum, but is func-tionally processed by Dicer, resulting in sequence specific genesilencing. Furthermore, through Ba0 and Ab0 loop–loop interactions,Ba0 pRNA–aptamer chimeras and Ab0 pRNA–siRNA chimerasformed dimers, which allow the specific delivery of Cy3-labeledsiRNAs to cells expressing gp120. This dual functional pRNA–apt-

Fig. 1. The pRNA–aptamer mediated targeted delivery of siRNA. (A) Schematic of theresponsible for binding to HIV-1 gp120 protein and the Ba0 pRNA is shown with black. (highlighted with blue and red, respectively. (C) Schematic of the dimer of Ba0 pRNA–aptamis marked with a red circle.

Please cite this article in press as: J. Zhou et al., Methods (2011), doi:10.1016/

amer chimera not only represents a potential HIV-1 inhibitor, butalso provides a cell-type specific siRNA delivery vehicle for deliveryof anti-HIV siRNAs in systemic anti-HIV therapy.

2. Materials and methods

2.1. Materials

Unless otherwise noted, all chemicals were purchased from Sig-ma–Aldrich, all restriction enzymes were obtained from New Eng-land BioLabs (NEB) and all cell culture products were purchasedfrom GIBOC (Gibco BRL/Life Technologies, a division of Invitrogen.).Recombinant human Dicer enzyme kit (Genlantis); lipofectamine2000 (Invitrogen); HEK 293 (ATCC); CHO-WT gp160, CHO-EE cells[34,35], and the HIV-1 IIIB virions were obtained from the AIDS Re-search and Reference Reagent Program. Specific reagents and notesare listed in Supplementary Table 1.

2.1.1. siRNAssiRNA and anti-sense strand RNA were purchased from Inte-

grated DNA Technologies (IDT). Site I (tat/rev) 27 mer:Sense sequence: 50-GCG GAG ACA GCG ACG AAG AGC UCA UCA-30.Anti-sense: 50-UGA UGA GCU CUU CGU CGC UGU CUC CGC

dTdT-30.

2.2. Description of methods

2.2.1. Nomenclatures of pRNAThe 120-nt full-length pRNA contains two functional domains,

the loop–loop interlocking interaction domain and the 50/30 endhelical domain. The former is located at the central region [36–39] comprising bases 23–97, while the latter is located at the 50/30 paired ends [40]. The pRNA molecules were named as previouslydescribed [24]. Specifically, the pRNA molecules were identified bythe R and/or L loop sequences. A particular R loop sequence is as-signed an uppercase letter (i.e., A, B. . .), and a particular L loop se-

Ba0 pRNA–aptamer RNA chimera. The region of the anti-gp120 aptamer (blue) isB) Schematic of the Ab0 pRNA–siRNA chimera. The sense and anti-sense strand areer/Ab0 pRNA–siRNA. The interlocking interaction through left- and right-hand loops

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quence is assigned a lowercase letter with a prime (i.e., a0, b0. . .).The same set of letters (i.e., A and a0) designates complementaryR/L loop sequences, while different letters indicate non-comple-mentary loop sequences. The intermolecular interaction domainsmediate the interactions among pRNA subunits through hand-in-hand loop interactions, facilitating the formation of pRNA dimer,trimer, and hexamer. In our case, pRNA A-b0 represents pRNAwhere right loop A (50G45G46A47C48) is complementary to left loopa0 (30C85C84U83G82) of pRNA B-a0. And left loop b0 (30U85G84C83G82)of A-b0pRNA is complementary to the right loop B (50A45C46G47C48)of pRNA B-a0.

2.2.2. Generation of aptamer and chimera RNAs by in vitrotranscription

Regular aptamer, pRNA–aptamer chimera and pRNA–siRNA chi-mera RNAs were generated by in vitro T7 transcription as previ-ously described [22,23,41]. Briefly, Transcriptions were carriedout in a mixture containing 0.1 lM DNA template, 0.75 mM of eachNTP, 0.375 U/ll of T7 RNA polymerase, 40 mM Tris–HCl (pH 7.5),15 mM MgCl2, 5 mM DTT, 2 mM spermidine, and 0.01% (v/v) TritonX-100. The mixture was incubated at 37 �C for 3 h. The transcrip-tion products were recovered by ethanol precipitation, and purifiedusing denaturing gels. 20-F modified aptamer, pRNA–aptamer chi-mera and pRNA–siRNA chimera RNAs were also transcribedin vitro using DuraScribe� T7 Transcription kit (EPICENTRE�

Biotechnologies).The sense strands of the pRNA–siRNA chimeras are underlined.

The italic UU is the linker between the aptamer and siRNA portions.The bold nucleotides indicate aptamer sequence.

A-1 apatamer (81 nt):50-GGG AGG ACG AUG CGG AAU UGA GGG ACC ACG CGC UGC

UUG UUG UGA UAA GCA GUU UGU CGU GAU GGC AGA CGA CUCGCC CGA-30.

Ba0 pRNA–aptamer chimera (pRNA-A-1 D3) (152 nt):50-GGU UGA UUG UCC GUC AAU CAU GGC GGG AGG ACG AUG

CGG AAU UGA GGG ACC ACG CGC UGC UUG UUG UGA UAA GCAGUU UGU CGU GAU GGC AGA CGA CUC GCC CGU CAU GUG UAUGUU GGG GAU UAA CGC CUG AUU GAG UUC AGC CCA CAU AC-30.

Ba0 pRNA–aptamer chimera (pRNA-A-1 D4) (198 nt):50-GGG AGG ACG AUG CGG AAU UGA GGG ACC ACG CGC UGC

UUG UUG UGA UAA GCA GUU UGU CGU GAU GGC AGA CGA CUCGCC CGA GGA AUG GUA CGG UAC UUC CAU UGU CAU GUG UAUGUU GGG GAU UAA CGC CUG AUU GAG UUC AGC CCA CAU ACUUUG UUG AUU GUC CGU CAA UCA UGG CAA AAG UGC ACG CUACUU UCC-30.

