The RNA interference (RNAi) era, started by Fire and Mello in 1998 completely changed the vision of research involving cell control and therapeutic intervention. RNAi machinery starts from a long dsRNA and pre-microRNA (pre-miRNA) that is cleaved by the ‘Dicer’ enzyme in a short dsRNA fragments called small interfering RNA (siRNA) and miRNA, respectively. This, in turn, inhibits gene expression of specific host messenger RNA (mRNA) sequences by preventing translation via post-transcriptional gene silencing. These structured RNA elements have been discovered to play critical roles in variety of diseases, including viral infections, neurological disorders and cancer.

Dicer, the ribonuclease enzyme involved in cleaving the dsRNA, is encoded in humans by the DICER1 gene. It has also been responsible for the production of all mature miRNAs and therefore, inactivation results in loss of mature miRNAs and accumulation of miRNA precursors, which make it a powerful approach to analysis of miRNAs’ global roles.

Molecules binding to such structured RNA including their Dicer substrate often suffer from poor selectivity due to RNA’s negatively charged backbone and structural redundancy. The discovery of molecules that are capable of binding RNA structures with high affinity and specificity has proven to be quite challenging.

Aptamers as promising agents 

A potential solution to this problem is the use of aptamers that have emerged as bona fide therapeutics. Aptamers are functionally capable of binding to these structured RNA molecules with high affinity and specificity, with the ability to internalize the target cell via endocytosis (Figure 1).

Figure 1. Schematic representation showing delivery of aptamer siRNA chimera (AsiC) into a target cell via endocytosis and recognized by RNAi machinery. The ribonuclease Dicer binds to the siRNA part of the AsiC, cleaves the aptamer and load it into the RNA – induced silencing complex (RISC) further mediating the specific degradation of mRNA (Adapted from Krupse and Giangrande, 2017).

Aptamers are comparable to antibodies with several distinct advantages such as:

  • Smaller size for better cell penetration
  • Enhanced stability, therefore less susceptible to cell nucleases
  • Flexible structure and modular design characteristics which allows them to be conjugated with various functional groups for enhanced activity

Numerous studies have demonstrated the potential of aptamers to modulate the generation of specific miRNA or siRNA molecules along with their dicer substrates by forming aptamer –siRNA/miRNA chimeras. These aptamer-RNA chimeras have also acted as effective delivery vehicles of such RNAi molecules for downstream therapeutic applications. Two of such examples are reviewed below.

Aptamers as carrier of siRNA for HIV- 1 therapy

HIV infection is initiated by the interactions between the external envelope glycoprotein gp120 of HIV and the human cell surface receptor CD4, subsequently leading to fusion of the viral membrane with the target cell membrane. Preventing the virus from binding to its primary receptor is one of the most crucial and direct ways to prevent infection.

In this context aptamers represent a good strategy for siRNA conjugation that is composed of an aptamer and siRNA or Dicer substrate siRNA (DsiRNA) in order to increase specific cell delivery and internalization of siRNA molecules (Figure 2).

Figure 2. Schematic representation of an aptamer linked with a Dicer substrate siRNA.

Zhou et al., 2009 successfully isolated several novel 2’F RNA aptamers against gp120 protein and constructed them with the siRNA conjugates which showed specific internalization into cells expressing HIV gp120. The aptamer-siRNA chimeras (Ch A-1 and Ch B-68) were then linked with recombinant human dicer substrate (anti-tat rev siRNA) that together helped in delivery of the dicer to the target cells and provided more potent inhibition of HIV in vitro (Figure 3). The results demonstrated that the aptamers were functional in three ways:

  • They acted as effective delivery vehicles for anti-HIV dicer substrate siRNAs into HIV infected cells
  • They acted as lead inhibitors for potential anti-HIV therapeutic applications
  • They delivered other small HIV inhibitory agents.

Figure 3. The design and dual inhibition activity of aptamer – siRNA delivery system. (A) Schematic representation of aptamer-siRNA chimera. (B and C) The chimera showed significant cell-type specific binding affinity to the target CHO-gp160 cells as compared CHO-EE control cells as assessed by flow cytometry and are internalized into the cytoplasm by confocal microscopy. (D) In vitro Dicer processing suggested that Dicer preferentially entered N-1 using 3’ – end of the sense strand. (E) The siRNAs delivered by aptamers down-regulated target gene expression in the HIV infected CEM cells (HIV-1 tat/rev, TNPO3 and CD4).

Specific inhibition of miRNA processing using RNA aptamers

miRNAs are small, non-coding RNAs that act as post-transcriptional regulators of gene expression and alterations in their expression patterns can contribute to the pathogenesis of human disease. Inhibition of miRNA biogenesis using aptamers that target the unique structures of pre- miRNA represents a promising therapeutic strategy for a variety of diseases including cancer. Such inhibitors also represent new biochemical tools for interrogating pre- miRNA function and can potentially be utilized as affinity reagents for imaging and diagnostic applications.

In this view, Sczepanski et al., 2015 demonstrated the ability of RNA aptamers composed of L-RNA (aptamiRs) to tightly bind to the dicer mediated RNA structures and be classified as new miRNA inhibitors. Three human pre- miRNA targets were chosen for the study:

  • pre-miR10b and pre-miR155 – prototypical oncogenic miRNAs and their overexpression has been associated with various types of cancer.
  • miR33a – involved in cholesterol homeostasis and is a potential target for treatment of atherosclerosis.

The aptamiRs bound specifically to their respective dicer mediated miRNA targets (Figure 4). Moreover, the binding of L-aptamer to the distal stem-loop of a pre- miRNA inhibited Dicer-mediated cleavage with an IC50 of 87 nM, thus blocking formation of the mature miRNA. Given that L-RNA are intrinsically resistant to nuclease degradation, the results suggested that the L-RNA aptamer have potential to act as novel miRNA inhibitors.

Figure 4. Top image: Binding affinity and specificity of the pre- miRNA – aptamiR complexes studied by electrophoretic mobility shift assay (EMSA). Bottom image: Inhibition of Dicer cleavage of pre- miRNA by corresponding aptamiRs were analyzed by PAGE and values were determined for each concentration of aptamiR based on a linear fit of the data. IC50 values were determined accordingly.

Therefore, in addition to high affinity and selectivity, aptamers are capable of modulating RNA function by inhibiting Dicer mediated cleavage and blocking RNA – protein interactions. Such novel strategies can allow efficient expression of the aptamer RNA fusions that target such structured RNAs and proteins simultaneously.  These can be applicable for gene therapy for a number of therapeutic areas.

At Aptamer Group Ltd (AGL) we offer the advantage of designing aptamers to be optimized for the conditions in your end applications. This way they are engineered to bind to their target with high specificity and affinity. Using our highly defined technology, researchers at AGL have successfully isolated and characterised aptamers against miRNA targets. Moreover, AGL continuously aims to conduct further research in prevention, diagnosis and treatment of various therapeutic areas. If you would like to know more about aptamers and their applications, please contact us using the form below.

References:

Kruspe S, Giangrande PH. Aptamer-siRNA Chimeras: Discovery, Progress, and Future Prospects. Biomedicines. 2017;5(3):45. Published 2017 Aug 9. doi:10.3390/biomedicines5030045

Sczepanski JT, Joyce GF. Specific Inhibition of MicroRNA Processing Using L-RNA Aptamers. J Am Chem Soc. 2015;137(51):16032-16037. doi:10.1021/jacs.5b06696

Zhou J, Swiderski P, Li H, et al. Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells. Nucleic Acids Res. 2009;37(9):3094-3109. doi:10.1093/nar/gkp185