|When DNA Needs to Stand Up and Be Counted|
DNA microarrays are small metal, glass, or silicon chips covered with patterns of short single-stranded DNA (ssDNA). These "DNA chips" are revolutionizing biotechnology, allowing scientists to identify and count many DNA sequences simultaneously. They are the enabling technology for genomic-based medicine and are a critical component of advanced diagnostic systems for medical and homeland security applications. Like digital chips, DNA chips are parallel, accurate, fast, and small. These advantages, however, can only be realized if the fragile biomolecules survive the attachment process intact. Furthermore, biomolecules must be properly oriented to perform their biological function. In other words, the DNA literally must stand up to be counted. Understanding both the attachment and orientation of DNA on gold surfaces was the goal of recent experiments performed at ALS Beamline 8.0.1 by an international collaboration of scientists.
The cornerstone of molecular biology is the study of the structure and function of biomolecules in solution. Likewise, the modified behavior of biomolecules on the surface of a chip or nanoparticle is the foundation of many areas of bio- and nanotechnology. However, characterizing the interactions between biomolecules and surfaces requires new measurement techniques, because most current bioanalytical methods are hindered by the inherently small number of molecules bound to a surface. Complementary surface-sensitive spectroscopies come to the rescue. X-ray photoelectron spectroscopy (XPS) provides quantitative information about elemental composition and surface chemistry. Fourier-transform infrared (FTIR) spectroscopy adds molecular fingerprints and orientational information. Near-edge x-ray absorption fine structure (NEXAFS) spectroscopy probes electronic transitions between core levels and valence orbitals.
Short strands of synthetic ssDNA typically used in bio- and nanotechnology are called oligonucleotides or oligos. Oligos with "trivial" sequences composed of only one letter of the DNA alphabet (A, C, G, or T) provide simplified spectroscopic signatures while maintaining realistic DNA structure. Thymine (T) has the simplest nucleobase structure and provides nitrogen atoms and carbonyl groups suitable for complementary measurements by XPS, FTIR, and NEXAFS. Furthermore, features in thymine spectra allow one to distinguish DNA strands lying down on the surface from those standing up.
In excellent agreement, all three techniques showed that strands of five Ts (T5), synthesized without special "linker" groups for surface attachment, lay flat against the surface, while strands modified with a thiol (T5-SH and T25-SH) stood upright, anchored by strong sulfur–gold bonds. Furthermore, signatures of internal molecular ("secondary") structure could be observed by NEXAFS for the upright T5-SH and T25-SH strands. The nitrogen π* orbitals within the T bases could be selectively excited by photons with energies around the nitrogen absorption edge. The polarization dependence of the nitrogen π* intensities then provided information about the orbital orientations, from which the T-base orientations could be deduced. Only minimal preferential orientation was observed for Ts in T25-SH films, consistent with a random-coil-like secondary structure. The Ts in T5-SH, however, showed a surprisingly strong orientation parallel to the surface. This result was corroborated by XPS and FTIR.
The significance of these results goes beyond the structure of a few DNA oligos. For DNA, RNA, and particularly proteins, secondary structure strongly affects function and contains valuable information about molecular interactions. Therefore, the researchers are already extending these analysis methods to larger biomolecules, such as proteins, on surfaces.
The consistent structural information from all three ex situ methods can be best understood if it reflects the common initial in situ structure, thus providing an affirmative answer to the long-standing question, "Can ex situ measurements provide relevant information about biomolecules on surfaces?" Furthermore, both FTIR and NEXAFS with fluorescence detection (as used in this work) can be performed in situ, opening the possibility of studying biomolecules on surfaces using a label-free method that provides a revolutionary combination of chemically specific, structurally sensitive, quantitative results.
Research conducted by D.Y. Petrovykh (University of Maryland and Naval Research Laboratory); A. Opdahl, H. Kimura-Suda, and M.J. Tarlov (National Institute of Standards and Technology [NIST]); V. Pérez-Dieste and F.J. Himpsel (University of Wisconsin); J.M. Sullivan (Northwestern University and Naval Research Laboratory); and L.J. Whitman (Naval Research Laboratory).
Research funding: Air Force Office of Scientific Research, Office of Naval Research, National Research Council postdoctoral program at NIST, U.S. Department of Energy, Office of Basic Energy Sciences (BES). Operation of the ALS is supported by BES.
Publication about this research: D.Y. Petrovykh, V. Pérez-Dieste, A. Opdahl, H. Kimura-Suda, J.M. Sullivan, M.J. Tarlov, F.J. Himpsel, and L.J. Whitman, "Nucleobase orientation and ordering in films of single-stranded DNA on gold," J. Am. Chem. Soc. 128, 2 (2006).