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This research focused on optimizing the preparations of pDNA-loaded calcium phosphate (CaP) nanoparticles by employing a 3-factor, 3-level Box-Behnken design. Results indicated that a Ca/P ratio of 189.56, pH of 7.82, and a stirring speed of 528.83 rpm were the optimum conditions for preparation of the nanoparticles. The size of the optimized CaP/pDNA nanoparticles was 61.3 ± 3.64 nm, with a polydispersity index of 0.341 and an encapsulation efficiency of up to 92.11%. The optimized CaP/pDNA nanoparticles had high transfection efficiency and demonstrated good biocompatibility in vitro. Therefore, the Box-Behnken design method was successful in providing desirable CaP nanoparticle pDNA delivery systems by optimizing the experimental factors., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.

Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications.

We demonstrate an accurate, quantitative, and label-free optical technology for high-throughput studies of receptor-ligand interactions, and apply it to TATA binding protein (TBP) interactions with oligonucleotides. We present a simple method to prepare single-stranded and double-stranded DNA microarrays with comparable surface density, ensuring an accurate comparison of TBP activity with both types of DNA. In particular, we find that TBP binds tightly to single-stranded DNA, especially to stretches of polythymine (poly-T), as well as to the traditional TATA box. We further investigate the correlation of TBP activity with various lengths of DNA and find that the number of TBPs bound to DNA increases >7-fold as the oligomer length increases from 9 to 40. Finally, we perform a full human genome analysis and discover that 35.5% of human promoters have poly-T stretches. In summary, we report, for the first time to our knowledge, the activity of TBP with poly-T stretches by presenting an elegant stepwise analysis of multiple techniques: discovery by a novel quantitative detection of microarrays, confirmation by a traditional gel electrophoresis, and a full genome prediction with computational analyses., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We utilize spectral self-interference fluorescent microscopy (SSFM) to measure fluorophore height with sub-nm precision to precisely quantify DNA orientation. A novel polymeric 3D scaffold is used to functionalize the sensor surface and to control orientation of the surface anchored DNA.

Quantitative determination of the density and conformation of DNA molecules tethered to the surface can help optimize and understand DNA nanosensors and nanodevices, which use conformational or motional changes of surface-immobilized DNA for detection or actuation. We present an interferometric sensing platform that combines (i) dual-color fluorescence spectroscopy for precise axial co-localization of two fluorophores attached at different nucleotides of surface-immobilized DNA molecules and (ii) independent label-free quantification of biomolecule surface density at the same site. Using this platform, we examined the conformation of DNA molecules immobilized on a three-dimensional polymeric surface and demonstrated simultaneous detection of DNA conformational change and binding in real-time. These results demonstrate that independent quantification of both surface density and molecular nanoscale conformation constitutes a versatile approach for nanoscale solid-biochemical interface investigations and molecular binding assays. [ABSTRACT FROM AUTHOR]

We demonstrate the application of a novel smart surface to modulate the orientation of immobilized double stranded DNA (dsDNA) and the conformation of a polymer scaffold through variation in buffer pH and ionic strength. An amphoteric poly(dimethylacrylamide) based coating containing weak acrylamido acids and bases, which are copolymerized together with the neutral monomer, is covalently bound to the surface. The coating can be made to contain any desired amount of buffering and titrant ionogenic monomers, allowing control of the surface charge when the surface is bathed in a given buffer pH. Spectral self-interference fluorescence microscopy (SSFM) is utilized to precisely quantify both the DNA orientation and the polymer conformation with subnanometer resolution. It is possible to utilize the polymer scaffold to functionalize a variety of common materials used in microfabrication, making it a general purpose building block for the next generation of nanomachines and biosensors. [ABSTRACT FROM AUTHOR]