Ab0 pRNA-Tat/rev siRNA chimera sense strand:50-GGU AUG UUG GGG AUU AGG ACC UGA UUG AGU UCA GCC

CAC AUA CUU UGU UGA UUG CGU GUC AAU UU GCG GAG ACAGCG ACG AAG AGC UCA UCA UU-30.

Tat/rev Anti-sense strand.50-UGA UGA GCU CUU CGU CGC UGU CUC CGC UU-30.

2.2.3. 50-End P32 labelingThe transcribed aptamer and pRNA–aptamer chimeras were

treated by CIP to remove the initiating 50-triphosphate and labeledwith T4 polynucleotide kinase and c-32P-ATP. Ten picomoles of CIPtreated RNAs were heat at 95 �C for 5 min and then chilled on theice. Subsequently, add 2 lL of PNK buffer, 1 lL of T4 polynucleotidekinase, 1 lL of gamma-P32-ATP and water to 20 lL. After incuba-tion at 37 �C for 30 min, add 20 lL of water and reaction was puri-fied by G-50 column. Finally, 40 lL of labeled RNA was obtained atfinal concentration of 250 nM. The labeled aptamer or pRNA-apt-amer chimera was refolded in 1� HBS buffer (10 mM HEPES, pH7.4, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2.7 mM KCl), heatedto 95 �C for 3 min and then slowly cooled to 37 �C. Incubationwas continued at 37 �C for 10 min. Store refolded RNA at �20 �C


until assay. The P32-labeled pRNA–siRNA chimera was obtainedby annealing end P32-labeled siRNA anti-sense to the other pieceby heating up to 80 �C for 5 min and then slowly cooled to roomtemperature. The annealed pRNA–siRNA chimeras were purifiedby native polyacrylamide gel and stored at �20 �C.

2.2.4. Determination of binding affinity by gel shift assaysPrepare a 25 mL 5% polyacrylamide gel by mixing 2.5 mL of 10�

TBE buffer, with 3.125 mL of 40% acrylamide/bis solution,19.375 mL water, 150 lL of 10% ammonium persulfate (APS) solu-tion, and 30 lL TEMED. The gel should polymerize in about 30 min.Carefully remove the comb and use a 30-mL syringe fitted with aneedle to wash the wells with running buffer (1� TBE). Completethe assembly of the gel unit and connect to a power supply. Thegel can be pre-run for 1 h at 180 V at 4 �C.

The HIV-1Bal gp120 protein was serially diluted with bindingbuffer to the desired concentrations. The reaction final concentra-tions of gp120 were 0, 1, 5, 10, 20, 40, 80, 160, 320, 640 nM. A con-stant amount of 50-P32-end-labeled RNA (10 nM) was incubatedwith increasing concentrations of gp120 protein in the bindingbuffer (total 20 lL reaction) on a rotating platform at room tem-perature for 30 min. After incubation, 20 lL of binding reactionwas mixed with 5 lL native loading buffer and loaded into a 5%non-denaturing polyacrylamide gel. Prepare a native loading buffer(4�) containing 10 mM Tris–HCl, pH 7.5; 1 mM EDTA, 0.1% bromo-phenol blue, 0.1% xylene cyanol FF, 0.1% orange G, 40% glycerol.Store in aliquots at �20 �C. Following electrophoresis (180 V at4 �C for 2 h, until the secondary dye runs in the middle of thegel), the gel was exposed to a Phosphor image screen and theradioactivity quantified using a Typhoon scanner. The dissociationconstants were calculated using non-linear curve regression with aGraph Pad Prism.

2.2.5. Preparation of fluorescent RNAsFluorescent aptamer and chimeras were generated using the Si-

lencer siRNA labeling kit. Add the following reagents in order:22.5 lL nuclease-free water; 5 mL 10� labeling buffer; 15 lLRNA (5 lg); 7.5 lL labeling dye. Total 50 lL labeling reaction wasincubated at 37 �C for 1 h. After incubation, add 5.0 lL (0.1 vol.)5 M NaCl and 125 lL (2.5 vol.) cold 100% EtOH, and mix thor-oughly. Incubate at �20 �C for 60 min then centrifuge at top speedat 4 �C for 20 min. Remove the supernatant and wash the pelletwith 175 lL 70% EtOH. Air dries the pellet in the dark. Suspend la-beled RNA in 15 lL of nuclease-free water. Measure the absor-bance of the labeled RNA at 260 nm and at the absorbancemaximum for the fluorescent dye. Calculate the base:dye ratioand RNA concentration according to the calculator provided byhttp://www.ambion.com/techlib/append/base_dye.html. Cy3-la-beled chimeras sense stand and anti-sense strands were mixedand refolded in refolding buffer as described above.

2.2.6. Cell cultureHEK 293 cells were cultured in DMEM supplemented with 10%

FBS. CHO-WT and CHO-EE cells were grown in GMEM-S medium(glutamine-deficient minimal essential medium with 400 lMmethionine sulfoximine). Peripheral blood mononuclear sampleswere obtained from healthy donors. PBMCs were isolated fromwhole blood by centrifugation through a Ficoll-Hypaque solution(Histopaque-1077, Sigma). CD8 cells (T-cytotoxic/suppressor cells)were depleted from the PBMCs by CD8 Dynabeads (Invitrogen, CA)according to the manufacturer’s instructions. CD8+ T cell-depletedPBMCs were washed twice in PBS and resuspended in culture med-ia (RPMI 1640 with 10% FBS, 1� PenStrep and 100 U/mL interleu-kin-2). Cells were cultured in a humidified 5% CO2 incubator at37 �C.

