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. 2010 Jan 26;18(4):796–802. doi: 10.1038/mt.2009.321

Rational Design of Micro-RNA-like Bifunctional siRNAs Targeting HIV and the HIV Coreceptor CCR5

Ali Ehsani 1, Pål Saetrom 2,3,4, Jane Zhang 5, Jessica Alluin 1, Haitang Li 1, Ola Snøve Jr 1,3, Lars Aagaard 1, John J Rossi 1,5
PMCID: PMC2862539  PMID: 20104214

Abstract

Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3′ untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

Introduction

Small-interfering RNAs (siRNAs) mediate sequence-specific gene silencing by directing site-specific cleavage of targeted mRNAs harboring perfect or near-perfect complementarity to the siRNA.1 Like endogenous micro-RNAs (miRNAs),2 siRNAs can also trigger translational suppression of messages harboring partial complementarity to the “seed sequence” within the 3′ untranslated region (UTR).3,4,5 For siRNAs, translational suppression is normally considered an unwanted source of off-target effects, as siRNAs are designed to have perfect complementarity to a single target gene.6,7,8,9,10 Translational suppression by miRNA or siRNA guide strands is generally driven by base-pairing between the mRNA 3′ UTR and nucleotides 2–8 from the guide strand's 5′ end. This region, the seed region, is critical for miRNA targeting,11,12,13,14,15,16,17,18,19 but factors that characterize a seed site's sequence context, such as accessibility,20,21 location in 3′ UTR,22,23 and additional base pairs between a miRNA and mRNA,19 influence the regulatory potential of each site. Moreover, a flanking base pair at miRNA position 8 or an Adenosine at position 116 enhances downregulation.23

The number of seed sites within a 3′ UTR is another important determinant for miRNA regulatory potential. Multiple target sites within a 3′ UTR give synergistic downregulation,4 but only if the distance between the start of the seed sites is in an optimal range of about 14–46 nucleotides.23,24 Moreover, different miRNAs can also cooperate and give synergistic downregulation as long as their sites are located within this optimal range.23,24 Consequently, pairs of target sites located within an optimal distance have a much higher regulatory potential than individual isolated sites.

We speculated that the high regulatory potential of multiple, optimally spaced seed sites could be employed for rational design of siRNAs that cause both targeted cleavage and translational suppression. Such bifunctional siRNAs would be useful in HIV therapy, as the siRNAs' combinatorial targeting could help reduce the frequency of viral-mutant escape.25,26

Results

To test this hypothesis, we identified all siRNA sequences that (i) were predicted to be highly effective against genes in the pNL4-3 HIV-1 plasmid (GenBank accession M19921),27 and (ii) had multiple 6mer seed-site occurrences within the plasmid's annotated 3′ UTR (Figure 1a; annotation based on HIV-1 genome sequence—GenBank accession NC_001802). This procedure identified several candidate siRNAs, but further filtering requiring that the siRNAs' seed sites were optimally spaced reduced the list to two candidate siRNAs (Figure 1b and Supplementary Figure S1a). Both siRNAs target the Gag-Pol polyprotein transcript. The siRNAs also share the same 3′ UTR seed sites, but the complementarity between the siRNA 3′ ends and mRNA differ. Therefore, we selected for further validation the siRNA candidate with the best 3′ complementarity against both 3′ UTR sites (siRNA CU2 in Figure 1b). A dual luciferase-based reporter screen showed that this siRNA had the intended double function, as it could strongly downregulate the Gag-Pol site via an siRNA mechanisms and also downregulate target expression through a miRNA mechanism in the 3′ UTR sites (Figure 2). We also tested a Gag-Pol-targeting siRNA that has three nonoverlapping 3′ UTR seed sites (siRNA CU3 in Supplementary Figure S1b), but this siRNA did not give any downregulation of the 3′ UTR reporter (Figure 2).

Figure 1.

