The binding of guanine-guanine mismatched DNA to naphthyridine dimer immobilized sensor surfaces: kinetic aspects.
Naphthyridine dimer composed of two naphthyridine chromophores and a linker connecting them strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. The kinetics for the binding of the G-G mismatch to the naphthyridine dimer was investigated by surface plasmon resonance assay. The sensor surface was prepared by immobilizing naphthyridine dimer through a long poly(ethylene oxide) linker with the ligand density of 9.1 x 10(-12) fmolnm(-2). The kinetic analyses revealed that the binding of the G-G mismatch was sequence dependent on the flanking base pairs, and the G-G mismatches flanking at least one G-C base pair bound to the surface via a two-step process with a 1:1 DNA-ligand stoichiometry. The first association rate constant for the binding of the G-G mismatch in the 5'-CGG-3'/3'-GGC-5' sequence to the naphthyridine dimer-immobilized sensor surface was 3.2 x 10(3)M(-1)s(-1) and the first dissociation rate constant was 1.4 x 10(-2)s(-1). The association and dissociation rate constants for the second step were insensitive to the flanking sequences, and were almost of the same order of magnitude as the first dissociation rate constant. This indicates that the second step had only a small energetic contribution to the binding. The association constant calculated from kinetic parameters was 2.7 x 10(5)M(-1), which is significantly smaller than the apparent association constants obtained from experiments in solution. Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry on the complex produced from the G-G mismatch and naphthyridine dimer showed the formation of the 1:1 complex and a 1:2 DNA-ligand complex in solution. The latter complex became the dominant complex when a six-fold excess of naphthyridine dimer was added to DNA.
Nakatani
Kazuhiko
K
PRESTO, Japan Science and Technology Agency (JST), Kyoto 615-8510, Japan. nakatani@sbchem.kyoto-u.ac.jp
Kobori
Akio
A
Kumasawa
Hiroyuki
H
Goto
Yuki
Y
Saito
Isao
I
D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D006147:Guanine / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D015394:Molecular Structure; D009287:Naphthyridines / Q000138:chemical synthesis / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance / Q000295:instrumentation* / Q000379:methods*
D009287:Naphthyridines; D012996:Solutions; D006147:Guanine; D004247:DNA