Aptamer-enabled uptake of small molecule ligands

www.nature.com/scientificreports OPEN Received: 13 March 2018 Accepted: 3 October 2018 Published: xx xx xxxx Aptamer-enabled uptake of small molecule ligands Supipi Liyamali Auwardt1,2,3, Yeon-Jung Seo1, Muslum Ilgu1,2,3,4, Judhajeet Ray1,2,5, Robert R. Feldges2, Shambhavi Shubham1,2,6, Lee Bendickson1,2, Howard A. Levine1 & Marit Nilsen-Hamilton1,2,3 The relative ease of isolating aptamers with high specificity for target molecules suggests that molecular recognition may be common in the folds of natural RNAs. We show here that, when expressed in cells, aptamers can increase the intracellular concentrations of their small molecule ligands. We have named these aptamers as DRAGINs (Drug Binding Aptamers for Growing Intracellular Numbers). The DRAGIN property, assessed here by the ability to enhance the toxicity of their ligands, was found for some, but not all, aminoglycoside aptamers. One aptamer protected cells against killing by its ligand. Another aptamer promoted killing as a singlemer and protected against killing as a tandemer. Based on a mathematical model, cell protection vs. killing is proposed as governed by aptamer affinity and access to the inner surface of the cell membrane, with the latter being a critical determinant. With RNA molecules proposed as the earliest functional polymers to drive the evolution of life, we suggest that RNA aptamer-like structures present in primitive cells might have selectively concentrated precursors for polymer synthesis. Riboswitches may be the evolved forms of these ancient aptamer-like “nutrient procurers”. Aptamers with DRAGIN capability in the modern world could be applied for imaging cells, in synthetic cell constructs, or to draw drugs into cells to make “undruggable” targets accessible to small molecule inhibitors. Controlling the entrance and exit of small molecules through the cell membrane is critical to cell survival. In modern cells, transmembrane protein transporters promote the entry of essential nutrients for cell proliferation. However, protein transporters were not available to early forms of life that, once surrounded by a hydrophobic membrane, depended on the availability of precursors for their expansion by macromolecular synthesis. Experimental results and logical deductions support the hypothesis that the earliest forms of life relied on RNA molecules to perform replication and other catalysis1–3. Initially, life may have evolved within confined inorganic cavities that were possibly located in mineral precipitates such as of iron monosulphide2 or on clay, which promotes encapsulation by lipids4. It is not clear how long the period of RNA dominance lasted before proteins took over most catalytic functions. But, primitive cells are believed to have evolved through several eras, which included the sequential introduction of functional RNAs, RNPs and proteins culminating in the last universal common ancestor (LUCA), which existed prior to the divergence of the domains of Bacteria, Archaea, and finally the appearance of Eukarya2,5. These primitive forms are presumed to have faced the same challenge, as do modern cells, which is to selectively concentrate the precursor molecules required for cell replication and function. The proposed membrane composition of the very early evolving cells, believed to consist of a mixture of fatty acids, glycerol esters and amphiphiles, is more permeable than a membrane of homogeneous lipid composition6–8. Small hydrophilic molecules might also have been flipped into the primitive cell by association with charged head-groups of the early membrane constituents9. However, neither diffusion nor flipping provides much selectivity to the distribution of small molecules that enter the cell and would have limited the rate of RNA replication in these primitive cells. Once cell membranes evolved to include hydrocarbon chains and ether/ester bonds on a glycerophosphate backbone, the permeability to hydrophilic molecules would have decreased and the cells then needed to evolve transporters to efficiently move needed metabolic precursors into the cell10. Aptamers are small nucleic acids with high affinities and specificities for a particular target molecule or a group of related molecules. They can be obtained in vitro by repeated selection and amplification of nucleic acid populations that bind a chosen molecular target11–14. Aptamer-like activities are also found as the recognition elements 1 Iowa State University, Ames, IA, USA. 2Ames National Laboratory, Ames, IA, USA. 3Aptalogic Inc., Ames, IA, USA. Present address: Middle East Technical University, Ankara, Turkey. 5Present address: Cornell University, Ithaca, NY, USA. 6Present address: Integrated DNA Technologies, Coralville, IA, USA. Correspondence and requests for materials should be addressed to M.N.-H. (email: ) 4 SCIENTIfIC ReportS | (2018) 8:15712 | DOI:10.1038/s41598-018-33887-w 1 www.nature.com/scientificreports/ in riboswitches, which are portions of RNAs that regulate transcription or translation differently when unbound compared with when bound to their target ligand. These RNA elements may date back to a period prior to the divergence of the Bacteria and Archaea domains. This is suggested by the sequence of the TPP riboswitch which is closely related in eubacteria and archaebacteria15. Here we consider that aptamers may have been “transporters” of RNA precursors during the earlier era of RNA predominance and later once a primitive membrane had evolved such as in LUCA. As the cellular metabolism and architecture became more complex, aptamers would have also evolved into components of modern-day riboswitches by which they regulate transcription and translation. We show, experimentally and by mathematical modeling, that RNA aptamers present inside a cell are capable of increasing both the total amounts and the free intracellular concentrations of the small molecules to which they bind. A previous mathematical model based on partial differential equations (PDE), by which we predicted this effect, required that the intracellular aptamer be mobile16. Here we describe a related compartmental model in which mobile aptamers have access to their small molecule ligands located near the surface of the inner cell membrane (Math Model, Supplementary Information). By capturing and moving their ligands from the cell membrane where they could readily exit the cell, the aptamers drive the intracellular free ligand concentrations up. With the development of the cell membrane, a primitive cell would have been advantaged by the ability of aptamers to capture and accumulate substrates for continued replication. The capability of selectively retaining the appropriate small molecule precursors and making them available to the replicating RNA or DNA would have given an evolutionary advantage over other cells. Curiously, many of the identified riboswitches recognize molecules chemically related to nucleic acid precursors. As well as the (...truncated)