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A-1 4D1-A-ANRP3D1-A-ANRP

0 5 10 20 40 80 160 320 640 0 1 5 10 20 40 80 160 320 640 0 1 5 10 20 40 80 160 320 640

A-1 pRNA-A-1 D3 pRNA-A-1 D4 KD 47.91 ± 7.55 48.60 ± 7.84 78.72 ± 7.34 nM

Gel shift assay

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Fig. 2. The design and binding activity of the pRNA–aptamer chimeras. (A) Schematic of Ba0 pRNA–Aptamer RNAs (pRNA-A-1 D3 and pRNA-A-1 D4) and RNA sequences. TheA-1 aptamer sequence was inserted into the 30/50 double helical domain (23 nt fragment) and loop domain (97 nt fragment) of Ba0 pRNA to obtain pRNA-A-1 D3 construct. Theintact Ba0 pRNA sequence was directly appended to the 30-end of the A-1 aptamer to obtain pRNA-A-1 D4 construct. The pRNA fragments (50/30 double-stranded helicaldomain; right hand loop and left hand loop) are highlighted by a clear box; the intermolecular interacting domain is highlighted by a gray box. (B) Binding affinity detected bya gel shift assay. The 50-end P32 labeled aptamers or Ba0 pRNA–aptamer chimeras were incubated with increasing amounts of gp120 protein. The binding reaction mixtureswere analyzed by a gel mobility shift assay and the Kd determinations are indicated. The Ba0 pRNA–aptamers showed a comparable binding affinity with the target protein aswell as aptamer alone. (C) Cell-type specific binding studies of pRNA-aptamer chimeras. Cy3-labeled Ba0 pRNA–aptamers were incubated with CHO-WT gp160 cells and CHO-EE control cells and cell-surface binding of Cy3-labeled chimeras was assessed by flow cytometry. (D and E) Internalization and localization analysis. CHO-WT gp160 cells (D)or CHO-EE cells (E) were grown in 35 mm plates and incubated with a 100 nM concentration of Cy3-labeled pRNA-A-1 D4 chimera in culture media for real-time live-cell-confocal microscopy analysis. The images were collected at 20 min intervals using 40� magnification. After 5 h incubation with 100 nM of Cy3-labeled chimera, cells weresubsequently stained with Hoechst 33342 (nuclear dye for live cells) and then imaged using real-time confocal microscopy.



28 January 2011

2.2.7. Cell-surface binding studies by flow cytometryThe CHO-WT gp160 or CHO-EE control cells were washed with

pre-warmed washing buffer (DPBS), trypsinized and detached from


the plates. After washing cells twice with 500 lL binding buffer(10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,2.7 mM KCl and 0.01% BSA), cell pellets were resuspended in

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(D)

(E)

10 min incubation

5 hours incubation

Before Nuclear staining After Nuclear staining

CHO-WT gp160 cells + Cy3 pRNA-A-1 D-4

CHO-EE control cells + Cy3 pRNA-A-1 D-4

10 min incubation

5 hours incubation

Before Nuclear staining After Nuclear staining

Flow Cytometry

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Sh

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CHO-WT gp160

CHO-EE(C)

Fig. 2 (continued)



28 January 2011

binding buffer and incubated at 37 �C for 30 min. Cells were thenpelleted and resuspended in 50 lL of pre-warmed binding buffercontaining 400 nM Cy3-labeled experimental RNAs. After incuba-tion at 37 �C for 40 min, cells were washed three times with500 lL of pre-warmed binding buffer, and finally resuspended in350 lL of binding buffer pre-warmed to 37 �C and immediatelyanalyzed by flow cytometry.

2.2.8. Cell internalization and localization studies by real-time confocalmicroscopy

The CHO-WT gp160 and CHO-EE cells were grown in 35 mmplate (Glass Bottom Dish, MatTek, Ashland, MA) with seeding at0.3 � 106 in GMEM-S medium to allow about 70% confluence in24 h. On the day of the experiments, cells were washed with1 mL of pre-warmed PBS and were incubated with 1 mL of pre-warmed complete growth medium for 30 min at 37 �C. Cy3-labeledRNAs at a 100 nM final concentration were added to the media andincubated for live-cell confocal microscopy in a 5% CO2 microscopyincubator at 37 �C. The images were collected every 20 min using a


Zeiss LSM 510 Meta Inverted 2 photon confocal microscope underwater immersion at 40�magnification. After 5 h of incubation andimaging, the cells were stained by treatment with 0.15 mg/mLHoechst 33342 (nuclear dye for live cells, Molecular Probes, Invit-rogen, CA) according to the manufacturer’s instructions. Theimages were collected as described previously.

2.3.9. In vitro HIV-1 challenge and p24 antigen assayHIV-1 NL4-3 virus was obtained from the AIDS Research and

Reference Reagent Program. After propagation of virus, store in ali-quots at�80 �C. Human PBMCs were infected with HIV NL4-3 virus(MOI 0.001). After 24 h of infection, cells were gently washed withPBS three times to remove free virus. Continue to culture the in-fected cells in a 5% CO2 microscopy incubator at 37 �C for 4 days.

Prior to RNA treatments the infected cells were gently washedwith PBS three times to remove free virus. 2 � 104 infected cellsand 3 � 104 uninfected cells were incubated with refolded experi-mental RNAs at 400 nM final concentration in 96-well plates at37 �C (100 lL per well, triplex assay). The culture supernatants

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(10 lL per well) were collected at different times (4 d, 6 d, 8 d and10 d) and stored at �20 �C until p24 assay. The p24 antigen analy-ses were performed using a HIV-1 p24 Antigen ELISA kit.

2.2.10. Serum stability assayFive micrograms of both regular and 20 F modified pRNA–siRNA

chimera were incubated at 37 �C in 100 lL RPMI 1640 Mediumcontaining 10% (v/v) FBS, resulting in a concentration of 50 ng/lLRNA. At times 0 min to 12 h, 10 lL aliquots of the reaction waswithdrawn and 2 lL of 6� loading buffer added to each sample.Twelve microliters of each mixture was analyzed by electrophore-sis in an 8% native polyacrylamide gel and the RNAs werevisualized following ethidium bromide staining using a UV-transilluminator.