Figure 1

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Rational design of bifunctional siRNAs. (a) Flow diagram showing the bifunctional design algorithm. An small-interfering RNA (siRNA) efficacy prediction algorithm27 screens all 19-mer subsequences from the cleavage target sequence to identify effective siRNA candidates. Then, a second screen identifies the candidates that have multiple seed sites in the 3′ UTR target sequence. A final filter identifies the siRNAs that have optimally spaced seed sites that can give synergistic downregulation of the target 3′ UTR. Numbers in braces are locations of seed sites within the input 3′ UTR—the pNL4-3 3′ UTR was used as an illustration. (b) Bifunctional siRNA candidate CU2 targeting the pNL4-3 Gag-Pol polyprotein transcript and two optimally spaced seed sites in pNL4-3′s 3′ UTR. (c) Bifunctional siRNA candidate CCR5-5 targeting CCR5's 3′ UTR for cleavage and pNL4-3's 3′ UTR for translational suppression.

Figure 2.

Figure 2

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Bifunctional siRNA candidates potently downregulate reporters bearing siRNA cleavage sites and guide moderate downregulation of reporters bearing miRNA-like seed sites. The psiCHECK2.2 vector (Promega) bearing an 892 nt Gag-Pol fragment (CDS; black) or the complete pNL4-3 3′ UTR (3′ UTR; gray) in the Renilla luciferase 3′ UTR was cotransfected with 50 nmol/l of bifunctional siRNA candidates, CU3 or CU2. Relative changes in Renilla luciferase levels compared to Firefly luciferase levels were normalized to an irrelevant control (IRR). A positive control (U2), designed to have high complementarity to both of CU2's seed sites in the 3′ UTR reporter, shows that CU2's 3′ UTR target sites are amendable for miRNA targeting. P values above 3′ UTR bars are from two-tailed, two-sample Student's t-tests comparing the CU2 and U2 siRNAs to the irrelevant controls (n = 2).

To address whether the bifunctional siRNA CU2 would function when expressed within the context of a miRNA precursor, we placed the siRNA into a miRNA-based expression system. The expression cassette was based on miR-126 and its upstream and downstream genomic flanking sequences, and the passenger strand of CU2 was modified such that the resulting precursor transcript mimicked the miR-126 secondary structure (Figure 3a). We then electroporated the miR-126-mimick expression system into CEM T cells and established stably transfected cell lines constitutively expressing the CU2 miRNA mimic (Figure 3b). A subsequent challenge of the stably expressing CEM T cells with the HIV-1 4-3 strain showed that CU2 siRNA gave long-term protection against the virus (Figure 3c). Viral replication, as measured by p24 antigen levels, increased rapidly in unprotected cells, whereas viral replication remained suppressed for a period of 1 month in the cells transfected with the bifunctional miRNA mimic.

Figure 3.

Figure 3

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Design and antiviral activity of miR-126-mimicking bifunctional siRNA with siRNA and miRNA activity against strains of HIV-1. (a) Predicted hairpin structure of hsa-mir-126 (top; mature duplex in red) and the CU2 miRNA-mimic bifunctional siRNA (bottom; CU2 guide strand in green and blue). (b) Northern blot analysis confirmed stable expression and efficient processing of the CU2 miRNA-mimic in CEM cells. “CEM” are wild-type CEM cells; “CU2” are CEM cells stably expressing the CU2 miRNA-mimicking bifunctional siRNA. (c,d) Antiviral activity of CU2 miRNA mimic. CEM cells stably expressing the CU2 mimic were challenged with the HIV 4-3 (c) or IIIB (d) strain at multiplicity of infection of 0.01. The culture supernatant was collected once a week from day 7 to day 28 and analyzed for p24 antigen expression. “CEM” (red squares) are wild-type CEM cells; “CU2” (blue diamonds) are CEM cells stably expressing the CU2 miRNA-mimicking bifunctional siRNA. Curves and error bars are averages and standard deviations of log10 p24 levels (n = 2). (e,f) CU2's cleavage site is disrupted in the IIIB strain. (e) Alignment between CU2's cleavage sites in pNL4-3 and IIIB. CU2 has two mismatches against the site in IIIB. (f) Normalized luciferase expression of reporters harboring the pNL4-3 and IIIB (red and blue) cleavage and 3′ UTR sites (CU2 CDS and CU2 3′UTR; n = 2).

