Eric Henderson

Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine·guanine base pairs

Structural properties of DNA oligonucleotides corresponding to the single-stranded molecular terminus of telomeres from several organisms were analyzed. Based on physical studies including nondenaturing polyacrylamide gel electrophoresis, absorbance thermal denaturation analysis, and 1H and 31P nuclear magnetic resonance spectroscopy, we conclude that these molecules can self-associate by forming non-Watson-Crick, guanine.guanine based-paired, intramolecular structures. These structures form below 40 degrees C at moderate ionic strength and neutral pH and behave like hairpin duplexes in nondenaturing polyacrylamide gels. Detailed analysis of the hairpin structure formed by the telomeric sequence from Tetrahymena, (T2G4)4, shows that it is a unique structure stabilized by hydrogen bonds and contains G residues in the syn conformation. We propose that this novel form of DNA is important for telomere function and sets a precedent for the biological relevance of non-Watson-Crick base-paired DNA structures.

An overhanging 3' terminus is a conserved feature of telomeres.

The reactivity of single-stranded thymidines with osmium tetraoxide was used to demonstrate the existence of a terminal overhang of the G-rich strand of telomeres from two distantly related eucaryotes, the ciliated protozoan Tetrahymena spp. and the acellular slime mold Didymium spp. Conservation of a G-strand overhang at the molecular terminus of telomeres is consistent with our suggestion that an unusual DNA structure formed by the G-strand overhang is important for telomere function (E. Henderson, C. C. Hardin, S. K. Wolk, I. Tinoco Jr., and E. H. Blackburn, Cell 51:899-908, 1987).

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

Studies using biophysical techniques suggest a highly structured organization of calcium channels at the presynaptic transmitter release face (Llinás et al., 1981; Stanley, 1993), but it has not as yet proved possible to localize identified channels at the required nanometer level of resolution. We have used atomic force microscopy on the calyx-type nerve terminal of the chick ciliary ganglion to localize single calcium channels tagged via biotinylated omega-conotoxin GVIA to avidin-coated 30 nm gold particles. Calcium channels were in low (modal value approximately < or = 1 per micron 2) and high (modal value approximately 55 per micron 2) density areas and exhibited a prominent interchannel spacing of 40 nm, indicating an intermolecular linkage. Particles were observed in clusters and short linear or parallel linear arrays, groupings that may reflect calcium channel organization at the transmitter release site.

Imaging of living cells by atomic force microscopy

Abstract The atomic force microscope (AFM, also referred to as the scanning force microscope, SFM) generates images of samples by measuring force interactions between the sample and a small, sharp tip on the end of a flexible cantilever. The general nature of this method permits the investigator to examine a large variety of sample types, including biological specimens. This review will focus on the application of AFM to imaging living cells.

Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.

Microfabricated quill-type surface patterning tools for the creation of biological micro/nano arrays.

Novel quill-type cantilever-based surface patterning tools (SPTs) were designed and constructed for use in controlled placement of femtoliter volumes of biological molecules on surfaces for biological applications. These tools were fabricated from silicon dioxide using microelectromechanical systems (MEMS) techniques. They featured a 1 μm split gap, fluidic transport microchannels and self-replenishing reservoirs. Experimental trials were performed using these tools on NanoArrayerTM molecular deposition instrumentation. Cy3-streptavidin was loaded as a biological sample and patterned on an amine-reactive dithiobis-succinimidyl undecanoate (DSU) monolayer on gold. Results showed these tools were capable of generating high quality biological arrays with routine spot sizes of 2–3 μm. The spot size could potentially achieve sub-micron dimensions if these SPT designs are reduced in size by more precise microfabrication techniques. The geometric designs of these tools facilitated sample replenishment from the local reservoir on the cantilever which allowed printing of large numbers of spots without sample reloading.

THREE-DIMENSIONAL PROBE RECONSTRUCTION FOR ATOMIC FORCE MICROSCOPY

Colloidal gold particles are used as hard, spherical imaging targets to assist in the three‐dimensional reconstruction of the atomic force probe apex. Probe reconstructions are shown to be accurate to 1 nm resolution and dynamically change as the sample is scanned, emphasizing the utility of colloidal gold particles as in situ calibration standards for image reconstruction of a coadsorbed specimen.

Detection and Quantification of Protein Biomarkers from Fewer than 10 Cells

The use of antibody microarrays continues to grow rapidly due to the recent advances in proteomics and automation and the opportunity this combination creates for high throughput multiplexed analysis of protein biomarkers. However, a primary limitation of this technology is the lack of PCR-like amplification methods for proteins. Therefore, to realize the full potential of array-based protein biomarker screening it is necessary to construct assays that can detect and quantify protein biomarkers with very high sensitivity, in the femtomolar range, and from limited sample quantities. We describe here the construction of ultramicroarrays, combining the advantages of microarraying including multiplexing capabilities, higher throughput, and cost savings with the ability to screen very small sample volumes. Antibody ultramicroarrays for the detection of interleukin-6 and prostate-specific antigen (PSA), a widely used biomarker for prostate cancer screening, were constructed. These ultramicroarrays were found to have a high specificity and sensitivity with detection levels using purified proteins in the attomole range. Using these ultramicroarrays, we were able to detect PSA secreted from 100 LNCaP cells in 3 h and from just four LNCaP cells in 24 h. Cellular PSA could also be detected from the lysate of an average of just six cells. This strategy should enable proteomic analysis of materials that are available in very limited quantities such as those collected by laser capture microdissection, neonatal biopsy microspecimens, and forensic samples.

Label-Free Protein and Pathogen Detection Using the Atomic Force Microscope

The atomic force microscope (AFM) uses a sharp micron-scale tip to scan and amplify surface features, providing exceptionally detailed topographical information with magnification on the order of ×106. This instrument is used extensively for quality control in the computer and semiconductor industries and is becoming a progressively more important tool in the biological sciences. Advantages of the AFM for biological application include the ability to obtain information in a direct, label-free manner and the ability to image in solution, providing real-time data acquisition under physiologically relevant conditions. A novel application of the AFM currently under development combines its surface profiling capabilities with fixed immuno-capture using antibodies immobilized in a nanoarray format. This provides a distinctive platform for direct, label-free detection and characterization of viral particles and other pathogens.

Imaging and manipulating chromosomes with the atomic force microscope.

Polytene chromosomes from the salivary gland cells of Drosophila melanogaster were examined by atomic force microscopy. The atomic force microscope (AFM) was capable of resolving chromosomal features down to the limits of the tip sharpness, about 500 Å for pyramidal-shaped tips. Resolution was increased to 300 Å by using electron beam deposited (EBD) tips with high aspect ratios. This significantly exceeds the resolution obtainable with conventional optical microscopes, but at the cost of compromising the structural integrity of the sample. A reasonable compromise was achieved by using oxide-sharpened tips. In this case high resolution was obtained without sample degradation, but when desired these tips were also capable of sample disintegration with increased scanning force and rate. Thus, oxide-sharpened tips were used to precisely dissect defined chromosomal regions to illustrate their potential use in genetic mapping efforts. This study illustrates the utility of the AFM in the characterization and manipulation of chromosomes and chromosomal DNA.