2.2.11. The siRNA function detection by psiCHECK dual luciferase assayBefore day one of the assay, the HEK293 cells were grown in 24-

well plate with seeding at 0.8 � 105 in 400 lL DMEM medium toallow about 70% �80% confluence in 24 h. Nucleic acid mixturescontaining 100 ng of siCHECK target derivative, 25 nM of experi-mental RNAs in 50 lL OptiMEM were cotransfected into HEK 293cells. After 24 h of post-transfection, the reporter gene expressionwas tested by Dual-luciferase Reporter Assay System (Promega,USA) according to the manufacturer’s instructions. All sampleswere cotransfected in triplicate and the experiment performed aminimum of three times. For each replicate, the Renilla-luc targetreading was normalized internally to the Firefly-luc value, and anaverage value was calculated from the replicates.

2.2.12. In vitro Dicer assaysThe anti-sense strands were end-labeled with T4 polynucleotide

kinase and c-32P-ATP. Subsequently, corresponding chimera sensestrands are annealed with equimolar amounts of 50-end-labeledanti-sense strands in HBS buffer to form the chimeras. The annealedpRNA–siRNA chimeras (1 pmol) were incubated at 37 �C for 40 minin the presence or in the absence of 1 U of human recombinant Di-cer enzyme following the manufacturer’s recommendations (Ambi-on, Austin, TX). Reactions were stopped by phenol/chloroformextraction and the resulting solutions electrophoresed in a denatur-ing 15% polyacrylamide gel. The gels were subsequently exposed toX-ray film.

HIV-1 Challenge (PBM

1.00E+05

1.50E+05

2.00E+05

2.50E+05

3.00E+05

3.50E+05

4.00E+05

4.50E+05

5.00E+05

4 day 6 day

Days of Post

HIV

-1 p

24 le

vel (

pg/m

L)

Fig. 3. The inhibition of HIV-1 infection mediated by pRNA-aptamer chimeras. Both aninfected human PBMCs (NL4-3 strain) culture. Data represent the average of triplicate mehuman PBMCs with comparable inhibitory potency as original A-1 aptamer. Aptamer A-5control. P values for the effects of A-1 and pRNA–aptamer chimera are indicated and w


2.2.13. Formation of dimer of pRNA–aptamer chimera/pRNA–siRNAchimera (gel shift assay)

Eight percent of native polyacrylamide gels were prepared inTBM buffer (89 mM Tris, 200 mM boric acid, 1 mM MgCl2, pH7.6). Ba0 pRNA-A-1 chimeras and Ab0 pRNA–siRNA chimeras wererefolded in 1� HBS buffer as described above. Equal amounts ofthe refolded Ba0 pRNA-A-1 chimeras and Ab0 pRNA–siRNA chimeraswere mixed and directly analyzed by electrophoresis in an 8% na-tive polyacrylamide gel. After running at 4 �C for 2 h using TBMrunning buffer, the RNAs were visualized following ethidium bro-mide staining using a UV-transilluminator.

2.2.14. Statistical methods for HIV-1 challenge p24 assayWe tested for differences in the mean and areas under the curve

with the Student t-test. To determine if the Ba0 pRNA-gp120 apt-amer chimeras would also block HIV infectivity in cell culture,we tested for parallel slopes with the Chow test for heterogeneouslinear regression.

3. Results

3.1. Ba0 pRNA-gp120 aptamer chimeras bind to the HIV-1 gp120protein and the cells expressing HIV gp160

Two different chimeric Ba0 pRNA–anti-gp120 aptamer con-structs (pRNA-A-1 D3 and pRNA-A-1 D4) were designed as shownin Fig. 2A. Specifically, for the pRNA-A-1 D3 construct, the A-1 apt-amer sequence was linked to the base 23 and 97 of Ba0 pRNA sincethe bases 23–97 are the minimum fragment required for stable di-mer formation [41]. The constructed chimeric pRNA–aptamer se-quence has the new 50/30 end relocated to bases 71/75 of thepRNA [42]. For the pRNA-A-1 D4 construct, the A-1 aptamer wasdirectly appended to the 50-end of the Ba0 pRNA sequence. Bothconstructs are shown to fold properly and maintain the capabilityto interact with its Ab0 pRNA partners.

To enhance the stability of these chimeric RNAs in cell cultureand in vivo, the Ba0 pRNA–aptamer contained nuclease-resistant20-fluoro UTP and 20-Fluoro CTP and were synthesized from thecorresponding double stranded DNA templates by in vitro T7 bac-teriophage transcription. The binding affinities of the Ba0 pRNA–

C CD4 and NL4-3)

8 day 10 day

-treatment

Buffer control 27 mer siRNAAptamer A-1pRNA-A-1 D4Aptamer A-5Infected PBMC

pRNA-A-1 D4 vs. Buffer control: P = 0.0302Aptamer A-1 vs. Buffer control: P = 0.0055

ti-gp120 aptamer and pRNA-aptamer chimera neutralized HIV-1 infection in HIVasurements of p24. Ba0 pRNA-gp120 aptamer chimera D4 inhibits HIV-1 infection ofthat has previously been shown to have poor affinity for gp120 is used as a negative

ere calculated as described in Section 2.

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28 January 2011

aptamer chimeric RNAs for HIV-1 gp120 protein was assessed byusing a gel shift assay and flow cytometry.

First, the dissociation constants (Kd) for Ba0 pRNA-aptamer chi-meras with the target protein HIV-1Bal gp120 were calculated froma native gel mobility shift assay (Fig. 2B). They showed good bind-ing kinetics to the gp120 protein. The apparent Kd values of the A-1aptamer alone, pRNA-A-1 D3 and pRNA-A-1 D4 were about 48 nM,49 nM and 79 nM, respectively.

