Force spectroscopy and fluorescence microscopy of dsDNA-YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region

Chandrashekhar U Murade, Vinod Subramaniam, Cees Otto, Martin L Bennink

    Research output: Contribution to JournalArticleAcademicpeer-review

    Abstract

    When individual dsDNA molecules are stretched beyond their B-form contour length, they reveal a structural transition in which the molecule extends 1.7 times its contour length. The nature of this transition is still a subject of debate. In the first model, the DNA helix unwinds and combined with the tilting of the base pairs (which remain intact), results in a stretched form of DNA (also known as S-DNA). In the second model the base pairs break resulting effectively in two single-strands, which is referred to as force-induced melting. Here a combination of optical tweezers force spectroscopy with fluorescence microscopy was used to study the structure of dsDNA in the overstretching regime. When dsDNA was stretched in the presence of 10 nM YOYO-1 an initial increase in total fluorescence intensity of the dye-DNA complex was observed and at an extension where the dsDNA started to overstretch the fluorescence intensity leveled off and ultimately decreased when stretched further into the overstretching region. Simultaneous force spectroscopy and fluorescence polarization microscopy revealed that the orientation of dye molecules did not change significantly in the overstretching region (78.0 degrees +/- 3.2 degrees). These results presented here clearly suggest that, the structure of overstretched dsDNA can be explained accurately by force induced melting.

    Original languageEnglish
    Pages (from-to)3423-31
    Number of pages9
    JournalNucleic Acids Research
    Volume38
    Issue number10
    DOIs
    Publication statusPublished - Jun 2010

    Keywords

    • Benzoxazoles
    • DNA
    • Fluorescence Polarization
    • Fluorescent Dyes
    • Intercalating Agents
    • Microscopy
    • Microscopy, Fluorescence
    • Nucleic Acid Conformation
    • Optical Tweezers
    • Quinolinium Compounds
    • Journal Article
    • Research Support, Non-U.S. Gov't

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