Different HIV strains have great sequence diversity. We took advantage of CU2's cleavage site not being conserved among different HIV strains to demonstrate and segregate CU2's bifunctionality. CU2's cleavage site in the 4-3 strain is not perfectly conserved in the HIV-1 IIIB strain (GenBank accession A04321; Figure 3e), but both 4-3 and IIIB strains contain the two optimally distanced seed sites in the viral mRNA 3′ UTR. Consequently, CU2 would only exert a miRNA-like effect against the IIIB strain. As expected, CU2 produced significantly better downregulation of the 4-3 cleavage site than of the corresponding IIIB site (Figure 3f, CU2 CDS; P = 0.001, two-tailed, two-sample Student's t-test; n = 2). Challenging the CU2-expressing CEM cells with IIIB generated reduced viral replication, compared to control cells (Figure 3d), and viral challenge of CEM T cells transduced by a lentiviral vector to stably expressing CU2 showed similar results (Figure 4). Thus, although CU2's perfectly complementary site in Gag-Pol gave the primary viral protection, CU2's miRNA-like target sites in the 3′ UTR also gave protection.

Figure 4.

Figure 4

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Anti-HIV-1 activity of the CU2 shRNA construct. Stably transduced CEM T cells expressing an empty lentiviral vector (pHIV7) or the CU2 shRNA vector (CU2) were challenged with HIV-IIIB at a multiplicity of infection of 0.001. Culture supernatants were collected at various time points and analyzed by a p24 enzyme-linked immunosorbant assay. CU2 values were calculated relative to the positive control (pHIV7). Data points are reported as means ± SD.

Combinatorial targeting of HIV genes and host genes important for viral integration, such as CCR5, is another effective approach for long-term inhibition of viral replication and infection.28 We, therefore, used our bifunctional siRNA design approach to identify siRNAs that could both cleave CCR5 mRNA and suppress translation of HIV-1 transcripts. None of the resulting siRNA candidates targeted optimally spaced seed sites in pNL4-3. We, therefore, selected four siRNAs that had their 3′ UTR target sites at near-optimal distances to the two bifunctional siRNAs that only targeted HIV (Supplementary Figure S1). Two of these siRNAs targeted CCR5's coding sequence (CDS); the other two targeted the 3′ UTR.

One reason why our design approach did not find bifunctional siRNA candidates that targeted highly effective cleavage sites in CCR5 and optimally spaced seed sites in pNL4-3 was our design algorithm's high stringency when identifying highly effective cleavage sites. An analysis of the siRNA efficacy prediction algorithm's27 ability to identify highly effective standard siRNAs showed that less stringent thresholds not only give increasing numbers of siRNA candidates but also reduce the probability of the siRNAs giving highly effective cleavage (Supplementary Figure S2). Although these lower-scoring sequences are less likely to be highly effective standard 21-mer siRNAs, the sequences may still be effective bifunctional siRNAs. To test this possibility, we selected for further validation one of the bifunctional siRNA candidates rejected by our original screen. This bifunctional siRNA candidate CCR5-5 targeted the CCR5 3′ UTR for cleavage and the same optimally spaced HIV 3′ UTR seed sites as our HIV-only bifunctional siRNA CU2 (Figure 1c).

Although a reporter-based screen identified CCR5-5 to be a potent bifunctional candidate (Figure 5), we nevertheless designed miRNA-mimicking versions of all five candidates, placed these in our miR-126-based expression system (Figure 6 and Supplementary Figure S3), and used these to stably transfect HOS-CD4-CCR5 cells. Two of the four cell lines (CCR5-2 and CCR5-5) expressed levels of miRNA-mimicking siRNAs that were detectable by northern blot (Figure 6b). The HOS-CD4-CCR5 cells stably expressing the CCR5-2 and CCR5-5 miR-126 mimics demonstrated good inhibition of the JR-FL HIV-1 strain (Figure 6c) and by day 6 the protected cells were 100% confluent. Both cell lines showed significant CCR5 downregulation (Figure 7). Whereas JR-FL requires CCR5 for cell entry, plasmid transfection bypasses the virus' CCR5 dependence. To investigate the miRNA-like activity of the anti-CCR5 bifunctional siRNAs on HIV itself, we transfected the stably expressing HOS-CD4-CCR5 cells with the pNL4-3 proviral DNA. Compared with the control, the anti-CCR5 bifunctional siRNAs gave reduced viral titers as expected from only a miRNA effect confirming their dual targeting (Figure 6d).