Next, CHO-WT cells stably expressing the HIV envelope glyco-protein gp160 (of which gp120 is a component) were used to testfor binding of the pRNA–aptamer chimeras. These cells do not pro-cess gp160 into gp120 and gp41 since they lack the gag encodedproteases required for envelope processing. As a control we usedthe parental CHO-EE cell line which does not express gp160. Theanti-gp120 aptamer A-1 and the Ba0 pRNA–aptamer chimeraswere labeled with Cy3 to follow their binding and uptake. Flowcytometric analyses (Fig. 2C) revealed that the aptamer andchimeras specifically bind to the CHO-gp160 cells. These data indi-

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Fig. 4. The design and evaluation of Ab0 pRNA–siRNA chimeras. (A) Schematic of the Aexample here, the siRNA targets HIV-1 tat/Rev. The chimeric pRNA–siRNA sense strandsense strand to get the whole pRNA–siRNA chimera. A linker (UU) between the aptamer aFBS of the 20-F modified pRNA–siRNA chimeras vs. unmodified pRNA–siRNA chimerasluciferase assays of psiCHECK sense and anti-sense targets are shown. All RNAs were normthe averages of triplicate assays.


cate that the Ba0 pRNA–aptamer chimeras maintain approximatelythe same binding affinities as the parental aptamers alone. In or-der to determine if the chimeras were internalized in the gp160expressing cells, we also carried out real-time live-cell Z-axis con-focal microscopy with the CHO-WT gp160 cells incubated with theCy3-labeled pRNA-A-1 D4 chimera (Fig. 2D). These cells expressthe precursor for gp120, gp160 in which the gp41 portion hasnot been processed by the HIV-1 gag protease since the cells onlyare transfected with the envelope gene. The gp120 glycoprotein isexposed on the cell surface whereas the gp41 segment is withinthe cell membrane. After 5 h of incubation, the Cy3-labeled con-struct was selectively internalized within the CHO-WT gp160 cells,but not the CHO-EE control cells (Fig. 2E). To visualize the nucleus,the cells were stained with the nuclear dye Hoechst 33342 afterincubation with the Cy3 labeled chimera. Fig. 2D showed thatthe chimera aggregated within the cytoplasm suggesting thatthe gp120 aptamers maybe enter cells via receptor-mediatedendocytosis.

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b0 pRNA–siRNA chimera. The siRNA part of the chimera consists of 27 bps. As anwas transcribed in vitro with T7 RNA polymerase and then annealed with the anti-nd siRNA is indicated. (B) The stability assay in cell culture medium containing 10%

. (C) Gene silencing activity and strand selectivity of pRNA–siRNA chimeras. Dualalized to the value of the corresponding buffer control. All the data were represent

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3.2. Ba0 pRNA-gp120 aptamer chimeras inhibit HIV-1 infection ofhuman PBMCs

In a previous study investigators demonstrated that anti-gp120aptamers provided significant anti-HIV function via binding to theviral envelope and preventing viral interaction with the cellularCD4 receptor [43–45]. We therefore carried out an assay to deter-mine if the Ba0 pRNA-gp120 aptamer chimeras would also blockHIV infectivity in cell culture. In this assay, the aptamer and chime-ras were incubated with HIV-1 infected primary human PBMCsfour days after the cells were challenged with the virus. At differentdays post treatment with the aptamers, aliquots of the media wereassayed for viral p24 antigen levels (Fig. 3). The results showedthat the Ba0 pRNA-gp120 aptamers (pRNA-A-1 D4) inhibited HIV-1 p24 production and provided comparable inhibitory potency aswell as the original A-1 aptamer, reaching statistical significance(P = 0.0302 and P = 0.0055, respectively).

3.3. Ab0 pRNA-siRNA chimeras improve RNase resistance andspecifically knock-down target gene expression

It has been demonstrated that the double-stranded helical do-main at the 50/30 end and the intermolecular interaction domain(loop–loop region) in the pRNA can fold independently of eachother, and altering the primary sequences of the 50/30 helical do-main in the pRNA does not affect its folding structure as long asthe two strands are base-paired [24,27]. Because of this tolerabil-ity, the double-stranded helical region of the pRNA can be replacedwith a duplex RNA (e.g. siRNA) (Fig. 1B), to introduce additionalfunctionalities to pRNA nanoparticles. Since the synthetic Dicersubstrate duplexes of 25–30 nt have been shown to enhance RNAipotency and efficacy [46], we chose a 27 mer Dicer substrate RNAas the siRNA portion of the chimeric molecule. The 27 mer siRNAportion of chimeras targets the HIV-1 tat/rev common exon se-quence. In the design we inserted a two nucleotide linker (UU) be-tween the Ab0 pRNA and siRNA portion to minimize stericinterference of the pRNA portion with Dicer.

As described above, the Ab0 pRNA and sense strand segment ofthe siRNAs contained nuclease-resistant 20-Fluoro UTP and 20-Flu-oro CTP and were synthesized from corresponding dsDNAtemplates by in vitro T7 transcription. To prepare the siRNA con-taining chimeras, in vitro transcribed chimeric Ab0 pRNA-

3029

23

29 nt fragm

21 nt fragm23 nt fragm

- + Dicer

Ladder

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chimera

Regular pRNA-siRNA

chimera

30 29 23 nt - + Dicer

Fig. 5. In vitro Dicer processing. Dicer cleavage of 50-end P32 anti-sense labeled RNAs [47molar equivalents of 50-end P32-labeled complementary anti-sense RNA strands. The cdenaturing polyacrylamide-gel electrophoresis.


sense strand polymers were annealed with equimolar concentra-tions of an unmodified anti-sense strand RNA (Fig. 4A). In thedesign, 20-Fluoro modified chimeras were stable in cell-culturemedia containing 10% FBS for up to 12 h whereas the unmodi-fied chimeric RNAs were degraded within several minutes(Fig. 4B).

Because of competition by the sense (passenger) strand withthe anti-sense (guide) strand for RISC entry, the strand selectivityis an important factor for evaluating siRNAs. Therefore, we evalu-ated the Ab0 pRNA-tat/rev siRNA chimera RNA using the pSiCheckreporter system, which readily allows screening of the potenciesof candidate sh/siRNAs. The gene silencing of both the sense target(corresponding to the mRNA) and the anti-sense target were testedindependently and the selectivity ratios calculated as a measure ofthe relative incorporation of each strand into the RISC. The compar-ison (Fig. 4C) demonstrated that the Ab0 pRNA-tat/rev siRNA chi-mera mediated �83% knockdown of the sense target; however,knockdown of the anti-sense target is much less (�63%), indicativeof good strand selection (R = 2.3).

