High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element.
Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed.
Roy
Subhadeep
S
Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213-3890, USA.
Tanious
Farial A
FA
Wilson
W David
WD
Ly
Danith H
DH
Armitage
Bruce A
BA
D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry*; D054856:G-Quadruplexes; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013696:Temperature; D013816:Thermodynamics
D015342:DNA Probes; C000715470:MAS1 protein, human; D020135:Peptide Nucleic Acids; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; D004247:DNA