Figure 5.

Figure 5

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Screening of bifunctional siRNA candidates reveals varying downregulation of CCR5 and pNL4-3 reporters. The psiCHECK2.2 vector (Promega) bearing a 1,047 nt CCR5 CDS fragment (CCR5-C; black), a 1,053 nt CCR5 3′ UTR fragment (CCR5-U; gray), or the complete pNL4-3 3′ UTR (3′ UTR; slash patterns) in the Renilla luciferase 3′ UTR was cotransfected with 50 nM of bifunctional siRNA candidates, CCR5-1 to CCR5-5. Relative changes in Renilla luciferase levels, compared to Firefly luciferase levels, were normalized to an irrelevant control (IRR; n = 2).

Figure 6.

Figure 6

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Design and antiviral activity of bifunctional siRNAs that possess cleavage activity against the HIV receptor CCR5 and microRNA activity against strains of HIV. (a) Predicted hairpin structure of hsa-mir-126 (top; mature duplex in red) and the CCR5-2 and CCR5-5 miRNA-mimic bifunctional siRNAs (middle and bottom; guide strands in green and blue). (b) Northern blot analysis confirmed stable expression and processing of the CCR5-2 (left) and CCR5-5 (right) miRNA-mimics in stably expressing HOS-CD4-CCR5 cells. Open and filled arrowheads indicate mature CCR5-2 and CCR5-5 siRNAs; see Supplementary Table S2 for probes. “HOS-CD4-CCR5” are wild-type cells; “Δ-126” are cells stably expressing the empty vector; “CCR5-1,” “CCR5-2,” “CCR5-4,” and “CCR5-5” are cells stably expressing the corresponding miRNA-mimicking bifunctional siRNAs (see Supplementary Figure S3 for details on CCR5-1 and CCR5-4). (c,d) Combined (c) and miRNA (d) antiviral activity of CCR5-2 and CCR5-5 miRNA mimics. HOS-CD4-CCR5 cells stably expressing the CCR5-2 or CCR5-5 mimics were challenged with the HIV JR-FL strain at multiplicity of infection of 0.1 (c) or transfected with the pNL4-3 provirus plasmid (d). The culture supernatants were collected at days 1, 3, 4, and 6 and analyzed for p24 antigen expression (c) or collected 1, 2, 3, and 4 days post-transfection and analyzed for viral mRNA expression by the branched DNA assay (d). “HCC” (blue diamonds) are wild-type HOS-CD4-CCR5 cells; “Δ-126” (red squares) are cells transduced with empty vector; “CCR5-2” and “CCR5-5” (yellow triangles and green x) are cells transduced with the corresponding miRNA-mimicking bifunctional siRNAs.

Figure 7.

Figure 7

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Anti-CCR5-expressing cell lines significantly downregulate CCR5. Graphs show expression levels of CCR5 measured by qRT-PCR in the HOS-CD4-CCR5 cells stably expressing the CCR5-2 and CCR5-5 miR-126 mimics; CCR5 expression levels were normalized to the Histone 2A mRNA, and the siRNA downregulation was normalized to the CCR5 level in the cells expressing the empty vector (Δ-126). P values above bars are from two-tailed, two-sample Student's t-tests comparing the CCR5-2- and CCR5-5-expressing cells to the empty vector cells (n = 3).

