Khin Moh Moh Aung

SPR study of DNA hybridization with DNA and PNA probes under stringent conditions.

Surface plasmon resonance (SPR) spectroscopy has been used for studying on-chip DNA hybridization to a PNA probe and its counterpart DNA probe of a 22-mer sequence. Two stringency control strategies are used for single base mismatch discrimination, namely (1) adding a denaturant, i.e. formamide (FA), into hybridization buffer and (2) coupling negative potentials for selective dehybridization of mismatch DNA. These two strategies have either not been used before or been less-well studied in SPR detection. An end-point SPR measurement protocol (no real-time hybridization profile recorded) is developed for detecting DNA hybridization in the presence of FA, to circumvent the problem that the refractive index of FA is out of the detectable range of the SPR equipment. The missing of real-time measurement of hybridization profile is compensated with QCM measurement. Under optimal conditions, i.e. 10mM PBS with 30% FA and 1mM PBS with 50% FA, single base mismatch DNA is detected with 1.7 and 2.8 times less hybridization signals compared with the perfect match DNA, with the DNA probe and PNA probe, respectively. Under negative potential of -0.2 to -0.4V (vs. Ag/AgCl), mismatch DNA dissociates more than perfect match DNA by 1.7-2.5 times from the DNA probe and 2.1-3.5 times from the PNA probe. The higher mismatch discrimination efficiency of the PNA probe under stringent conditions would be attributable to its higher intrinsic sequence selectivity.

Femtomol SPR detection of DNA–PNA hybridization with the assistance of DNA-guided polyaniline deposition

The inability of surface plasmon resonance (SPR) spectroscopy to detect extremely small refractive index changes has hindered its applications in ultrasensitive DNA analysis. In this study we report a signal amplification strategy that uses DNA-templated polyaniline deposition, suitable for DNA hybridization analysis with charge neutral peptide nucleic acid (PNA) being probes. Under acidic conditions, protonated aniline monomers are adsorbed on DNA backbones through electrostatic interaction. The microenvironment provided by the DNA facilitates oxidative aniline polymerization initialized by H(2)O(2) in the presence of horseradish peroxide. Under optimal conditions, the detection limit is lowered from 5nM for conventional SPR detection to 0.1pM. The significant sensitivity improvement is attributed to the in-situ polymer chain growth along DNA strands, which introduces drastic refractive index increases. This signal amplification approach does not involve secondary hybridization processes. The detection sensitivity obtained is much better than that of gold nanoparticle-based amplification involving a secondary hybridization process and labeled DNA detection probes.

Studying forkhead box protein A1–DNA interaction and ligand inhibition using gold nanoparticles, electrophoretic mobility shift assay, and fluorescence anisotropy

Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. Conventional analytical methods-electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA)-as well as a gold nanoparticles (AuNPs)-based assay were used to study DNA binding properties of FoxA1 and ligand interruption of FoxA1-DNA binding. In the AuNPs assay, the distinct ability of protein-DNA complex to protect AuNPs against salt-induced aggregation was exploited to screen sequence selectivity and determine the binding affinity constant based on AuNPs color change and absorbance spectrum shift. Both conventional EMSA and FA and the AuNPs assay suggested that FoxA1 binds to DNA in a core sequence-dependent manner and the flanking sequence also played a role to influence the affinity. The EMSA and AuNPs were found to be more sensitive than FA in differentiation of sequence-dependent affinity. With the addition of a spin filtration step, AuNPs assay has been extended for studying small molecular ligand inhibition of FoxA1-DNA interactions enabling drug screening. The results correlate very well with those obtained using FA.