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Biomedical subjects

L A Bottomley

Publications and source records attributed to L A Bottomley.

6 recordsLinked to original sources
PubMed2001

Scanning force microscopy of nucleic acid complexes.

SFM is a viable effective method for determining the mode of binding, the extent of binding, and the site of binding of intercalators to nucleic acids. Establishing the presence of a groove-bound ligand can be achieved either by competitive binding experiments with a well-defined intercalator (minor groove) or by changes in apparent contrast (major groove). In our opinion, SFM has an important role in resolving the structural polymorphisms for small molecule-DNA complexes. Application of these assays in the study of polyintercalator molecules is currently underway in our laboratory. SFM is an important, new tool in the study of protein-DNA complexes. New insights into the structure and function of these complexes are enabled by real-time visualization. Currently the temporal resolution of the SFM limits the degree to which definitive rate data can be determined. Several binding and unbinding events could take place in the time it takes to acquire one image. New developments in SFM technology will allow faster scanning and will improve the temporal resolution of so-called SFM movies. To this end, the Hansma group is developing small cantilevers and improved optical deflection systems to enable intermittent imaging at scanning rates of 1.7 sec per image. These improvements will enable SFM visualization of complex biological processes as they occur, one molecule at a time.

Binding Sites↗
PubMed1996

A novel assay for drug-DNA binding mode, affinity, and exclusion number: scanning force microscopy.

Determining the mode-of-binding of a DNA ligand is not always straightforward. Here, we establish a scanning force microscopic assay for mode-of-binding that is (i) direct: lengths of individual DNA-ligand complexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an observed increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding. Mode-of-binding, binding affinity, and site-exclusion number are readily determined from scanning force microscopy measurements of the changes in length of individual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2,5-bis(4-amidinophenyl) furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of aromatic dicationic drugs that show significant activity in the treatment of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.

Amidines↗
PubMed1992

Immobilization of DNA for scanning probe microscopy.

Reproducible scanning tunneling microscope and atomic force microscope images of entire molecules of uncoated plasmid DNA chemically bound to surfaces are presented. The chemically mediated immobilization of DNA to surfaces and subsequent scanning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with scanning probe microscopes, can be prevented.

DNA, Bacterial↗
PubMed1992

Scanning tunneling microscopy of DNA: a novel technique using radiolabeled DNA to evaluate chemically mediated attachment of DNA to surfaces.

pBS+ plasmid deoxyribonucleic acid (DNA) was imaged by scanning tunneling microscopy (STM) after mounting microdroplets by aerosol deposition onto heated epitaxial gold surfaces. However, the instability of the adsorbate to forces exerted by the tunneling tip points out the need for more aggressive bonding of molecules to surfaces. We describe a sensitive assay for the qualitative and quantitative evaluation of chemical agents to influence binding of DNA to surfaces using 32P-labeled pBS+ plasmid DNA. We propose that such an assay can make an important contribution to immobilization techniques prior to STM imaging.

DNA↗