Discussion

Our results show that the combinatorial targeting of rationally designed bifunctional siRNAs can give robust knockdown that prevents HIV replication for a prolonged period, either by simultaneously targeting multiple regions in HIV RNAs or by simultaneously targeting CCR5 and HIV mRNAs. Our results show that bifunctional siRNAs can be useful agents in HIV therapy, but we were not certain whether or not the bifunctional design approach would be generally applicable against other targets. To address this question, we applied our approach to design bifunctional siRNAs for all well-annotated human protein-coding genes. Using standard parameter settings, our approach found bifunctional siRNA candidates that had both a predicted effective cleavage site and optimally spaced seed sites for 77% of the transcripts in a previously described nonredundant set of 17,448 RefSeq genes.24 Lowering the efficacy threshold increased coverage, such that at most 92% of the genes in the set can be targeted. As expected, the remaining 8% of the genes have very short 3′ UTRs (median length of 75 nt)—the probability of finding optimally spaced seed pairs depends upon the length of the 3′ UTR (Supplementary Figure S4). Finally, as more than 90% of human protein-coding genes harbor pairs of optimally spaced seed sites, our design approach can likely find bifunctional siRNAs against most combinations of pairs of genes. Indeed, we found that 86% of randomly selected pairs of transcripts could be targeted by bifunctional siRNAs such that one transcript of the pair contained a predicted effective cleavage site and the other transcript of the pair contained optimally spaced seed sites (n = 10,000). Thus, the bifunctional design approach is generally applicable against both individual transcripts and combinations of pairs of transcripts.

We have shown that bifunctional siRNAs—just as many other combinatorial approaches26,28,29,30—can be useful agents in HIV therapy. Unlike other combinatorial approaches, however, bifunctional siRNAs have only one single active molecule causing the combinatorial targeting. This is beneficial, as compared with combinatorial approaches such as multi-shRNA expression systems,29,30 a single bifunctional siRNA should have reduced risk of seed-based off-targeting and of saturating endogenous miRNA pathways. This is because multi-shRNA expression systems produce multiple molecules, with each of them having separate off-target effects and competing with endogenous miRNAs for miRNA pathway complexes. Our experiments also show that bifunctional siRNAs can be designed to target most human protein-coding genes—either individually or in pairs—where one gene is targeted for cleavage and another is targeted for translational suppression. Future experiments will reveal whether bifunctional siRNAs can be effective combinatorial treatments of other human diseases as well.

Materials and Methods

siRNA efficacy prediction

The siRNA efficacy prediction algorithm has previously been described.27 Briefly, the algorithm assigns a score between –1 and 1 to each 19-mer siRNA candidate such that higher-scoring siRNAs have a higher probability of being highly effective (≥80% target knockdown) than lower-scoring siRNAs have. Our bifunctional siRNA design algorithm uses a default threshold of 0.2 to select siRNA candidates. With this threshold, 8 of 10 siRNAs are expected to be highly effective (Supplementary Figure S4).

Design of luciferase reporter constructs

In a standard PCR reaction, the 455 nucleotide 3′ UTR of the 4-3 strain of HIV-1 was amplified from the provirus plasmid, pNL4-3, using forward primer 4-3′ UTR-F and reverse primer 4-3′ UTR-R (Supplementary Table S1) containing the Spe1 and Not1 restriction endonucleases. The PCR product and psiCheck 2.2 plasmid (Promega, Madison, WI) were digested with appropriate enzymes and gel purified. The two fragments were ligated to insert the 3′ UTR of the 4-3 strain of HIV-1 in the multiple cloning site of the psiCheck vector. The engineered construct (pU) contains the 3′ UTR of the 4-3 strain of HIV-1 as the 3′ UTR of the Renilla luciferase. A similar strategy was applied for cloning of the 900 nt coding-region of the 4-3 strain, the CCR5 coding, and 3′ UTR region fragments, and the IIIB 3′ UTR and coding regions in the 3′ UTR of the Renilla luciferase in the psiCheck 2.2 plasmid. The resulting constructs encompass the CU3 and CU2 sites in the 4-3 strain of HIV-1; the CCR5-2 and CCR5-4 cleavage sites in the CCR5 CDS; the CCR5-1 and CCR5-3 cleavage sites in the CCR5 3′ UTR; the CCR5-5 cleavage site in the CCR5 3′ UTR; the CU2 sites in the IIIB 3′ UTR; and the CU2 site in the IIIB CDS (primer sets 4-3 CDS-F/R, CCR5-2-T/B, CCR5-1-T/B, CCR5-5-T/B, b3′ UTR-F/R, and bCDS-F/R and bCDS-FA/RA in Supplementary Table S1). Construction of the IIIB CDS reporter required two primer sets and two annealing temperatures in the PCR reaction. Primer set 1 consisted of bCDS-F and bCDS-R with restriction endonuclease sites, Spe I and Not I. Primer set 2 consisted of bCDS-FA and bCDS-RA. The 188 nucleotide PCR product required five PCR cycles with an annealing temperature of 52 °C followed by 25 cycles with an annealing temperature of 61 °C.

