Design and Characterization of Asparagine-and Lysine-Containing Alanine-Based Helical Peptides That Bind Selectively to A·T Base Pairs of Oligonucleotides Immobilized on a 27 MHz Quartz Crystal Microbalance

摘要

We have systematically designed and synthesized six kinds of 16- 17 mer alanine-based peptides containing four to six lysine (K) and one to four asparagine (N) residues to achieve the selective binding to A .T base pairs of DNA duplexes. The position and number of K and N residues were changed in the helical structure according to common features of the DNA-binding proteins, in which K and N residues are expected to interact electrostatically with phosphate groups and to interact with A .T base pairs by hydrogen bonding, respectively. The time courses of binding of these peptides to dA30.dT 30 and dG30.dC30 duplexes immobilized on a 27 MHz quartz crystal microbalance (QCM) were studied in 10 mM phosphate buffer (pH 7.5) and 40 mM NaCl at 10 °C. The maximum binding amounts (~mmax) on a nanogram scale and binding constants (Ka) could be obtained from the frequency decrease (mass increase) of the oligonucleotide-immobilized QCM. The conformation changes of the peptides upon binding to DNAs were monitored by circular dichroism (CD) spectroscopy. The four properly arranged N residues in the six-cationic K peptide, K6N4(d), resulted in a 5-fold higher affinity for A.T base pairs (Ka = 5.9 X 105 M-I) than for G.C base pairs (Ka = 1.2 X 105 M-I), and a.-helices were clearly promoted by the binding to A .T base pairs from CD spectral changes.