3.4. Ab0 pRNA–siRNA chimeras are processed by Dicer

We next detected whether or not the siRNAs could be processedfrom the chimeric pRNA–siRNA in vitro by recombinant human Di-cer. This set of experiments used a 50-end P32 labeled anti-sensestrand to follow Dicer processing (Fig. 5). The size of the P32 labeledcleavage product(s) indicates the direction Dicer enters the siRNAand cleaves [47]. When pRNA-siRNA chimeras were incubatedwith the recombinant human Dicer we observed that the 29 ntanti-sense strand (containing 2 nt 30-overhang) was processed intoa 21–23 nt siRNA. This result suggests Dicer processing preferen-tially enters from the 50 end of the anti-sense strand and cleaves21 to 23 nt downstream, leaving the 30 end of the anti-sense strandintact. These data suggest that the Ab0 pRNA-siRNA chimeras canbe processed by Dicer and result in an siRNA that guides targetgene silencing.

3.5. Ba0 pRNA-aptamer chimeras and Ab0 pRNA-siRNA chimeras formdimers through Ba0 and Ab0 loop interaction

In order to determine whether or not the Ba0 pRNA–aptamerchimeras and Ab0 pRNA–siRNA chimeras can form dimers, we car-

UU3’Sense

Antisense

UU5’3’UU

pRNA-siRNA chimera

P325’

Dicer processing

UU3’ Sense

Antisense

5’

3’P325’

21-23 mer siRNA

ent

entent

]. The RNA sense strands (20-F modified or regular RNA) were annealed with equalleavage products or un-cleaved, denatured strands were visualized following 15%

j.ymeth.2010.12.039


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1) 2’-F Ab’ pRNA-siRNA chimera (124 nt)2) 2’-F Ab’ pRNA (120 nt)3) 2’-F Ba’ pRNA (120 nt)4) 2’-F Ba’ pRNA-aptamer chimera D3 (152 nt)5) 2’ F Ba’ pRNA-aptamer chimera D4 (198 nt)

6) 2’-F Ab’ pRNA-siRNA chimera + D3 7) 2’-F Ab’ pRNA-siRNA chimera + D48) 2’-F Ab’ pRNA + 2’-F Ba’ pRNA9) Regular Ab’ pRNA-siRNA chimera + D3

2’-F modified pRNAmonomer

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sense +antisense

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Fig. 6. Dimerization study of Ba0 pRNA–aptamer and Ab0 pRNA–siRNA chimeras. (A) Formation of dimmers composed of Ba0 pRNA–aptamer chimeras and Ab0 pRNA–siRNAchimeras through the interaction of right and left hand loops of Ba0 and Ab0 in gel shift analysis at 1 mM Mg2+. (B) In vitro Dicer processing of dimers derived from 50-end P32

RNAs [47]. Ba0 pRNA–aptamer D4 and Ab0 pRNA–siRNA chimers were mixed to form dimers and incubated with Dicer. ‘‘D’’ means dimer; ‘‘M’’ means monomer; ‘‘+’’ meanswith Dicer; ‘‘�’’ means without Dicer. The cleavage products (arrows) or un-cleaved, denatured strands were visualized following 15% denaturing polyacrylamide-gelelectrophoresis. (C) Flow cytometry assay for cell-type specific binding of dimer. Ba0 pRNA–aptamers or Ab0 pRNA–siRNA chimeras with Cy3 label at either the pRNA-sense oranti-sense strands were tested for binding to CHO-WT gp160 cells and CHO-EE control cells.



28 January 2011

ried out the gel shift assay to monitor dimerization. In this study,the mixing of one pair of pRNAs with interlocking loops was car-ried out to test whether the Ba0 pRNA–aptamer and Ab0 pRNA–siR-


NA chimeras were still able to form dimers through loop–loopinteractions. As shown in Fig. 6A, dimers could form betweentwo loops (Ab0 pRNA and Ba0 pRNA) at different concentration of

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28 January 2011

Mg2+ (1 mM is shown as example). Compared with the pRNA alone,the additional aptamer or siRNA segments in the chimeras some-what reduced the efficiency of dimerization (Fig. 6A).

The in vitro Dicer assay [47] was also performed to verify thatDicer can process the siRNA portion after dimer formation(Fig. 6B). Similarly, the dimers of Ba0 pRNA–aptamer and Ab0

pRNA–siRNA chimeras were preferentially cleaved from the 50-end labeled anti-sense side resulting in the 32P-labeled 21–23 ntspecies.

Next, to examine whether or not the Ba0 pRNA–aptamer selec-tively delivers Ab0 pRNA–siRNA chimeras to the cells expressinggp160, we performed the flow cytometric analysis (Fig. 6C) as pre-vious described. As expected, the Cy3-labeled Ba0 pRNA–aptamerspecifically bound to the CHO-gp160 cells but did not bind to thecontrol CHO-EE cells. The Ab0 pRNA-siRNA chimeras in whicheither the chimera pRNA-sense or anti-sense strands were labeledwith Cy3 and subsequently used in formation of the chimeras, didnot show differences between the two cell lines. However, thecombination of the Ba0 pRNA-aptamer D4 with the Cy3-labeledAb0 pRNA–siRNA chimera resulted in a threefold binding specificitywith the CHO-gp160 cells as compared with the CHO-EE controlcells. These results indicate that the pRNA–siRNA chimera isdelivered by the pRNA–aptamer to gp160 expressing cells viadimerization.

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4. Discussion

The essential feature of the RNAi mechanism is the sequence-specificity, which derives from complementary Watson–Crick basepairing of a target messenger RNA (mRNA) and the guide strand ofthe siRNA [48]. Within the past decade, RNA interference hasshown great potential as a therapeutic modality for various dis-eases. HIV-1 became one of the first infectious agents targeted byRNAi due to its well-understood life cycle and pattern of geneexpression. However, getting the technique to work in vivo will re-quire targeted delivery of siRNAs to virally infected cells or cellswhich are targets for HIV-1 infection.