Dual-luciferase assay

Equal numbers of HEK-293 cells were seeded in a 24-well dish 1 day prior to transfection. The next day the appropriate reporter plasmids and synthetic siRNAs were complexed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and cotransfected in the HEK-293 cells, and 24 hours post-transfection the cells were lysed using the passive lysis buffer according to the manufacturer's suggested protocol. Subsequently, the lysates were analyzed for luciferase activity using the Dual-luciferase Reporter Assay System (Promega) and the Veritas microplate luminometer (Turner Biosystems, Madison, WI).

siRNA expression cassette design and engineering of the stably expressed cell lines

We used hsa-mir-126 as a template for designing the bifunctional expression cassette using the pcDNA3.1 (Invitrogen) expression plasmid. More specifically, we replaced hsa-miR-126's mature duplex with the bifunctional siRNA duplex, but to ensure that the resulting sequence shared hsa-mir-126's predicted secondary structure (Figure 2a), we modified the siRNA passenger strand and added one additional base pair at the 3′ end of the guide. To generate individual miRNA mimics, we designed pairs of siRNA-specific overlapping oligonucleotides (Supplementary Table S1) and PCR extended each pair. The extended product contained the bifunctional miRNA-mimic precursor hairpin (pre-miR) and the stem downstream of the pre-miR. The final expression plasmids were either electroporated in the CEM T cells using the Amaxa Nucleofection Kit C according to the manufacturer's suggested protocol or transfected into HOS-CD4-CCR5 cells using Lipofectamine 2000 (Invitrogen). And 48 hours postelectroporation the CEM T cells were diluted 1:6 and subjected to 1.2 mg/ml neomycin (G418) drug selection for a period of 6 weeks. The cells were then maintained in the 600–800 µg/ml G418 containing medium. The HOS-CD4-CCR5 cells were transfected with the expression plasmids using Lipofectamine 2000; 24 hours post-transfection the cells were divided at the ratio of 1:5 and subjected to 900-µg/ml G418 and 1-µg/ml puromycin-containing medium. After a period of 3 weeks, the control untransfected cells were depleted of all the cells, and the transfected dishes were maintained in the -00 µg/ml G418 and 1-µg/ml Puromycin media.

Northern blot analysis of the stably expressed miR-126 mimics

Total cellular RNA was isolated from either the CEM T cells or the HOS-CD4-CCR5 cells stably expressing the bifunctional siRNAs using RNA Stat-60 according to the manufacturer's suggested protocol (Tel-Test). The total cellular RNA was resolved on a 15% polyacrylamide gel at approximately 400–500 V for 1.5–2 hours. The gel was blotted on a Hybond-N (Amersham) nylon membrane. The nylon membrane was incubated in the prehybridization solution for ~2–3 hours at 42 °C. Subsequently, the end-labeled probe specific to each bifunctional siRNA (Supplementary Table S2) was added sequentially to the hybridization solution. The hybridization was carried overnight at 42 °C. The next day the membrane was washed once with 6x SSC/0.1% SDS for 10 minutes and twice with 2 × SSC/0.1% SDS for 5 and 3 minutes. Finally, the membrane was subjected to autoradiography for detection of expressed siRNAs.

