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May 23, 2006

 

 

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.


dna microarray

Schematic of DNA structures in various conformations on a gold surface. Differences in overall structure and orientation are emphasized by color-coding of DNA structural elements: phosphate backbone (red), nitrogen (blue), oxygen (green), and sulfur linker (cyan). Upright orientation is required for efficient hybridization with a complementary strand from solution.

Looking Inside a DNA Chip

A simple test in the doctor's office reveals not only what is making a patient sick, but also what drug will work best to treat the disease. A similar test quickly checks a suspicious powder or a city's water supply for dangerous biowarfare agents. If this sounds like science fiction, it is only because the latest biotech marvel —the "DNA chip"—still needs a few more years to become part of everyday life.

DNA chips start with a piece of glass or silicon (sometimes coated with a thin layer of gold) about the size of a light-sensitive chip from a digital camera. Instead of electronic circuitry, however, DNA chips are covered with up to half a million "pixels" of precisely positioned microscopic droplets of DNA. The "probe DNA" in each pixel is designed to recognize a particular "target DNA" signature via a process called hybridization, which occurs when matching pairs of probe and target DNA combine to form the famous double helix.

For this process to work, each strand of probe DNA must stand up on the surface of the chip while being firmly attached to it by one end tagged with a sticky "linker" chemical. Petrovykh et al. have found a way to adapt methods normally used to study clean solid surfaces to obtain detailed information about how DNA strands are attached to DNA chips. This ability to scrutinize the details of DNA chips will become increasingly important for the development of future generations of smaller and more complex chips and related devices.
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.

diagram of xps and nexafs techniques

XPS and NEXAFS techniques use incident x rays to probe the core levels of nitrogen atoms (blue) in nucleobases (thymine shown). FTIR spectroscopy adds vibrational fingerprints of submolecular structures such as the C=O groups. NEXAFS and FTIR use linearly polarized incident photons and thus are sensitive, via the dipole selection rule, to the orientation of nitrogen π* orbitals (yellow) and C=O ligands, respectively.

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.

fluorescence yield

Fluorescence yield NEXAFS was used to determine the structure of DNA on gold surfaces. The changes in intensity of the nitrogen π* peaks as a function of the incident angle θi (inset) indicate strong preferential orientation of thymine bases in T5-SH monolayers but a nearly random distribution of orientations in T25-SH monolayers. For thymine nucleotides chemisorbed directly on gold—the dominant structure in T5 monolayers—the spectral features are shifted to lower photon energies (dashed gold lines, dT-Au).

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).

ALSNews Vol. 265, May 31, 2006

 

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