Unusual Structures in Super-Long CAG Repeat DNA Figure 6. Convoluted DNA is selectively digested by EcoP15I. (A, B) AFM images of DNA with 216 CAG repeats (total length 740 bp) before (A) and after (B) incubation with EcoP15I. (C, D) DNA with 360 CAG repeats (total length 1211 bp) before (C) and after (D) incubation with EcoP15I. Arrows in (D) indicate DNA molecules bearing enzyme bound at the ends. Arrowheads indicate residual tightly-wound tangles. All scale bars, 500 nm. (E, F) Frequency distributions of lengths of linear DNA in untreated (E) and EcoP15I-treated (F) samples of 360-repeat DNA. doi:10.1371/journal.pone.0017119.g006 tag. The cantilever was then lowered onto the antibody-coated surface, retracted, and the pulling forces between the cantilever and the surface measured. A typical pulling trace for the 114- repeat DNA had multiple force peaks, a final rupture force of 200 pN, and a pulling distance before rupture of 41 nm (Figure 7D). With the 360-repeat DNA, multiple force peaks were again seen, and in the case shown, the final rupture force was 159 pN, and the pulling distance before rupture was 117 nm (Figure 7E). The mean pulling distance before rupture was greater for the long- repeat DNA (72639 nm; S.D.; n = 22) than for the short-repeat DNA (32615 nm; n = 14; P,0.01). The mean number of peaks over a series of pulling events was 2.0060.75 for the short-repeat DNA and 2.8260.85 for the long-repeat DNA. The mean final rupture forces were very similar for the two types of DNA (126678 pN for the short-repeat DNA, and 125655 pN for the long-repeat DNA). Since both types of DNA had similar numbers of peaks per pulling event, despite the fact that only the long-repeat DNA has a significant number of convolutions, the multiple peaks likely represent the rupture of several DNA molecules conjugated at different points on the cantilever. The fact that the rupture forces are the same for the two types of DNA indicates that they represent the breakage of the same bond. This is likely to be the DIG-anti-DIG interaction, since antigen-antibody bonds have lower rupture forces (e.g. [40]) than the biotin-streptavidin bond (e.g. [41]). The fact that the rupture distance for the long-repeat DNA (72 nm) is much shorter than the total length of the DNA (370 nm) suggests that convoluted DNA is being attached to the cantilever. The distance across a typical convoluted molecule is about 100 nm (see Figure 2C, for example). Although the long- repeat DNA typically showed a rupture distance of less than 100 nm, in a few cases, a very long pulling distance was detected. PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e17119