A Novel Solid-Phase Assembly for Identifying Potent and Selective RNA Ligands
Nathan W. Luedtke and Yitzhak Tor
Abstract
Replication of the human immunodeficiency virus (HIV-1)
requires an ordered pattern of viral gene expression. This process is dependent
upon the association of Rev, an essential viral regulatory protein, with its
respective RNA binding site, the Rev-response-element (RRE). Small molecules that specifically bind the RRE and preclude or
competitively displace the Rev protein are therefore promising antiviral
candidates. New approaches that
allow the rapid determination of both the RNA affinity and specificity of small
molecules will assist in the discovery of new lead compounds and advance the
understanding of RNA recognition. To
this end, we report the assembly of an immobilized RNA-protein complex and
demonstrate its application to the discovery and characterization of new RNA
binders. We have developed an assay
that identifies small molecules that specifically interfere with Rev-RRE
binding. To examine the versatility
of the assembly-based assay the competitive binding of polycyclic aromatic
amidines to the RRE has also been examined. This family of ligands is more potent than
aminoglycosides. Their
previously reported trend in RRE affinity is again accurately reproduced. A tenfold increase in
IC50 values in the presence of competing DNA
indicates that these compounds also have a relatively high affinity for
double-stranded DNA. This result clearly illustrates how the observed potency of
an inhibitor is influenced by its selectivity.
Both selectivity and affinity are crucial for the design and evaluation
of new RNA-protein inhibitors.