Nucleic Acids Research, Vol 24, Issue 6 1029-1036, Copyright © 1996 by Oxford University Press
A Geiger, P Burgstaller, H von der Eltz, A Roeder and M Famulok
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.
ARTICLES
RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity
Institut fur Biochemie, Genzentrum der Ludwig-Maximilians-Universitat Munchen, Germany.
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