RNA Aptamers That Bind l-Arginine with Sub-Micromolar Dissociation Constants and High Enantioselectivity

Albert Geiger 1 Petra Burgstaller 1 Herbert von der Eltz 0 1 Albert Roeder 0 1 Michael Famulok 1 0 Boehringer Mannheim GmbH, Research and Development , Werk Penzberg, Nonnenwald 2, 82372 Penzberg, Germany 1 Institut fr Biochemie, Genzentrum der Ludwig-Maximilians-Universitt Mnchen , Wrmtalstrae 221, 81375 Mnchen, Germany A completely randomized RNA pool as well as a degenerate pool comprised of an RNA sequence which binds citrulline with a dissociation constant of 60 m M were used to select for tight binding arginine specific RNA aptamers. A modified in vitro selection scheme, based on affinity chromatography was applied to allow the enrichment of high affinity solution binders. The selection scheme included a negative selection with the noncognate ligand citrulline, and a heat denaturation step prior to affinity elution with an excess of the cognate ligand arginine. After 20 cycles the majority of the pools bound specifically to the arginine matrix even after denaturation/renaturation in the presence of 20 mM of a non-cognate amino acid. When denatured and eluted in the presence of 20 mM arginine, the selected RNAs quantitatively washed off the column. These RNA aptamers were cloned and sequenced. Equilibrium dialysis performed with the most abundant clone among the selected sequences revealed Kd values of 330 nM for the RNA/arginine affinity, which is nearly a 200-fold improvement over the tightest binding arginine binding RNAs known to date. Arginine recognition by this RNA is highly enantioselectice: L-arginine is bound 12 000-fold better than D-arginine. Chemical modification analysis revealed that the secondary structure of the aptamer might contain a pseudoknot motif. Our tight binding arginine aptamers join a number of natural and in vitro selected RNAs which recognize arginine. The RNAs described here compare in their binding affinity with the tightest binding RNA aptamers for low molecular weight molecules isolated in other in vitro selection experiments. - Interactions between amino acids and RNA play substantial roles in a number of biological systems (1). For example, arginine inhibits * To whom correspondence should be addressed the self-splicing reaction of the group I intron of Tetrahymena by substituting for two H-donor sites of the guanosine cofactor which contact the G264C311 base pair in the ribozymes guanosine binding site (2,3). Recently, the editing reactions of aminoacyl tRNA synthetases have been viewed as an example of RNA dependent amino acid recognition (4). These editing reactions involve RNA dependent steps which eliminate errors of amino acid activation and aminoacylation (5). A third example is the interaction of the HIV-1 TAT protein with a stemloop structure of TAR RNA, located at the 5-end of HIV-1 mRNA. Critical for the recognition of TAT and TAR is a single arginine within a basic region of TAT (6). Short oligopeptides resembling the basic region as well as free arginine bind specifically to TAR, although weaker than within the context of the whole protein (7). The TATTAR interaction provided the first example to show that in proteinRNA recognition RNA structures are involved which interact with individual amino acid side chains in the protein. It seems likely that other, yet undiscovered RNAprotein interactions exist in which single amino acid side chains within a protein or peptide form specific contacts to structural elements provided by RNA to largely determine specificity, functionality and strength of binding (1). The isolation and characterization of RNA sequences which specifically recognize individual amino acids might facilitate a better understanding of biologically relevant proteinRNA or RNA amino acid interactions. A powerful tool to obtain amino acid binding RNAs is in vitro selection (811). RNA aptamers which specifically recognize amino acids, such as immobilized tryptophan (12), arginine (13,14), citrulline (14) and valine (15) have been extracted from pools of up to 1015 different RNA sequences. The reported affinities ranged from 60 m M (14) to 12 mM (15) with a high level of discrimination against other amino acids being obtained in each case. Among these amino acid binding RNAs, the arginine specific aptamers might be especially relevant to proteinRNA recognition because arginine side chains carrying a positive charge at neutral pH seem to be particularly suited to form specific contacts with a negatively charged nucleic acid (16). For example, the HIV-1 Rev protein contains a basic region in which 10 out of 17 amino acids between positions 34 and 50 are arginines. A corresponding 17mer peptide binds to the Rev responsive element (RRE) RNA IIB hairpin in the same way as within the context of the full-length protein (17) with four arginines being important for specificity (18). In the BIV-TAR/Tat complex an arginine and an isoleucine residue were found to be critical for binding and specificity (19). Furthermore, the Rex-protein of HTLV-I might interact with its natural RNA binding element XBE through arginine residues (20). To explore the range of affinities which can be achieved in RNAarginine recognition we set out to obtain RNA sequence motifs which tightly bind to arginine. We started with a completely randomized pool and a partially randomized pool of RNAs comprised of a citrulline binding sequence which was previously isolated by in vitro selection (14). Previously, arginine binding sequences which differed from the citrulline motif in three base positions were identified from the latter pool (14). In the present study, a modified in vitro selection scheme based on affinity chromatography was applied to extract tight binding arginine specific RNA aptamers from the two pools. A number of sequences were identified which bound tightly to arginine and did not share significant sequence homologies to each other or any of the other previously identified arginine aptamers (13,14). One of these sequences was shown to bind L-arginine with high enantioselectivity and with affinities in the order of magnitude of the tightest binding RNA aptamers for low molecular weight molecules obtained so far (2124). Our study shows that the problem of arginine recognition can be achieved by many different RNA sequences over a broad range of binding affinities. MATERIALS AND METHODS L-Arginine, D-arginine, L-citrulline, agmatine and 1-cyclohexyl3-(2-(morpholino-ethyl)-carbodiimide metho-p-toluenesulphonate (CMCT) were purchased from Sigma. L-Homoarginine and dimethyl sulfate (DMS) were from Aldrich, kethoxal (KE) from ICN. L-[2,3,4,5-3H]arginineHCl, [a -32P]ATP and [g -32P]ATP (3 Ci/m mol) from Amersham. dNTPs, NTPs, glycogen and all enzymes and cloning plasmids used were from Boehringer Mannheim unless otherwise noted. Sequencing was performed with a digoxigenin labelled primer (DIG Taq DNA sequencing; Boehringer Mannheim). PCR primers were synthesized on an Millipore Expedite oligonucleotide synthesizer using standard phos (...truncated)