Commandeering a biological pathway using aptamer-derived molecular adaptors

Abstract

Induction of molecular proximity can mediate a discrete functional response in biological systems. Therefore, creating new and specific connectivity between non-interacting proteins is a means of imposing rational control over biological processes. According to this principle, here we use composite RNA aptamers to generate molecular adaptors that link various 'target' molecules to a common 'utility' molecule, with the utility molecule being an entry point to a pathway conscripted to process the target molecule. In particular, we created a bi-functional aptamer that simultaneously binds to the green fluorescent protein (serving as a surrogate extracellular target) and the opsonin C3b/iC3b (serving as the utility molecule). This bi-functional aptamer enabled us to commandeer the C3-based opsonization-phagocytosis pathway to selectively transport an extracellular target into the lysosome for degradation. This novel strategy has the potential for powerful therapeutic applications with extracellular proteins involved in tumor development or surface markers on cancer cells as the target molecules.

Figure 1.

RNA aptamers for C3 and its derivatives. (A) Secondary structure of full length AptC3-1 with its minimized version encircled. This structure was predicted using the mfold program (48) and supported by mutational analyses. (B) Affinity of AptC3-1 and AptC3-2 for C3 as measured by filter binding. Values represent the average of three independent assays. Error bars indicate standard deviations. (C) EMSA results for AptC3-1 and AptC3-2 binding to C3, C3b or iC3b.

Figure 2.

Bi-functional aptamer and formation of the triple complex. (A) Predicted secondary structure of Apt[C3-GFP]. Individual aptamers in the composite molecule are enclosed in boxes. (B) Binding of Apt[C3-GFP] to C3 (or its derivatives) or GFP (or its derivatives). The ‘low’ and ‘high’ concentrations are 50 and 250 nM for the fluorescent proteins, C3b and iC3b; 20 and 100 nM for C3; and 500 nM and 1 µM for BSA. (C) Schematic diagram of the protocol used to demonstrate triple complex formation. (D) Detection of triple complex formation.

Figure 3.

Aptamer-dependent GFP association with unfixed THP-1 cells. Representative micrographs of cells at two different magnifications, using differential interference contrast (DIC) or a green fluorescence filter. Scale bars indicate 50 and 5 µm, respectively. In the experimental panel, cells were incubated with Apt[C3-GFP], GFP and iC3b. iC3b was omitted in the control. Cells were washed with ice-cold serum-free RPMI medium and kept on ice before photography.

Figure 4.

Clearance of opsonized particles by the commandeered pathway. (A) Co-localization of GFP with iC3b molecules. iC3b molecules were visualized using anti-iC3b antibodies. The experimental panel shows cells that were incubated with Apt[C3-GFP], GFP and iC3b. iC3b was omitted in the control. (B) Co-localization of GFP with lysosomes. Lysosomes were visualized using the LysoTracker dye. The cells in the experimental panel were incubated with Apt[C3-GFP], GFP, iC3b and Lysotracker. iC3b was omitted in the control. Cells were fixed with 4% paraformaldehyde. Scale bars in both panels indicate 5 µm.

Figure 5.

Destruction of target molecules in lysosomes. (A) Rapid disappearance of the d2EGFP signal compared to that of GFP. The THP-1 cells were incubated with Apt[C3-GFP], iC3b and either GFP or d2EGFP. Live cells were photographed as in Figure 3. (B) Bi-color tracking of GFP-mCherry fusion protein. An early (15 min) and a late (48 h) stage are shown. Cells in the experimental panel were incubated with Apt[C3-GFP], GFP-mCherry (GFP-MC) and iC3b. iC3b was omitted in the control. Scale bars in both panels indicate 5 µm.