Progressing single biomolecule force spectroscopy measurements for the screening of DNA binding agents
| Autor: | Stephanie Allen, Claire Madden, Wenke Zhang, Alison Woolford, Romina Barbagallo, Clive J. Roberts |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Streptavidin
chemistry.chemical_classification Mechanical Engineering Biomolecule Force spectroscopy Bioengineering General Chemistry Small molecule chemistry.chemical_compound Crystallography chemistry Mechanics of Materials Biotinylation Biophysics General Materials Science A-DNA Electrical and Electronic Engineering Proflavine DNA |
| Zdroj: | Nanotechnology. 16:2325-2333 |
| ISSN: | 1361-6528 0957-4484 |
| DOI: | 10.1088/0957-4484/16/10/055 |
| Popis: | Recent studies have indicated that the force-extension properties of single molecules of double stranded (ds) DNA are sensitive to the presence of small molecule DNA binding agents, and also to their mode of binding. These observations raise the possibility of using this approach as a highly sensitive tool for the screening of such agents. However, particularly for studies employing the atomic force microscope (AFM), several non-trivial barriers hinder the progress of this approach to the non-specialist arena and hence also the full realization of this possibility. In this paper, we therefore address a series of key reproducibility and metrological issues associated with this type of measurement. Specifically, we present an improved immobilization method that covalently anchors one end (5' end) of a dual labelled (5'-thiol, 3'-biotin) p53 DNA molecule onto a gold substrate via gold-thiol chemistry, whilst the biotinylated 3' end is available for 'pick-up' using a streptavidin modified AFM tip. We also show that co-surface immobilization of DNA with 6-mercapto-1-hexanol (MCH) can also lead to a further increase the measured contour length. We demonstrate the impact of these improved protocols through the observation of the cooperative transition plateau in a DNA fragment of approximately 118 bp, a significantly smaller fragment than previously investigated. The results of a comparative study of the effects of a model minor groove binder (Hoechst 33258) and an intercalating drug (proflavine), alone, as a mixture and under different buffer conditions, are also presented. |
| Databáze: | OpenAIRE |
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