In Vitro Selection of RNA Molecules That Inhibit the Activity of Ricin A-chain

Abstract

The cytotoxin ricin disables translation by depurinating a conserved site in eukaryotic rRNA. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no resemblance to the normal RTA substrate, the sarcin-ricin loop (SRL), and were not depurinated by RTA. An initial 80-nucleotide RNA ligand was minimized to a 31-nucleotide aptamer that contained all sequences and structures necessary for interacting with RTA. This minimal RNA formed high affinity complexes with RTA (K d ‫؍‬ 7.3 nM) which could compete directly with the SRL for binding to RTA. The aptamer inhibited RTA depurination of the SRL and could partially protect translation from RTA inhibition. The IC 50 of the aptamer for RTA in an in vitro translation assay is 100 nM, roughly 3 orders of magnitude lower than a small molecule inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower than the best known RNA inhibitor. The novel anti-RTA aptamers may find application as diagnostic reagents for a potential biological warfare agent and hold promise as scaffolds for the development of strong ricin inhibitors.Ribosome-inactivating proteins inhibit protein synthesis by disabling the translation machinery. Class I ribosome-inactivating proteins have a catalytic A-chain that recognizes and depurinates a universally conserved adenosine found in a GAGA tetraloop in the sarcin-ricin loop (SRL) 1 of eukaryotic 23-28 S rRNA (1, 2). Class II ribosome-inactivating proteins have an additional lectin B-chain that is required for cell surface attachment and subsequent endocytosis of the toxin domain (3).Ricin, from the castor bean plant Ricinus communis, is a class II ribosome-inactivating protein that has the potential of being used as a weapon in a biological attack (4, 5). Once inside a cell, ricin is extremely toxic; one resident molecule is sufficient to kill the cell, and the enzyme has been reported to inactivate 1777 ribosomes min Ϫ1 (2). Aerosolized ricin causes severe respiratory distress and is lethal when injected intravenously at a level of only 3-5 g/kg (6). Ricin has so far proved to be a relatively inefficient biological weapon but is prepared easily even by Third World nations and terrorist groups. Therefore, facile detection of the toxin and the development of antidotes remain priorities (6, 7).In vitro selection is a powerful molecular tool that can be used to generate ligands for a wide variety of targets, including both nucleic acid and non-nucleic acid-binding proteins (8, 9). Aptamers can be engineered readily to function as either therapeutics or sensors. For instance, RNA aptamers that bind to human immunodeficiency virus type I Rev also inhibit viral replication (10). Similarly, RNA aptamers that bind to the ␤ 2 integrin leukocyte function-associated antigen 1 specifically inhibit a signal transduction pathway when expressed in vivo (11). Consequently, aptamers that recognize and potentially inhibit ricin might be useful prophylactic or therapeutic agent...

The DNA aptamers that specifically recognize ricin toxin are selected by twoin vitro selection methods

“…Therefore, selection of aptamers which specifically recognize ricin and can be used as biocomponents in biosensors (aptasensors) should be paid more attention. Hesselberth et al [36] have gained aptamers which have high affinity with ricin A chain. But, they are RNA ligands, and have a very short lifetime in biological fluids.…”

Kinetic CE: Foundation for homogeneous kinetic affinity methods

“…1). In heterogeneous methods, L is separated from L?T on the surface of a solid substrate, such as a filter, chromatographic support, or a sensor [7][8][9]. In homogeneous methods, L is separated from L?T in solution based on differences in the mobility of L and L?T.…”

Aptasensors for biosecurity applications

“…[18] The RNA aptamers were generated via 9 selection cycles using the NC filter partitioning approach. Upon analysis of the obtained selected aptamers, it was observed that the sequences bear no resemblance to its usual target ribomsal RNA substrate.…”