We have capitalized upon the exquisite specificity of an anti-gp120 aptamer to deliver anti-HIV-1 siRNAs to HIV-1 infected cells,thereby resulting in a dual inhibitory function of replication andspread of HIV by the combined action of the aptamer and siRNAtargeting the tat/rev common exon of HIV-1. This system providesa highly selective approach for inhibiting HIV-1 replication thatcombines siRNA delivery with the inhibitory properties of ananti-HIV-1 envelope aptamer. On the other hand, pRNA has beenreported to have novel applications in nanotechnology and nano-medicine [13]. Through the interaction of two reengineered inter-locking loops, pRNAs are able to form nanoscale dimers, trimers,hexamers and patterned superstructures. This unique ability ofpRNA makes it a versatile polyvalent vehicle to combine siRNAsand other therapeutic molecules and may be applied as a therapeu-tic nano-platform which can improve drug solubility and pharma-cokinetic (PK)/distribution properties.

In the present study, we therefore took advantages of the gp120aptamer binding affinity for HIV-1 gp120 and the ability of pRNA toform dimers to explore the potential use of chimeric pRNA–apta-mers for delivery of anti-HIV siRNAs into HIV-1 infected cells. Inthis delivery system, the Ba0 pRNA and Ab0 pRNA were successfullyfused with an anti-gp120 aptamer and an HIV siRNA against HIV-1tat/rev, respectively. The resultant chimeric pRNA–aptamer andpRNA–siRNA did not interfere with the function of either moiety.We designed two different pRNA–aptamer chimeras D3 and D4(as shown in Fig. 2A). Although the linkage strategy is different,both constructs are shown to fold properly and maintain the capa-bility to interact with its Ab0 pRNA partners. Moreover, our results


demonstrate that the pRNA–aptamer chimera specifically binds tocells expressing HIV gp120 and provides inhibition of HIV-1 repli-cation comparable to the original anti-gp120 aptamer, suggestingfusion of the pRNA with aptamers does not impede the formationof dimers or interfere with binding affinity of the aptamer.

In our design of the Ab0 pRNA–siRNA chimera 20-Fluoro back-bone modifications of pyrimidines were incorporated into thepRNA, aptamer and siRNA sense strand. Although the siRNA anti-sense has no chemical modification, the pRNA–siRNA chimeraswere stabilized by virtue of the non-modified guide strand basepairing to the modified sense strand. In contrast, unmodifiedpRNA–siRNA chimeras were quickly degraded in several minutesin sera. Moreover, the Ab0 pRNA–siRNA chimera also is functionallyprocessed by Dicer and specifically induced target gene silencingwith reasonably good strand selectivity. Through the Ba0 and Ab0

loop-loop interactions, the Ba0 pRNA–aptamer chimeras and Ab0

pRNA-siRNA chimeras formed dimers, which facilitate the specificdelivery of Cy3-labeled chimeric pRNA–siRNA to cells expressinggp120. However, compared with pRNA alone, the additional apt-amer or siRNA segments in the chimeras reduced somewhat theefficiency of dimerization. Although our results indicate the poten-tial of pRNA–aptamer chimera as an anti-HIV agent and a targeteddelivery vector for siRNA, further optimization and evaluation, forexample using crosslinking of the dimers to improve stability willbe necessary to optimize the dual function inhibition afforded bythe aptamer and siRNA.

In conclusion, this dual functional pRNA–aptamer chimera notonly represents a potential HIV-1 inhibitor, but also provides acell-type specific siRNA delivery vehicle, showing considerablepromise for systemic anti-HIV therapy. Future work will aim atoptimizing the dimerized delivery system. This system might beprecisely tailored as a multifunctional nanomedicine with optimalsize and pharmacokinetic properties. Although the HIV-1 gp120aptamer and anti-HIV-1 siRNA were discussed as representativeexamples for fusion with the pRNAs, to take advantage of pRNA-assembled nanoparticles, this combinatorial approach also can begeneralized with different cell internalizing aptamers and thera-peutics for the treatment of other diseases.

Disclosures (competing interests)

J.R. and J.Z. have a patent pending on ‘‘Cell-type specific apt-amer–siRNA delivery system for HIV-1 therapy’’. USPTO, No. 12/328994, publication date: June 11, 2009. P.G. is the inventor of pat-ents on phi29 motor pRNA chimera and a co-founder of Kylin Ther-apeutics, Inc.

Acknowledgments

We thank Haitang Li and Jang Zhang for HIV-1 challenge assay.This work was supported by grants from the National Institutes ofHealth awarded to J.J.R and P.X.G. This work was supported by theNIH Nanomedicine Development Center on Phi29 DNA-packagingMotor for Nanomedicine, through the NIH Roadmap for MedicalResearch (PN2 EY 018230) as well as NIH Grants AI29329 to J.J.R.and EB003730 to P.G. The following reagents were obtainedthrough the NIH AIDS Research and Reference Reagent Program,Division of AIDS, NIAID, NIH: The CHO-EE and CHO-gp160 cell line;HIV NL4-3 virus; HIV-1BaL gp120.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ymeth.2010.12.039.

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References

[1] D.D. Richman, D.M. Margolis, M. Delaney, W.C. Greene, D. Hazuda, R.J.Pomerantz, Science 323 (2009) 1304–1307.

[2] D.H. Kim, J.J. Rossi, Nat. Rev. Genet. 8 (2007) 173–184.[3] A. de Fougerolles, H.P. Vornlocher, J. Maraganore, J. Lieberman, Nat. Rev. Drug

Discov. 6 (2007) 443–453.[4] J.J. Rossi, C.H. June, D.B. Kohn, Nat. Biotechnol. 25 (2007) 1444–1454.[5] L. Scherer, J.J. Rossi, M.S. Weinberg, Gene Ther. 14 (2007) 1057–1064.[6] S. Subramanya, S.S. Kim, N. Manjunath, P. Shankar, Expert Opin. Biol. Ther. 10

(2010) 201–213.[7] P. Kumar, H.S. Ban, S.S. Kim, H. Wu, T. Pearson, D.L. Greiner, A. Laouar, J. Yao, V.

