Kinetic characterization of TAR RNA-Tat peptide and neomycin interactions by acoustic wave biosensor.
The kinetics of binding of short Tat peptides and an aminoglycoside molecule to the human immunodeficiency virus-type 1(HIV-1) TAR RNA and to a bulge mutant analogue (MTAR) is studied in a biosensor format by monitoring the time course of the response in a series resonance frequency, using an acoustic wave biosensor. Association and dissociation rate constants are evaluated by fitting the experimental data to a simple 1:1 (Langmuir) model. Kinetic rate and equilibrium dissociation constants show that MTAR-peptide complexes are subject to a higher dissociation rate and are less stable compared to the corresponding TAR-peptide complexes. In addition, longer peptides display enhanced discrimination ability than a shorter peptide according to the equilibrium dissociation constants evaluated using this technique. K(D) values for TAR-Tat vs. MTAR-Tat complexes are 2.6 vs. 3.8 microM for Tat-12, 0.87 vs. 4.3 microM for Tat-18 and 0.93 vs. 1.6 microM for Tat-20. The equilibrium dissociation constant for TAR-neomycin complex is 12.4 microM and it is comparable to the values obtained from non-biosensor type assays. These findings are in parallel with those cited in the literature and the results from this study underline the potential of the acoustic wave sensor for detailed biophysical analysis of nucleic acid-ligand binding.
Tassew
Nardos
N
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, Canada M5S 3H6.
Thompson
Michael
M
D000162:Acoustics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D015696:Gene Products, tat / Q000737:chemistry / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009355:Neomycin / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015696:Gene Products, tat; D012367:RNA, Viral; D009355:Neomycin