In the present study, insight into the structural properties and target specificity of hIL-6R RNA aptamer AIR-3A was gained. Although this GQ forming aptamer has been dealt with in earlier publications and was subjected to intensive characterization, the focus lay now on clarification of GQ folding in dependence of cation presence and identity and the contribution of nucleotides to quadruplex formation as well as target binding interaction. It turned out that several features had been undiscovered so far. In this regard, it was found that no specific structuring can be determined, but that AIR-3A displays a polymorphic GQ and that it is likely to oligomerize in solution as well as in presence of target molecule hIL-6R.
Concerning structural elucidation of AIR-3 and AIR-3A, it can be assumed that, due to the polymorphic character of the GQ, further analyses should be conducted on less variable variants.
Design of aptamers derived from AIR-3A that fold into defined quadruplexes may be beneficial with regard to the possibility of broadening the scope of analysis methods applicable. As such, NMR and X-ray crystallography could yield valuable information on the 3D-structure as well as on the process of folding and the degree of association.
Comparison of different variants could then help to investigate the interaction surfaces of RNA and protein and gain knowledge on the properties that make hIL-6R a suitable target for GQs. With the polymorphic AIR-3A, these methods cannot be used, as signals of the different species overlay and impede analysis.
Regarding secondary structure analyses, AIR-3A variants could be subjected to chemical and enzymatic probing. Here, SHAPE could be another option to identify flexible nucleotide positions.250 Also probing with RNase U2,47 which cleaves A-specific, could be used as a complement to RNase T1.
Footprinting experiments in presence of hIL-6R could then also help to find out which nucleotides mediate target interaction.251, 252 Interaction could further be monitored by UV cross-linking experiments as presented in former works.197, 245 These approaches could be optimized for identification of amino acid residues involved in RNA binding.253-255
In this work, the proposed lysosomal fate of AIR-3A after cellular uptake197 was confirmed. For future investigations, the cellular whereabouts and trafficking, as well as stability of the aptamer are of interest. This could be examined in a time resolved manner. On the one hand, it could be achieved by co-localization with further cellular compartments using faster techniques compared to cLSM such as confocal spinning disc laser microscopy. Also, double labeled AIR-3A (two different fluorescent dyes
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conjugated to 5’- and 3’-end allowing for Förster resonance energy transfer (FRET)) could thereby be followed throughout the cell with the benefit of detecting degradation of the aptamer as a decay of FRET signal intensity and differentiation of the fluorescence of the two dyes. Another method yielding high spatial and time resolution is represented by in-liquid transmission electron microscopy (TEM).256 This novel technique was already applied for visualization of DNA functionalized gold nanoparticles as a proof that electron microscopy works in aqueous solution, and it is currently worked on for application in cellular detection. Against this background, coordination of cations with scattering potential (such as Ba2+) could serve as a labeling to visualize AIR-3A during endocytosis and inside the cell by in-liquid TEM. Further cations suitable in this respect, such as Sr2+, could be analyzed by CD detected titrations as well.
To quantify cellular uptake of AIR-3A as well as its stability, quantitative real time PCR could be applied.257 After incubation with AIR-3A, surface bound aptamer could be removed and cells cultured for different time periods, after which total RNA is isolated and analyzed by RT-PCR for abundance of AIR-3A. This could give information on the extent of lysosomal degradation and help to compare intracellular stability of derivatized AIR-3(A). Also, influence of aptamer treatment on the transcriptome could be investigated with respect to regulative properties.
Before further aptamer aided drug deliveries are established, stabilization of AIR-3A should be focused on. Next to 2’F modifications (which are only applicable for AIR-3, but not for AIR-3A), integration of 3’-inverted thymidine or 5’-PEG could increase the half-life.131
The efficiency of drug delivery and release could be improved by addition of endosomal escape agents.106 This would for example enable application of siRNA-AIR-3A conjugates. Targeted delivery could also be achieved by aptamer-functionalized drug-encapsulating liposomes.258 This would increase the drug load per aptamer molecule and result in a more pronounced cytotoxic effect.
Further targets of AIR-3A could be identified by intracellular crosslinking. However, in preparation of such experiments, optimization of the pull down experiments established in this study would be necessary to verify AIR-3A specificity and reproducibility of the assay.
To tackle the task of signaling inhibition, experiments could be designed to multimerize AIR-3A in a controlled manner. Alternatively, conjugation of AIR-3A and RAID3 would be possible. This could result in an aptamer derivative with higher affinity and slower off rate. Although neither RAID3 nor AIR-3A alone affect receptor interaction with hIL-6 or gp130, the conjugate might interfere with it by sheer size.
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Finally, the other aptamers with hIL-6R specificity could be subjected to further analysis. As such, identification of the binding site of AID-1 might give insight into the GQ binding potential of the receptor. Especially as AID-1 was shown to compete for binding with AIR-3A,259 and is therefore supposed to target the same site, this could yield valuable information. RAID3 showed no competition and therefore complements AIR-3A. Structural analyses could be performed to further characterize this aptamer. As RAID3 has already been stabilized and was shown to be internalized by target cells, it represents an alternative tool for aptamer mediated drug delivery using the hIL-6R system.
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