Dual inhibition on HIV-1 infection mediated by aptamer-based siRNA delivery system. (A) Both anti-gp120 aptamer, aptamer-siRNA chimeras (Ch A-1) and aptamer-stick-siRNA (N-1 and N-2) neutralize HIV-1 Bal infection in PBMCs. The chimeras Ch A-1, N-1 and N-2 were more potent inhibitors than the respective aptamers alone. Data represent the average of triplicate measurements of p24. (B) The siRNA delivered by aptamers down-regulates tat/rev gene expression in human PBMCs. Data represent the average of three replicates.

Dual inhibition of HIV-1 infection mediated by aptamer-siRNA chimeras. Both anti-gp120 aptamer and aptamer-siRNA chimeras neutralized HIV-1 infection in (A) CEM cells (IIIB strain) and (B) human PBMCs (NL4-3 strain) culture, respectively. Data represent the average of triplicate measurements of p24. The chimeras (Ch A-1 and Ch B-68) showed stronger inhibition than aptamer alone indicating that (C) the siRNA delivered by the aptamers down-regulated Tat/rev gene expression in the PBMCs. Data represent the average of three replicates.

Binding activity assay and predicted secondary structure of selected individual aptamers against HIV-1_BaL gp120. (A) Binding curve from a gel shift assay. The 5′-end P³²-labeled individual aptamers were incubated with the increasing amounts of gp120 protein. The binding reaction mixtures were analyzed by a gel mobility shift assay. Aptamer A-1 and B-68 showed the best binding affinity with the target protein. (B) The predicated secondary structures of the anti-gp120 aptamer A-1 and B-68 as predicted by QuickFOLD and the calculated K_d determinations are indicated. Data represent the average of four replicates.

The design and binding activity of the aptamer-siRNA delivery system. (A) Schematic aptamer-siRNA chimeric RNAs: the region of the anti-gp120 aptamer is responsible for binding to gp120 and the siRNA is targeting a common exon of HIV-1 tat/rev. A linker (UU) between the aptamer and siRNA is indicated in green. (B) The aptamer-siRNA chimeric RNAs that have comparable K_d values as well as parental aptamers specifically bind the HIV_BaL gp120 protein. Data represent the average of three replicates. (C) Cell-type specific binding studies of aptamers. Cy3-labeled RNAs were tested for binding to CHO-gp160 cells and CHO-EE control cells. Cell surface bindings of Cy3-labeled RNAs were assessed by flow cytometry. The selected aptamers showed cell-type specific binding affinity. The 2^nd RNA pool and irrelevant RNA were used as negative controls. Data represent the average of two replicates.

In vitro Dicer processing. (A) Dicer cleavage of 5′-end P³² sense or antisense labeled RNAs. The RNA strands were annealed with equal molar equivalents of 5′-end P³²-labeled complementary RNA strands. (B) The P³²-labeled RNAs were marked with asterisks. The cleavage products or uncleaved, denatured strands were visualized following 20% denaturing PAGE. According to path of Dicer entry different cleavage products were observed. For example, the 5′-end P³²-labeled antisense N-1 and 5′-P³² sense strand N-1 was cleaved into two products, one of which is 21–23 nt and the other a lower abundance 6 nt fragment. The results obtain suggest that Dicer preferentially enters N-1 using the 3′-end of the sense strand.

The selected anti-gp120 aptamers inhibit HIV-1 replication in cells previously infected with HIV-1. The 1.5 × 10⁴ HIV-1 infected CEM cells and 3.5 × 10⁴ uninfected CEM cells were incubated at 37°C with the various experimental RNAs at a final concentration of 400 nM. The culture supernatants were collected at different time points following addition of the aptamers (3 d, 5 d, 7 d, 9 d and 11 d) for p24 antigen analyses. Data represent the average of triplicate measurements of p24.

The HIV-1 inhibition mediated by the various siRNAs delivered using the ‘stick’ aptamer system. (A) Three different siRNAs which target the HIV-1 Tat/rev, human CD4 receptor and HIV-1 dependency factor TNPO3 RNAs, were annealed to aptamer A-1 via the ‘stick’ bridge. The resulting aptamer-stick-siRNAs (Tat/rev N-1, TNPO3 N-1 and CD4 N-2) neutralized HIV-1 infection in CEM cell culture, providing more potent inhibition than the aptamer alone. The combination of three siRNAs also suppressed the HIV-1 replication. Data represent the average of triplicate measurements of p24. (B) The siRNAs delivered by aptamers down-regulates target gene expression in the HIV infected CEM cells (HIV-1 tat/rev, TNPO3 and CD4). Data represent the average of three replicates.

Cell-type specific binding and uptake studies of aptamers. (A) Cell surface binding of Cy3-labeled RNAs was assessed by flow cytometry. Cy3-labeled RNAs were tested for binding to CHO-gp160 cells and CHO-EE control cells. The selected aptamers showed cell-type specific binding affinity. The 2nd RNA pool and irrelevant RNAs were used as negative controls. Data represent the average of three replicates. (B) Internalization analysis. CHO-gp160 cells were grown in 35 mm plates and incubated with a 100 nM concentration of Cy3-labeled A-1 in culture media for real-time live-cell-confocal microscopy analysis. The images were collected at 15 min intervals using 40× magnification. (C,D) Localization analysis. CHO-gp160 cells (C) and CHO-EE control cells (D) were grown in 35 mm plates. Before incubation with 100 nM of Cy3-labeled A-1, cells were stained with Hoechst 33342 (nuclear dye for live cells) and then analyzed using real-time confocal microscopy.

Cell-type specific aptamer-stick-siRNA delivery system. (A) Schematic aptamer-stick-siRNA system (N-1 and N-2). The anti-gp120 aptamer A-1 and the 27mer siRNA targeting HIV-1 tat/rev are shown. The siRNA is linked to the aptamer portion via the “stick” sequence which consists of 16 nt appended to the aptamer 3′-end allowing complementary interaction of one of the two siRNA strands with the aptamer. After a simple incubation of the A-1-stick and NS-1 or NS-2 siRNA configurations, they form stable base-pairing. A linker of seven three-carbons between the aptamer RNA and the stick portion is used to avoid steric interaction of the stick with the aptamer. (B) The aptamer-stick-siRNA system (N-1 and N-2) has comparable K_d values to the aptamer A-1 alone in binding assays with the HIV_Bal gp120 protein. Data represent the average of two replicates. (C) Cell-type specific binding studies of aptamer-stick-siRNA. Cy3-labeled RNAs were tested for binding to CHO-gp160 cells and CHO-EE control cells. Binding of the Cy3-labeled RNAs was assessed by flow cytometry. The selected aptamers show cell-type specific binding affinities. A 27 mer duplex RNA (NS-3) with a 5′-Cy3-labelled sense strand is used as a negative control. Data represent the average of two replicates. (D) Internalization and intracellular localization analyses. CHO-gp160 cells were grown in 35 mm plates and incubated in culture medium with a 100 nM concentration of N-1 containing a 5′-Cy3-labeled sense strand for real-time live-cell confocal microscopy analysis as previously described. After overnight incubation, cells were stained with Hoechst 33342 (nuclear dye for live cells) and then analyzed by confocal microscopy.