Holistic design

Treatment with D4 aptamer (left) inhibits RET-induced differentiation of NIH-3T3 cells (controls, right).

The promise of specific oligonucleotide ligands (aptamers) as potential regulators of biological processes is beginning to be realized, with several lead compounds in clinical trails. Laura Cerchia and colleagues now validate an aptamer selection protocol that targets transmembrane receptors in the form in which they exist on the surface of cells. The aptamers produced bind specifically to the RET receptor tyrosine kinase and inhibit its downstream signalling effects.

RET is mutated in multiple endocrine neoplasia type 2A and 2B syndromes and in familial medullary thyroid carcinoma. The C634Y mutation in the extracellular domain causes constitutive activation of the receptor. The authors used a 'systematic evolution of ligands by exponential enrichment' (SELEX) procedure that was modified to isolate aptamers using the RET^C634Y mutant expressed on PC12 cells. The authors reasoned that this in vivo procedure would select aptamers that bound only to transmembrane receptors in their natural physiological environment.

Initially, they incubated a library of 2'-fluoropyrimidine RNAs with parental PC12 cells to remove aptamers that bind non-specifically to the cell surface. To select for aptamers that specifically bound the mutant receptor, the supernatant was incubated with PC12–RET^C634Y cells. Unbound sequences were washed off, and the bound winning sequences cloned. The resulting aptamers did not bind to a recombinant extracellular C634Y RET fragment, highlighting the strength of the authors' whole-cell approach.

The winning aptamers were screened for their ability to inhibit the signalling of the mutant receptor and several blocked phosphorylation of RET^C634Y and of its downstream effector extracellular signal-regulated kinase (ERK). Surprisingly, the best inhibitor (D4) also binds to the wild-type human RET that is expressed naturally on neuroblastoma cells. D4 blocks phosphorylation of wild-type RET and ERK following stimulation of the receptor by its ligand glial-cell-derived neurotrophic factor (GDNF). The aptamer also inhibits RET-dependent changes in cell phenotype: the growth of neurites from PC12 cells expressing the wild-type RET receptor and activated with GDNF is blocked by D4, as are changes in morphology seen in NIH-3T3 cells following expression of the constitutively active RET^C634Y receptor (pictured).

So it seems that D4 is a highly selective aptamer for RET that can block the downstream effects of RET signalling on cell differentiation, and possibly transformation. The authors suggest that this differential whole-cell SELEX approach will be useful in the isolation of other lead therapeutic compounds and diagnostic cell-surface markers.