RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity

Abstract

A completely randomized RNA pool as well as a degenerate pool comprised of an RNA sequence which binds citrulline with a dissociation constant of 0 muM 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 non-cognate 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 sequence 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.

RNA–Amino Acid Binding: A Stereochemical Era for the Genetic Code

“…This is as anticipated-larger RNA sites bind GTP better (Carothers et al 2004) because larger sites are better prestructured for nucleotide binding (Carothers et al 2006), rather than because larger sites make more interactions. Though amino acids are somewhat different double-ended ligands with loose linkage, unlike nucleotides, a similar progression might be anticipated, and has been partially characterized for arginine (Geiger et al 1996).…”

“…A rigorous selection (Geiger et al 1996) for side chain selectivity and slow dissociation yields likely double-ended sites with low K D (=0.33 lM) and high enantioselection (K L/D = 12,000). If selection is relaxed, likely single-ended sites with K D of millimolar range are recovered (Connell et al 1993;Tao and Frankel 1996).…”

Isolation of high-affinity GTP aptamers from partially structured RNA libraries

“…efficiently recovered, elution was achieved by four successive incubations of 30 min each with elution buffer containing GTP, and the four resulting fractions were combined for amplification. In later rounds, weak complexes were selected against by a pre-elution step in which one column volume of elution buffer was incubated on the column for 0.5-10 min (the time was increased over the course of the selection), then replaced with fresh elution buffer for further pre-elution, or for the first long elution step (12). Pre-elution fractions were not included in reverse transcription and amplification.…”

“…Many aptamers have at least one internal stem-loop that appears to act as a structural anchor for recognition loops (for examples, see refs. [10][11][12][13]. The basepaired stems of these stem-loops can generally be of any sequence (10,11).…”

In vitro and in vivo characterization of novel mRNA motifs that bind special elongation factor SelB

“…Sequence of wild-type competitor: 5Ј-CTC GTG TCT GAC ACG GCC CAT CGG TTG CAG GTC TGC ACC AAT CGG TCG GTA ATG GCG CAA-3Ј; primers: 5Ј-TCT AAT ACG ACT CAC TAT AGG CTC GTG TCT GAC ACG GCC CA-3Ј and 5Ј-TTG CGC CAT TAC CGA CCG AT-3Ј. PCR amplification, reverse transcription, and in vitro transcription were performed as described (20). The complexity of the library was 5 ϫ 10 14 different molecules, determined as described (21).…”