Nucleic acid aptamers with binding affinities in the low to mid-nanomolar range have been utilized for flexible applications ranging from diagnostic to therapeutic assay formats. Moreover, aptamers that target specific cell surface proteins are employed as delivery molecules to target a distinct cell type, hence reducing off-target effects or other unwanted side effects. We have capitalized on the exquisite specificity of a gp120 aptamer to deliver anti-HIV siRNAs into HIV infected cells with the net result that the replication and spread of HIV is strongly inhibited by the combined action of the aptamer and a siRNA targeting the tat/rev common exon of HIV-1. Therefore, the anti-gp120 aptamers can function both as antiviral agents and as siRNA delivery reagents.
It is highly desirable to generate new aptamers to expand their diversity for in vivo
applications. In a therapeutic setting, anti-gp120 aptamers and various siRNAs targeting different genes may need to be interchanged to avert viral resistance. In the present work, a series of new 2′-F substituted RNA aptamers that specifically bind to the HIV-1BaL
gp120 protein with nanomolar affinity were successfully isolated from an 81
nt RNA library via
SELEX (Systematic Evolution of Ligands by EXponential enrichment). The selected aptamers are able to specifically bind gp120 and are internalized into cells expressing the HIV envelope. We observe an aggregation of aptamer within the cytoplasm as revealed by confocal microscopy, suggesting that the aptamers are internalized by gp120-mediated endocytosis. In addition, these aptamers have been shown to inhibit several different HIV-1 strains, indicating that anti-gp120 aptamers that inhibit the initial step of HIV-1 entry by blocking the binding of the gp120 and CD4 receptor may be useful in HIV-1 therapeutic applications. Compared with the previously published B40t77 aptamer that was selected against the HIV-1BaL
gp120 protein through BIAcore biosensor chip-based SELEX, our selected aptamers show somewhat better inhibitory activity. The fact that B40t77 did not bind with our target protein suggests that the different sources of target and selection methods can affect the aptamer-target protein binding epitopes.
On the basis of our previous results, two kinds of dual inhibitory functional anti-gp120 aptamer-based delivery systems were constructed and evaluated for HIV-1 inhibition. In the first design, we cotranscribed the aptamer–siRNA sense single strand, followed by annealing of the complementary siRNA antisense strand to complete the chimeric molecule. Specifically, the secondary design is a worthy point of discussion. A new anti-gp120 aptamer–stick–siRNA delivery system has been designed which allows mixing new siRNAs with the same aptamer, which could ultimately be useful for minimizing viral escape mutants. Both the aptamer and siRNA portions are chemically synthesized and subsequently annealed via
a complementary 16-nt ‘stick’ sequence which forms a stable base-pair via
the ‘sticky’ bridge. The linker, seven sets of three-carbon atoms (C3), between the RNA and the stick end provides molecular flexibility and minimizes steric hindrance which could impede annealing of the two RNAs. These design strategies not only guarantee tight conjugation of the aptamer with the siRNA portion, but also do not interfere with correct folding of the aptamer and the Dicer processing of the siRNA. Most importantly, since this kind of design is quickly assembled from two chemically synthesized RNA portions, each portion is easily changed to avert viral resistance. So far, the chemical synthesis of long RNAs is limited by the length and backbone modifications of the RNAs. Using the ‘sticky bridge’ strategy, we have been able to chemically synthesize a 2′-fluoropyrimidine modified aptamer (81
nt plus 16-base bridge) separately from the 27
bp (plus 16 base bridge) siRNA in milligram scale, followed by in vitro
joining of the two RNAs. This attractive ‘sticky’ bridge-based approach can potentially be used for mixing different siRNAs with a single aptamer to multiplex target down regulation, and in the case of HIV infection, to avert resistance to the siRNA component.
We demonstrate that our delivery systems specifically bind to the surface of cells expressing gp160 and are internalized, allowing functional processing of the siRNA into RISC, resulting in specific inhibition of HIV-1 replication and infectivity in cell culture. Both of the anti-gp120-based siRNA delivery systems serve as dual function inhibitors and therefore provide greater efficacy than either the aptamer or siRNA applied alone.
Interestingly, even though our designs (Ch A-1
) are able to down-regulate the target expression and show good strand selectivity, their RNAi potencies are different. Previous studies of Dicer substrate siRNAs (49
) used substrates in which Dicer entry on the blunt end of the DsiRNAs was completely blocked by placing two deoxy nucleotides at the 3′-end of the desired passenger strand, creating an absolute polarity for Dicer entry from the two base 3′ overhang. The present studies did not employ deoxys so entry from the blunt end of the Ch A-1
siRNA takes place relatively efficiently relative to the entry from the 2 base 3′ overhang, which is adjacent to the aptamer that creates a steric hindrance for Dicer entry (). Also, in contrast to our previous observations, (49
) the preference for guide strand selection in our chimeras does not necessarily favour the strand with the two base 3′ overhang (Supplementary Figures S6 and S7
), since the more potent configuration N-1
has the guide strand positioned with the 5′-end at the side of Dicer entry. In this context it should be noted that the strand selectivity of N-1
is not as good as that of N-2
(Supplementary Figure S6
). The difference in potency between N-1
most likely resides in the species of 21- to 23-mer produced, which differ between the two forms. Interestingly, Dicer does not process the ‘stick’ aptamer siRNAs at all from the blunt end side as it does with the Ch A-1. We attribute this to the lack of a free 3′OH in these chimeras, in which the end of the siRNA is covalently attached to the ‘stick’ sequence which in turn forms a 2′OMe modified duplex with the aptamer. The 2′OMe backbone modifications throughout the ‘stick’ also block Dicer cleavage (51
). Since the strand selectivity of Dicer generally favors the strand with the 2 base 3′ overhang (49
), it is important to design the Dicer substrates such that a potent siRNA against the desired target generated from the cleavage event. This will minimize the off target potential of the passenger strand.
In summary, we have demonstrated that the anti-gp120 aptamers not only provide a potential new drug therapy approach for combating HIV infection, but also act as delivery vehicles for siRNAs and perhaps other small RNA inhibitors. The ‘sticky’ bridge-base approach offers a major advantage in both chemical synthesis and the opportunity to mix and match the aptamer with different siRNAs in a non-covalent fashion. For HIV clinical applications, the mixing approach may be a requirement for averting viral escape mutants.