Why Develop Bispecific Aptamer Chimeras?
Cell membrane proteins play an important role in regulating cellular functions and are important therapeutic targets for cancer and immune-related diseases. Rapid and specific reduction in the abundance of specific membrane proteins would facilitate the development of therapeutic strategies for targeting membrane proteins. Strategies to control the abundance of membrane protein targets rely primarily on interference with gene expression, however, the application of these genetic engineering approaches is limited by time-consuming, unpredictable and low efficiency. Non-genetic strategies, such as the use of small molecules as inhibitors, still lack a sufficient number of therapeutic membrane protein-targeting agents. Proteolysis methods (e.g. PROTAC) developed to date have been mainly focused on cytoplasmic proteins and there is still a need to develop new methods to degrade cytosolic membrane-bound proteins.
Design of Bispecific Aptamer Chimeras
Aptamers, as a “synthetic antibodies”, have the advantages of simple preparation, precise synthesis, and high stability of in vitro transportation compared with antibodies. The team has designed and synthesized the bispecific aptamer chimeras consisting of aptamer A1 targeting the cation-independent mannose-6-phosphate receptor (IGFIIR), aptamer A2 targeting membrane proteins, and a linker L for bridging and stabilizing two separate aptamers, which is named Aptamer1-Linker-Aptamer2 (A1-L-A2).
Three bispecific aptamer chimeras have been reported in the literature: D1 (single-stranded DNA with a linker of 10 bases), D2 (double-stranded DNA with a linker of 17 base pairs), and D3 (double-stranded DNA with a linker of 23 base pairs). Using A1-L-A2 to target both IGFIIR and the membrane protein, the target membrane protein enters the lysosome under IGFIIR-mediated degradation.
Biological Activity of Bispecific Aptamer Chimeras
Bispecific aptamer chimeras transport target membrane proteins to the lysosome. The researchers examined the content of mesenchymal epithelial transition factor receptor (Met) on the cell membrane surface after incubating the bispecific aptamer chimeras with HeLa cells, and the results showed that Met internalization increased with longer incubation time. Confocal results showed that the D3 fluorescent signal overlapped with that of the lysosomal dye, while the fluorescent signal of the Met alone was mainly located at the cell surface.
Experiments at the cellular level revealed that treatment of cells with bispecific aptamer chimera D3 for 1 h significantly reduced the levels of Met proteins (tumor-specific markers) on the cell membrane. At the same time, the bispecific aptamer chimera only targeted the protein of interest for degradation, and had no significant effect on the levels of non-target proteins, which had good targeting and specificity. Based on this bispecific aptamer chimera degradation method, it is universal and can effectively induce different membrane proteins (e.g. Met, PTK7) to lysosomes for degradation, and then inhibit the function and signaling of target proteins.
Source: https://ptc.bocsci.com/resource/bispecific-aptamer-chimeras-degrade-target-proteins-on-cell-membranes.html