DNA conformation on surfaces measured by fluorescence self-interference

Abstract

The conformation of DNA molecules tethered to the surface of a microarray may significantly affect the efficiency of hybridization. Although a number of methods have been applied to determine the structure of the DNA layer, they are not very sensitive to variations in the shape of DNA molecules. Here we describe the application of an interferometric technique called spectral selfinterference fluorescence microscopy to the precise measurement of the average location of a fluorescent label in a DNA layer relative to the surface and thus determine specific information on the conformation of the surface-bound DNA molecules. Using spectral self-interference fluorescence microscopy, we have estimated the shape of coiled single-stranded DNA, the average tilt of doublestranded DNA of different lengths, and the amount of hybridization. The data provide important proofs of concept for the capabilities of novel optical surface analytical methods of the molecular disposition of DNA on surfaces. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields.hybridization ͉ microarray ͉ spectroscopy D NA array technology has become a widespread tool in biological research with applications in expression screening, sequencing, and drug discovery, all benefiting greatly by the highly paralleled detection of the technique. One of the defining characteristics of a DNA array is the availability of the single-stranded probes for hybridization with the target. Immobilized molecules located farther away from the solid support are closer to the solution state and are more accessible for contact with dissolved analytes. The surface, especially a hydrophobic one, acts as a shield for probes positioned close to it because of the associated steric factors and lack of diffusion of the bound molecules (1-5). Thus, knowing the physical structure of DNA probes may prove useful not only in the future development and fabrication of microarrays, but also in designing new applications (6).Recently, advances have been made to characterize the structure of surface-bound DNA probes using such optical or contact methods as ellipsometry, optical reflectivity (7, 8), neutron reflectivity (9), x-ray photoelectron spectroscopy (10), FRET (11,12), SPR (13,14), and AFM (15-18). This previous work has helped in visualizing the structure of the surface-bound DNA depending on its density and surface charge. However, most experimental techniques characterize the DNA layer as a single entity (for instance, parameterizing its thickness or density) without examining the specific positions of internal elements of the DNA chain. Spectral selfinterference fluorescence microscopy (SSFM) can measure the vertical position of a fluorescent label above an optically structured silicon chip. We show that locating the label attached to a certain position within a DNA chain provides insight into the shape of DNA molecules boun...

Diamond‐based DNA sensors: surface functionalization and read‐out strategies

“…The values found here are comparable to those reported on Au surfaces: for 15 bp ds-DNA an average tilt angle of 55-60° with respect to the surface plane is found [92], and for 21-50 bp ds-DNA average tilt angles of 40-50° from the surface are measured [93]. Yet, our values are larger than those already mentioned, obtained by nano-shaving AFM experiments [62] (30-36° for 16 bp ds-DNA on top of a 15 b ss-DNA-tail).…”

Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications

“…Biacore measurements have recently expanded from four channels (Biacore T100) with imaging SPR technology, yet reported examples are still limited to a few hundred spots (17). Wavelength-dependent reflectance from layered surfaces has also been studied for optical detection of biomolecular interactions for many years (18)(19)(20)(21)(22)(23)(24). We argue that SRIB presents advantages when compared with these previous techniques.…”

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