Real-time RT-PCR

RNA was treated with Turbo DNA-Free Kit (Ambion, Austin, TX) and reverse-transcribed into complementary DNA using random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen). One RNA sample of each preparation was processed without Moloney murine leukemia virus RT to provide a negative control in subsequent real-time PCR reactions. Quantitative analysis of CCR5 expression was performed by real-time PCR SYBR Green I (Bio Rad, Hercules, CA) analysis (C1000 Thermal Cycler, Bio Rad). CCR5 expression was detected using 12.5 ng of complementary DNA, amplified with primers CCR5-M-F and CCR5-M-R.The internal control, Histone H2A expression, was detected using 12.5 ng of complementary DNA, with primers H2A-F2 and H2A-R2. Both CCR5 and H2A PCR reactions were amplified using PCR conditions of 95 °C for 5 minutes, followed by 40 cycles of 95 °C for 40 seconds, 56 °C for 40 seconds, and 72 °C for 1 minute (see Supplementary Table S1 for primers).

Viral challenge of the stably expressed bifunctional siRNAs cell lines

One million CEM T cells and CEM T cells stably expressing the CU2 bifunctional siRNA each were infected with HIV NL4-3 and HIV-IIIB strains of the virus at a multiplicity of infection of 0.01. After overnight incubation, the cells were washed three times with Hank's balanced salts solution and cultured in RPMI 1640 with 10% fetal bovine serum. At designated time points between day 7 and day 28, culture supernatants were collected weekly and analyzed for p24 viral antigen expression using the Alliance HIV-1 p24 ELISA kit (Perkin-Elmer, Waltham, MA), according to the manufacturer's suggested protocol, and viral RNA levels were analyzed by a branched DNA assay, using QuantiGene Regent System (Panomics, Fremont, CA), according to the manufacturer's instructions.

SUPPLEMENTARY MATERIALFigure S1. Additional bifunctional siRNAs.Figure S2. Number of bifunctional siRNA candidates increases with decreasing efficacy thresholds.Figure S3. Design of bifunctional siRNAs that possess cleavage activity against the HIV receptor CCR5 and microRNA activity against strains of HIV.Figure S4. Probability of finding optimally spaced seed pairs increases with 3′ UTR length.Table S1. Oligonucleotide sequences for polymerase chain reactions.Table S2. Probes for the northern blot detection.

Supplementary Material

Figure S1.

Additional bifunctional siRNAs.

Click here for supplemental data (167KB, doc)
Figure S2.

Number of bifunctional siRNA candidates increases with decreasing efficacy thresholds.

Click here for supplemental data (70KB, doc)
Figure S3.

Design of bifunctional siRNAs that possess cleavage activity against the HIV receptor CCR5 and microRNA activity against strains of HIV.

Click here for supplemental data (33.5KB, doc)
Figure S4.

Probability of finding optimally spaced seed pairs increases with 3′ UTR length.

Click here for supplemental data (69KB, doc)
Table S1.

Oligonucleotide sequences for polymerase chain reactions.

Click here for supplemental data (48.5KB, doc)
Table S2.

Probes for the northern blot detection.

Click here for supplemental data (26KB, doc)

Acknowledgments

This work was supported by NIH grants HL07470, AI42552, and AI29329 to J.J.R., O.S., and P.S. received support from the Norwegian Functional Genomics Program (FUGE) and the Leiv Eriksson program of the Norwegian Research Council; L.A. was supported by the Alfred Benzons Foundation. A.E., P.S., O.S., L.A., and J.J.R. conceived the study. P.S. designed siRNAs and conducted bioinformatics analyses. A.E. and H.L. conducted experiments. A.E., P.S., and J.J.R. wrote the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Additional bifunctional siRNAs.

Click here for supplemental data (167KB, doc)
Figure S2.

Number of bifunctional siRNA candidates increases with decreasing efficacy thresholds.

Click here for supplemental data (70KB, doc)
Figure S3.

Design of bifunctional siRNAs that possess cleavage activity against the HIV receptor CCR5 and microRNA activity against strains of HIV.

Click here for supplemental data (33.5KB, doc)
Figure S4.

Probability of finding optimally spaced seed pairs increases with 3′ UTR length.

Click here for supplemental data (69KB, doc)
Table S1.

Oligonucleotide sequences for polymerase chain reactions.

Click here for supplemental data (48.5KB, doc)
Table S2.

Probes for the northern blot detection.

Click here for supplemental data (26KB, doc)

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

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