Acridine-N peptide conjugates display enhanced affinity and specificity for boxB RNA targets.
Arginine-rich peptides and small-molecule intercalating agents utilize distinct molecular mechanisms for RNA recognition. Here, we combined these distinct binding modules in an effort to create conjugate ligands with enhanced affinity and specificity using the bacteriophage lambda N peptide-boxB interaction as a model system. We first designed and synthesized a series of peptide-acridine conjugates using portions of the RNA-binding domain of N protein (11- and 22- residue peptide segments) and then compared the binding affinity, specificity, salt dependence, and structural properties of the RNA-peptide and RNA-peptide-acridine conjugate complexes using steady-state fluorescence, CD spectroscopy, NMR, and native gel mobility shift assays (GMSAs). These analyses revealed that the full-length peptide-acridine conjugate displayed substantially improved RNA binding affinity ( approximately 80-fold; K(d) approximately 15 pM) relative to that of the peptide alone (K(d) approximately 1.2 nM). In accordance, we also observed specificity enhancement ( approximately 25-fold) as determined via comparison of the binding of the best conjugate to a cognate lambda boxB RNA with that to a noncognate P22 RNA hairpin (80-fold vs 3.2-fold enhancement). Furthermore, the observed binding enhancement was unique to the full-length conjugate with a flexible linker, implying that the structural context of the acridine presentation was critical. Taken together, our observations support the idea that peptide- and intercalation-based binding can be combined to create a new class of high-affinity, high-specificity RNA-binding ligands.
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Mail-code 147-75, Pasadena, California 91125, USA.
Xin
X
Qi
Tianbing
T
Xia
Richard W
RW
Roberts
D000166:Acridines / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*
D000166:Acridines; D008024:Ligands; D010455:Peptides; D012313:RNA