Probing Biomolecular Interactions with Dual Polarization Interferometry: Real-Time and Label-Free Coralyne Detection by Use of Homoadenine DNA Oligonucleotide

We incorporated the specific recognition of adenine-rich singled-stranded DNA (ssDNA) into dual polarization interferometry (DPI) measurements for direct, selective, and sensitive detection of the small molecule coralyne, and we simultaneously employed the real-time and label-free technique for detailed investigation of the interaction between coralyne and adenine-rich ssDNA. Data from UV-visible spectroscopy, circular dichroism (CD) spectroscopy, and DNA melting firmly confirmed that 48-mer homoadenine ssDNA oligonucleotide (A(48)) had highly specific recognition for coralyne, whereas 48-mer homothymine ssDNA oligonucleotide (T-48) as the control had no such recognition. The immobilization of ssDNA (A(48) or T-48) on a silicon oxynitride chip could be achieved through a preadsorbed poly(ethylenimine) (PEI) layer. Mass, thickness, and refractive index (RI) changes resolved by DPI during the whole process of ssDNA immobilization suggested that most ssDNA molecules were likely to lie on the PEI surface mainly in the form of a flat monolayer and insert themselves partly into the PEI layer. Qualitative and quantitative analysis of mass, thickness, and RI changes in A(48)/PEI layer upon addition of different concentrations of coralyne revealed that A(48) most likely underwent a conformational change from single-stranded to double-stranded structure. By evaluation of the binding curves from changes in mass, the association rate constant (k(a)), dissociation rate constant (k(d)), and association constant (K-A) between coralyne and A(48) were determined to be 4.95 X 10(3) M-1 s(-1), 0.031 s(-1), and 1.6 X 10(5) M-1, respectively. Good linear correlations between coralyne concentrations ranging from 0.5 to 12 mu M and three parameters (mass, thickness, and RI) resolved by the response to coralyne binding were obtained. The detection limits were 0.22 mu M for mass calibration, 0.14 mu M for thickness calibration, and 0.32 mu M for RI calibration. The high selectivity of the biosensor to coralyne at the A(48)/PEI interface was successfully confirmed by using the other two interfaces (T-48/PEI and PEI) and three typical intercalators (ethidium bromide, daunomycin, and methylene blue). It is expected that the biosensing platform may be extended to simultaneously detect and characterize the interactions of a variety of target molecules with functional DNA molecules with high sensitivity.