Haridas, K. Habiro, Y.G. Yang, J.H. Jeong, K.Y. Lee, Y.H. Kim, S.W. Kim, M. Peipp,G.H. Fey, N. Manjunath, L.D. Shultz, S.K. Lee, P. Shankar, Cell 134 (2008) 577–586.

[8] Z. Liu, M. Winters, M. Holodniy, H. Dai, Angew. Chem. Int. Ed. Engl. 46 (2007)2023–2027.

[9] N. Weber, P. Ortega, M.I. Clemente, D. Shcharbin, M. Bryszewska, F.J. de laMata, R. Gomez, M.A. Munoz-Fernandez, J. Control. Release 132 (2008) 55–64.

[10] A. Eguchi, B.R. Meade, Y.C. Chang, C.T. Fredrickson, K. Willert, N. Puri, S.F.Dowdy, Nat. Biotechnol. 27 (2009) 567–571.

[11] D. Castanotto, J.J. Rossi, Nature 457 (2009) 426–433.[12] K.A. Whitehead, R. Langer, D.G. Anderson, Nat. Rev. Drug Discov. 8 (2009) 129–

138.[13] P. Guo, Nat. Nanotechnol. 5 (2010) 833–842.[14] A.G. Dalgleish, P.C. Beverley, P.R. Clapham, D.H. Crawford, M.F. Greaves, R.A.

Weiss, Nature 312 (1984) 763–767.[15] D. Klatzmann, E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J.C.

Gluckman, L. Montagnier, Nature 312 (1984) 767–768.[16] P.D. Kwong, R. Wyatt, J. Robinson, R.W. Sweet, J. Sodroski, W.A. Hendrickson,

Nature 393 (1998) 648–659.[17] S. Ugolini, I. Mondor, Q.J. Sattentau, Trends Microbiol. 7 (1999) 144–149.[18] R. Wyatt, J. Sodroski, Science 280 (1998) 1884–1888.[19] D.C. Chan, P.S. Kim, Cell 93 (1998) 681–684.[20] W.S. Blair, P.F. Lin, N.A. Meanwell, O.B. Wallace, Drug Discov. Today 5 (2000)

183–194.[21] J.P. Moore, W. Doms, Proc. Natl Acad. Sci. USA 100 (2003) 10598–10602.[22] J. Zhou, H. Li, S. Li, J. Zaia, J.J. Rossi, Mol. Ther. 16 (2008) 1481–1489.

698


[23] J. Zhou, P. Swiderski, H. Li, J. Zhang, C.P. Neff, R. Akkina, J.J. Rossi, Nucleic AcidsRes. (2009).

[24] P. Guo, C. Zhang, C. Chen, K. Garver, M. Trottier, Mol. Cell 2 (1998) 149–155.[25] P.X. Guo, S. Erickson, D. Anderson, Science 236 (1987) 690–694.[26] S. Hoeprich, P. Guo, J. Biol. Chem. 277 (2002) 20794–20803.[27] D. Shu, L.P. Huang, S. Hoeprich, P. Guo, J. Nanosci. Nanotechnol. 3 (2003) 295–

302.[28] A. Khaled, S. Guo, F. Li, P. Guo, Nano Lett. 5 (2005) 1797–1808.[29] D. Shu, W.D. Moll, Z. Deng, C. Mao, P. Guo, Nano Lett. 4 (2004) 1717–1723.[30] P. Guo, J. Nanosci. Nanotechnol. 5 (2005) 1964–1982.[31] S. Guo, N. Tschammer, S. Mohammed, P. Guo, Hum. Gene Ther. 16 (2005)

1097–1109.[32] L. Li, J. Liu, Z. Diao, D. Shu, P. Guo, G. Shen, Mol. BioSyst. 5 (2009) 1361–1368.[33] J. Liu, S. Guo, M. Cinier, L.S. Shlyakhtenko, Y. Shu, C. Chen, G. Shen, P. Guo, ACS

Nano (2010).[34] C.D. Weiss, J.M. White, J. Virol. 67 (1993) 7060–7066.[35] M.A. Vodicka, W.C. Goh, L.I. Wu, M.E. Rogel, S.R. Bartz, V.L. Schweickart, C.J.

Raport, M. Emerman, Virology 233 (1997) 193–198.[36] R.J. Reid, J.W. Bodley, D. Anderson, J. Biol. Chem. 269 (1994) 5157–5162.[37] R.J. Reid, F. Zhang, S. Benson, D. Anderson, J. Biol. Chem. 269 (1994) 18656–

18661.[38] C. Chen, S. Sheng, Z. Shao, P. Guo, J. Biol. Chem. 275 (2000) 17510–17516.[39] K. Garver, P. Guo, RNA 3 (1997) 1068–1079.[40] C. Zhang, C.S. Lee, P. Guo, Virology 201 (1994) 77–85.[41] C. Chen, C. Zhang, P. Guo, RNA 5 (1999) 805–818.[42] C. Zhang, T. Tellinghuisen, P. Guo, RNA 3 (1997) 315–323.[43] M. Khati, M. Schuman, J. Ibrahim, Q. Sattentau, S. Gordon, W. James, J. Virol. 77

(2003) 12692–12698.[44] A.K. Dey, M. Khati, M. Tang, R. Wyatt, S.M. Lea, W. James, J. Virol. 79 (2005)

13806–13810.[45] C. Cohen, M. Forzan, B. Sproat, R. Pantophlet, I. McGowan, D. Burton, W. James,

Virology 381 (2008) 46–54.[46] D.H. Kim, M.A. Behlke, S.D. Rose, M.S. Chang, S. Choi, J.J. Rossi, Nat. Biotechnol.

23 (2005) 222–226.[47] S. Guo, F. Huang, P. Guo, Gene Ther. 13 (2006) 814–820.[48] A. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello, Nature 391

(1998) 806–811.

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